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Eukaryotic DNA replication

From Wikipedia, the free encyclopedia
https://en.wikipedia.org/wiki/Eukaryotic_DNA_replication

Eukaryotic DNA replication is a conserved mechanism that restricts DNA replication to once per cell cycle. Eukaryotic DNA replication of chromosomal DNA is central for the duplication of a cell and is necessary for the maintenance of the eukaryotic genome.

DNA replication is the action of DNA polymerases synthesizing a DNA strand complementary to the original template strand. To synthesize DNA, the double-stranded DNA is unwound by DNA helicases ahead of polymerases, forming a replication fork containing two single-stranded templates. Replication processes permit copying a single DNA double helix into two DNA helices, which are divided into the daughter cells at mitosis. The major enzymatic functions carried out at the replication fork are well conserved from prokaryotes to eukaryotes, but the replication machinery in eukaryotic DNA replication is a much larger complex, coordinating many proteins at the site of replication, forming the replisome.

The replisome is responsible for copying the entirety of genomic DNA in each proliferative cell. This process allows for the high-fidelity passage of hereditary/genetic information from parental cell to daughter cell and is thus essential to all organisms. Much of the cell cycle is built around ensuring that DNA replication occurs without errors.

In G₁ phase of the cell cycle, many of the DNA replication regulatory processes are initiated. In eukaryotes, the vast majority of DNA synthesis occurs during S phase of the cell cycle, and the entire genome must be unwound and duplicated to form two daughter copies. During G₂, any damaged DNA or replication errors are corrected. Finally, one copy of the genomes is segregated into each daughter cell at the mitosis or M phase. These daughter copies each contains one strand from the parental duplex DNA and one nascent antiparallel strand.

This mechanism is conserved from prokaryotes to eukaryotes and is known as semiconservative DNA replication. The process of semiconservative replication for the site of DNA replication is a fork-like DNA structure, the replication fork, where the DNA helix is open, or unwound, exposing unpaired DNA nucleotides for recognition and base pairing for the incorporation of free nucleotides into double-stranded DNA.

Initiation

Initiation of eukaryotic DNA replication is the first stage of DNA synthesis where the DNA double helix is unwound and an initial priming event by DNA polymerase α occurs on the leading strand. The priming event on the lagging strand establishes a replication fork. Priming of the DNA helix consists of the synthesis of an RNA primer to allow DNA synthesis by DNA polymerase α. Priming occurs once at the origin on the leading strand and at the start of each Okazaki fragment on the lagging strand.

Origin of replication

Replication starts at origins of replication. DNA sequences containing these sites were initially isolated in the late 1970s on the basis of their ability to support replication of plasmids, hence the designation of autonomously replicating sequences (ARS). Origins vary widely in their efficiency, with some being used in almost every cell cycle while others may be used in only one in one thousand S phases. The total number of yeast ARSs is at least 1600, but may be more than 5000 if less active sites are counted, that is, there may be an ARS every 2000 to 8000 base pairs.

Pre-replicative complex

Multiple replicative proteins assemble on and dissociate from these replicative origins to initiate DNA replication. with the formation of the pre-replication complex (pre-RC) being a key intermediate in the replication initiation process.

Association of the origin recognition complex (ORC) with a replication origin recruits the cell division cycle 6 protein (Cdc6) to form a platform for the loading of the minichromosome maintenance (Mcm 2–7) complex proteins, facilitated by the chromatin licensing and DNA replication factor 1 protein (Cdt1). The ORC, Cdc6, and Cdt1 together are required for the stable association of the Mcm2-7 complex with replicative origins during the G₁ phase of the cell cycle.

Eukaryotic origins of replication control the formation of several protein complexes that lead to the assembly of two bidirectional DNA replication forks. These events are initiated by the formation of the pre-replication complex (pre-RC) at the origins of replication. This process takes place in the G₁ stage of the cell cycle. The pre-RC formation involves the ordered assembly of many replication factors including the origin recognition complex (ORC), Cdc6 protein, Cdt1 protein, and minichromosome maintenance proteins (Mcm2-7). Once the pre-RC is formed, activation of the complex is triggered by two kinases, cyclin-dependent kinase 2 (CDK) and Dbf4-dependent kinase (DDK) that help transition the pre-RC to the initiation complex before the initiation of DNA replication. This transition involves the ordered assembly of additional replication factors to unwind the DNA and accumulate the multiple eukaryotic DNA polymerases around the unwound DNA. Central to the question of how bidirectional replication forks are established at replication origins is the mechanism by which ORC recruits two head-to-head Mcm2-7 complexes to every replication origin to form the pre-replication complex.

Origin recognition complex

The first step in the assembly of the pre-replication complex (pre-RC) is the binding of the origin recognition complex (ORC) to the replication origin. In late mitosis, the Cdc6 protein joins the bound ORC followed by binding the Cdt1-Mcm2-7 complex. ORC, Cdc6, and Cdt1 are all required to load the six protein minichromosome maintenance (Mcm 2–7) complex onto the DNA. The ORC is a six-subunit, Orc1p-6, protein complex that selects the replicative origin sites on DNA for initiation of replication and ORC binding to chromatin is regulated through the cell cycle. Generally, the function and size of the ORC subunits are conserved throughout many eukaryotic genomes with the difference being their diverged DNA binding sites.

The most widely studied origin recognition complex is that of Saccharomyces cerevisiae or yeast which is known to bind to the autonomously replicating sequence (ARS). The S. cerevisiae ORC interacts specifically with both the A and B1 elements of yeast origins of replication, spanning a region of 30 base pairs. The binding to these sequences requires ATP.

The atomic structure of the S. cerevisiae ORC bound to ARS DNA has been determined. Orc1, Orc2, Orc3, Orc4, and Orc5 encircle the A element by means of two types of interactions, base non-specific and base-specific, that bend the DNA at the A element. All five subunits contact the sugar phosphate backbone at multiple points of the A element to form a tight grip without base specificity. Orc1 and Orc2 contact the minor groove of the A element while a winged helix domain of Orc4 contacts the methyl groups of the invariant Ts in the major groove of the A element via an insertion helix (IH). The absence of this IH in metazoans explains the lack of sequence specificity in human ORC. Removing the IH from the ScORC causes it to lose its specificity for the A element, and to bind promiscuously and preferentially (83%) to promoter regions. The ARS DNA is also bent at the B1 element through interactions with Orc2, Orc5 and Orc6. The bending of origin DNA by ORC appears to be evolutionarily conserved suggesting that it may be required for the Mcm2-7 complex loading mechanism.

When the ORC binds to DNA at replication origins, it serves as a scaffold for the assembly of other key initiation factors of the pre-replicative complex. This pre-replicative complex assembly during the G₁ stage of the cell cycle is required prior to the activation of DNA replication during the S phase. The removal of at least part of the complex (Orc1) from the chromosome at metaphase is part of the regulation of mammalian ORC to ensure that the pre-replicative complex formation prior to the completion of metaphase is eliminated.

Cdc6 protein

Binding of the cell division cycle 6 (Cdc6) protein to the origin recognition complex (ORC) is an essential step in the assembly of the pre-replication complex (pre-RC) at the origins of replication. Cdc6 binds to the ORC on DNA in an ATP-dependent manner, which induces a change in the pattern of origin binding that requires Orc1 ATPase. Cdc6 requires ORC in order to associate with chromatin and is in turn required for the Cdt1-Mcm2-7 heptamer to bind to the chromatin. The ORC-Cdc6 complex forms a ring-shaped structure and is analogous to other ATP-dependent protein machines. The levels and activity of Cdc6 regulate the frequency with which the origins of replication are utilized during the cell cycle.

Cdt1 protein

The chromatin licensing and DNA replication factor 1 (Cdt1) protein is required for the licensing of chromatin for DNA replication. In S. cerevisiae, Cdt1 facilitates the loading of the Mcm2-7 complex one at a time onto the chromosome by stabilising the left-handed open-ring structure of the Mcm2-7 single hexamer. Cdt1 has been shown to associate with the C terminus of Cdc6 to cooperatively promote the association of Mcm proteins to the chromatin. The cryo-EM structure of the OCCM (ORC-Cdc6-Cdt1-MCM) complex shows that the Cdt1-CTD interacts with the Mcm6-WHD. In metazoans, Cdt1 activity during the cell cycle is tightly regulated by its association with the protein geminin, which both inhibits Cdt1 activity during S phase in order to prevent re-replication of DNA and prevents it from ubiquitination and subsequent proteolysis.

Minichromosome maintenance protein complex

The minichromosome maintenance (Mcm) proteins were named after a genetic screen for DNA replication initiation mutants in S. cerevisiae that affect plasmid stability in an ARS-specific manner. Mcm2, Mcm3, Mcm4, Mcm5, Mcm6 and Mcm7 form a hexameric complex that has an open-ring structure with a gap between Mcm2 and Mcm5. The assembly of the Mcm proteins onto chromatin requires the coordinated function of the origin recognition complex (ORC), Cdc6, and Cdt1. Once the Mcm proteins have been loaded onto the chromatin, ORC and Cdc6 can be removed from the chromatin without preventing subsequent DNA replication. This observation suggests that the primary role of the pre-replication complex is to correctly load the Mcm proteins.

The Mcm proteins on chromatin form a head-to-head double hexamer with the two rings slightly tilted, twisted and off-centred to create a kink in the central channel where the bound DNA is captured at the interface of the two rings. Each hexameric Mcm2-7 ring first serves as the scaffold for the assembly of the replisome and then as the core of the catalytic CMG (Cdc45-MCM-GINS) helicase, which is a main component of the replisome. Each Mcm protein is highly related to all others, but unique sequences distinguishing each of the subunit types are conserved across eukaryotes. All eukaryotes have exactly six Mcm protein analogs that each fall into one of the existing classes (Mcm2-7), indicating that each Mcm protein has a unique and important function.

Minichromosome maintenance proteins are required for DNA helicase activity. Inactivation of any of the six Mcm proteins during S phase irreversibly prevents further progression of the replication fork suggesting that the helicase cannot be recycled and must be assembled at replication origins. Along with the minichromosome maintenance protein complex helicase activity, the complex also has associated ATPase activity. Studies have shown that within the Mcm protein complex are specific catalytic pairs of Mcm proteins that function together to coordinate ATP hydrolysis. These studies, confirmed by cryo-EM structures of the Mcm2-7 complexes, showed that the Mcm complex is a hexamer with subunits arranged in a ring in the order of Mcm2-Mcm6-Mcm4-Mcm7-Mcm3-Mcm5-. Both members of each catalytic pair contribute to the conformation that allows ATP binding and hydrolysis and the mixture of active and inactive subunits presumably allows the Mcm hexameric complex to complete ATP binding and hydrolysis as a whole to create a coordinated ATPase activity.

The nuclear localization of the minichromosome maintenance proteins is regulated in budding yeast cells. The Mcm proteins are present in the nucleus in G₁ stage and S phase of the cell cycle, but are exported to the cytoplasm during the G₂ stage and M phase. A complete and intact six subunit Mcm complex is required to enter into the cell nucleus. In S. cerevisiae, nuclear export is promoted by cyclin-dependent kinase (CDK) activity. Mcm proteins that are associated with chromatin are protected from CDK export machinery due to the lack of accessibility to CDK.

Initiation complex

During the G₁ stage of the cell cycle, the replication initiation factors, origin recognition complex (ORC), Cdc6, Cdt1, and minichromosome maintenance (Mcm) protein complex, bind sequentially to DNA to form a head-to-head dimer of the MCM ring complex, known as the pre-replication complex (pre-RC). While the yeast pre-RC forms a closed DNA complex, the human pre-RC forms an open complex.^[47] At the transition of the G₁ stage to the S phase of the cell cycle, S phase–specific cyclin-dependent protein kinase (CDK) and Cdc7/Dbf4 kinase (DDK) transform the inert pre-RC into an active complex capable of assembling two bidirectional replisomes. CryoEM structures showed that two DDKs independently dock onto the interface of the MCM double hexamer straddling across the two rings. The sequential phosphorylation of multiple substrates on the NTEs of Mcm4, Mcm2 and Mcm6 is achieved by a wobble mechanism whereby Dbf4 assumes different wobble states to position Cdc7 over its multiple substrates. Phosphorylation of the MCM double hexamer, the Mcm4-NSD in particular, by DDK is essential for viability in yeast. The recruitment of Cdc45 and GINS follows after the activation of the MCMs by DDK and CDK.

Cdc45 protein

Cell division cycle 45 (Cdc45) protein is a critical component for the conversion of the pre-replicative complex to the initiation complex. The Cdc45 protein assembles at replication origins before initiation and is required for replication to begin in Saccharomyces cerevisiae, and has an essential role during elongation. Thus, Cdc45 has central roles in both initiation and elongation phases of chromosomal DNA replication.

Cdc45 associates with chromatin after the beginning of initiation in late G₁ stage and during the S phase of the cell cycle. Cdc45 physically associates with Mcm5 and displays genetic interactions with five of the six members of the Mcm gene family and the ORC2 gene. The loading of Cdc45 onto chromatin is critical for loading other various replication proteins, including DNA polymerase α, DNA polymerase ε, replication protein A (RPA) and proliferating cell nuclear antigen (PCNA) onto chromatin. Within a Xenopus nucleus-free system, it has been demonstrated that Cdc45 is required for the unwinding of plasmid DNA. The Xenopus nucleus-free system also demonstrates that DNA unwinding and tight RPA binding to chromatin occurs only in the presence of Cdc45.

Binding of Cdc45 to chromatin depends on Clb-Cdc28 kinase activity as well as functional Cdc6 and Mcm2, which suggests that Cdc45 associates with the pre-RC after activation of S-phase cyclin-dependent kinases (CDKs). As indicated by the timing and the CDK dependence, binding of Cdc45 to chromatin is crucial for commitment to initiation of DNA replication. During S phase, Cdc45 physically interacts with Mcm proteins on chromatin; however, dissociation of Cdc45 from chromatin is slower than that of the Mcm, which indicates that the proteins are released by different mechanisms.

GINS

The six minichromosome maintenance proteins and Cdc45 are essential during initiation and elongation for the movement of replication forks and for unwinding of the DNA. GINS are essential for the interaction of Mcm and Cdc45 at the origins of replication during initiation and then at DNA replication forks as the replisome progresses. The GINS complex is composed of four small proteins Sld5 (Cdc105), Psf1 (Cdc101), Psf2 (Cdc102) and Psf3 (Cdc103), GINS represents 'go, ichi, ni, san' which means '5, 1, 2, 3' in Japanese. Cdc45, Mcm2-7 and GINS together form the CMG helicase, the replicative helicase of the replisome. Although the Mcm2-7 complex alone has weak helicase activity Cdc45 and GINS are required for robust helicase activity

Mcm10

Mcm10 is essential for chromosome replication and interacts with the minichromosome maintenance 2-7 helicase that is loaded in an inactive form at origins of DNA replication. Mcm10 also chaperones the catalytic DNA polymerase α and helps stabilize the polymerase at replication forks.

DDK and CDK kinases

At the onset of S phase, the pre-replicative complex must be activated by two S phase-specific kinases in order to form an initiation complex at an origin of replication. One kinase is the Cdc7-Dbf4 kinase called Dbf4-dependent kinase (DDK) and the other is cyclin-dependent kinase (CDK). Chromatin-binding assays of Cdc45 in yeast and Xenopus have shown that a downstream event of CDK action is loading of Cdc45 onto chromatin. Cdc6 has been speculated to be a target of CDK action, because of the association between Cdc6 and CDK, and the CDK-dependent phosphorylation of Cdc6. The CDK-dependent phosphorylation of Cdc6 has been considered to be required for entry into the S phase.

Both the catalytic subunits of DDK, Cdc7, and the activator protein, Dbf4, are conserved in eukaryotes and are required for the onset of S phase of the cell cycle. Both Dbf4 and Cdc7 are required for the loading of Cdc45 onto chromatin origins of replication. The target for binding of the DDK kinase is the chromatin-bound form of the Mcm complex. High resolution cryoEM structures showed that the Dbf4 subunit of DDK straddles across the hexamer interface of the DNA-bound MCM-DH, contacting Mcm2 of one hexamer and Mcm4/6 of the opposite hexamer. Mcm2, Mcm4 and Mcm6 are all substrates of phosphorylation by DDK but only the N-terminal serine/threonine-rich domain (NSD) of Mcm4 is an essential DDK target. Phosphorylation of the NSD leads to the activation of Mcm helicase activity.

Dpb11, Sld3, and Sld2 proteins

Sld3, Sld2, and Dpb11 interact with many replication proteins. Sld3 and Cdc45 form a complex that associated with the pre-RC at the early origins of replication even in the G1₁ phase and with the later origins of replication in the S phase in a mutually Mcm-dependent manner. Dpb11 and Sld2 interact with Polymerase ɛ and cross-linking experiments have indicated that Dpb11 and Polymerase ɛ coprecipitate in the S phase and associate with replication origins.

Sld3 and Sld2 are phosphorylated by CDK, which enables the two replicative proteins to bind to Dpb11. Dpb11 had two pairs of BRCA1 C Terminus (BRCT) domains which are known as a phosphopeptide-binding domains. The N-terminal pair of the BRCT domains binds to phosphorylated Sld3, and the C-terminal pair binds to phosphorylated Sld2. Both of these interactions are essential for CDK-dependent activation of DNA budding in yeast.

Dpb11 also interacts with GINS and participates in the initiation and elongation steps of chromosomal DNA replication. GINS are one of the replication proteins found at the replication forks and forms a complex with Cdc45 and Mcm.

These phosphorylation-dependent interactions between Dpb11, Sld2, and Sld3 are essential for CDK-dependent activation of DNA replication, and by using cross-linking reagents within some experiments, a fragile complex was identified called the pre-loading complex (pre-LC). This complex contains Pol ɛ, GINS, Sld2, and Dpb11. The pre-LC is found to form before any association with the origins in a CDK-dependent and DDK-dependent manner and CDK activity regulates the initiation of DNA replication through the formation of the pre-LC.

Elongation

The formation of the pre-replicative complex (pre-RC) marks the potential sites for the initiation of DNA replication. Consistent with the minichromosome maintenance complex encircling double stranded DNA, formation of the pre-RC does not lead to the immediate unwinding of origin DNA or the recruitment of DNA polymerases. Instead, the pre-RC that is formed during the G₁ of the cell cycle is only activated to unwind the DNA and initiate replication after the cells pass from the G₁ to the S phase of the cell cycle.

Once the initiation complex is formed and the cells pass into the S phase, the complex then becomes a replisome. The eukaryotic replisome complex is responsible for coordinating DNA replication. Replication on the leading and lagging strands is performed by DNA polymerase ε and DNA polymerase δ. Many replisome factors including Claspin, And1, replication factor C clamp loader and the fork protection complex are responsible for regulating polymerase functions and coordinating DNA synthesis with the unwinding of the template strand by Cdc45-Mcm-GINS complex. As the DNA is unwound the twist number decreases. To compensate for this the writhe number increases, introducing positive supercoils in the DNA. These supercoils would cause DNA replication to halt if they were not removed. Topoisomerases are responsible for removing these supercoils ahead of the replication fork.

The replisome is responsible for copying the entire genomic DNA in each proliferative cell. The base pairing and chain formation reactions, which form the daughter helix, are catalyzed by DNA polymerases. These enzymes move along single-stranded DNA and allow for the extension of the nascent DNA strand by "reading" the template strand and allowing for incorporation of the proper purine nucleobases, adenine and guanine, and pyrimidine nucleobases, thymine and cytosine. Activated free deoxyribonucleotides exist in the cell as deoxyribonucleotide triphosphates (dNTPs). These free nucleotides are added to an exposed 3'-hydroxyl group on the last incorporated nucleotide. In this reaction, a pyrophosphate is released from the free dNTP, generating energy for the polymerization reaction and exposing the 5' monophosphate, which is then covalently bonded to the 3' oxygen. Additionally, incorrectly inserted nucleotides can be removed and replaced by the correct nucleotides in an energetically favorable reaction. This property is vital to proper proofreading and repair of errors that occur during DNA replication.

Replication fork

The replication fork is the junction between the newly separated template strands, known as the leading and lagging strands, and the double stranded DNA. Since duplex DNA is antiparallel, DNA replication occurs in opposite directions between the two new strands at the replication fork, but all DNA polymerases synthesize DNA in the 5' to 3' direction with respect to the newly synthesized strand. Further coordination is required during DNA replication. Two replicative polymerases synthesize DNA in opposite orientations. Polymerase ε synthesizes DNA on the "leading" DNA strand continuously as it is pointing in the same direction as DNA unwinding by the replisome. In contrast, polymerase δ synthesizes DNA on the "lagging" strand, which is the opposite DNA template strand, in a fragmented or discontinuous manner.

The discontinuous stretches of DNA replication products on the lagging strand are known as Okazaki fragments and are about 100 to 200 bases in length at eukaryotic replication forks. The lagging strand usually contains longer stretches of single-stranded DNA that is coated with single-stranded binding proteins, which help stabilize the single-stranded templates by preventing a secondary structure formation. In eukaryotes, these single-stranded binding proteins are a heterotrimeric complex known as replication protein A (RPA).

Each Okazaki fragment is preceded by an RNA primer, which is displaced by the procession of the next Okazaki fragment during synthesis. RNase H recognizes the DNA:RNA hybrids that are created by the use of RNA primers and is responsible for removing these from the replicated strand, leaving behind a primer:template junction. DNA polymerase α, recognizes these sites and elongates the breaks left by primer removal. In eukaryotic cells, a small amount of the DNA segment immediately upstream of the RNA primer is also displaced, creating a flap structure. This flap is then cleaved by endonucleases. At the replication fork, the gap in DNA after removal of the flap is sealed by DNA ligase I, which repairs the nicks that are left between the 3'-OH and 5'phosphate of the newly synthesized strand. Owing to the relatively short nature of the eukaryotic Okazaki fragment, DNA replication synthesis occurring discontinuously on the lagging strand is less efficient and more time-consuming than leading-strand synthesis. DNA synthesis is complete once all RNA primers are removed and nicks are repaired.

Leading strand

During DNA replication, the replisome will unwind the parental duplex DNA into a two single-stranded DNA template replication fork in a 5' to 3' direction. The leading strand is the template strand that is being replicated in the same direction as the movement of the replication fork. This allows the newly synthesized strand complementary to the original strand to be synthesized 5' to 3' in the same direction as the movement of the replication fork.

Once an RNA primer has been added by a primase to the 3' end of the leading strand, DNA synthesis will continue in a 3' to 5' direction with respect to the leading strand uninterrupted. DNA Polymerase ε will continuously add nucleotides to the template strand therefore making leading strand synthesis require only one primer and has uninterrupted DNA polymerase activity.

Lagging strand

DNA replication on the lagging strand is discontinuous. In lagging strand synthesis, the movement of DNA polymerase in the opposite direction of the replication fork requires the use of multiple RNA primers. DNA polymerase will synthesize short fragments of DNA called Okazaki fragments which are added to the 3' end of the primer. These fragments can be anywhere between 100 and 400 nucleotides long in eukaryotes.

At the end of Okazaki fragment synthesis, DNA polymerase δ runs into the previous Okazaki fragment and displaces its 5' end containing the RNA primer and a small segment of DNA. This generates an RNA-DNA single strand flap, which must be cleaved, and the nick between the two Okazaki fragments must be sealed by DNA ligase I. This process is known as Okazaki fragment maturation and can be handled in two ways: one mechanism processes short flaps, while the other deals with long flaps. DNA polymerase δ is able to displace up to 2 to 3 nucleotides of DNA or RNA ahead of its polymerization, generating a short "flap" substrate for Fen1, which can remove nucleotides from the flap, one nucleotide at a time.

By repeating cycles of this process, DNA polymerase δ and Fen1 can coordinate the removal of RNA primers and leave a DNA nick at the lagging strand. It has been proposed that this iterative process is preferable to the cell because it is tightly regulated and does not generate large flaps that need to be excised. In the event of deregulated Fen1/DNA polymerase δ activity, the cell uses an alternative mechanism to generate and process long flaps by using Dna2, which has both helicase and nuclease activities. The nuclease activity of Dna2 is required for removing these long flaps, leaving a shorter flap to be processed by Fen1. Electron microscopy studies indicate that nucleosome loading on the lagging strand occurs very close to the site of synthesis. Thus, Okazaki fragment maturation is an efficient process that occurs immediately after the nascent DNA is synthesized.

Replicative DNA polymerases

After the replicative helicase has unwound the parental DNA duplex, exposing two single-stranded DNA templates, replicative polymerases are needed to generate two copies of the parental genome. DNA polymerase function is highly specialized and accomplish replication on specific templates and in narrow localizations. At the eukaryotic replication fork, there are three distinct replicative polymerase complexes that contribute to DNA replication: Polymerase α, Polymerase δ, and Polymerase ε. These three polymerases are essential for viability of the cell.

Because DNA polymerases require a primer on which to begin DNA synthesis, polymerase α (Pol α) acts as a replicative primase. Pol α is associated with an RNA primase and this complex accomplishes the priming task by synthesizing a primer that contains a short 10 nucleotide stretch of RNA followed by 10 to 20 DNA bases. Importantly, this priming action occurs at replication initiation at origins to begin leading-strand synthesis and also at the 5' end of each Okazaki fragment on the lagging strand.

However, Pol α is not able to continue DNA replication and must be replaced with another polymerase to continue DNA synthesis. Polymerase switching requires clamp loaders and it has been proven that normal DNA replication requires the coordinated actions of all three DNA polymerases: Pol α for priming synthesis, Pol ε for leading-strand replication, and the Pol δ, which is constantly loaded, for generating Okazaki fragments during lagging-strand synthesis.

Polymerase α (Pol α): Forms a complex with a small catalytic subunit (PriS) and a large noncatalytic (PriL) subunit. First, synthesis of an RNA primer allows DNA synthesis by DNA polymerase alpha. Occurs once at the origin on the leading strand and at the start of each Okazaki fragment on the lagging strand. Pri subunits act as a primase, synthesizing an RNA primer. DNA Pol α elongates the newly formed primer with DNA nucleotides. After around 20 nucleotides, elongation is taken over by Pol ε on the leading strand and Pol δ on the lagging strand.
Polymerase δ (Pol δ): Highly processive and has proofreading, 3'->5' exonuclease activity. In vivo, it is the main polymerase involved in both lagging strand and leading strand synthesis.
Polymerase ε (Pol ε): Highly processive and has proofreading, 3'->5' exonuclease activity. Highly related to pol δ, in vivo it functions mainly in error checking of pol δ.

Cdc45–Mcm–GINS helicase complex

The DNA helicases and polymerases must remain in close contact at the replication fork. If unwinding occurs too far in advance of synthesis, large tracts of single-stranded DNA are exposed. This can activate DNA damage signaling or induce DNA repair processes. To thwart these problems, the eukaryotic replisome contains specialized proteins that are designed to regulate the helicase activity ahead of the replication fork. These proteins also provide docking sites for physical interaction between helicases and polymerases, thereby ensuring that duplex unwinding is coupled with DNA synthesis.

For DNA polymerases to function, the double-stranded DNA helix has to be unwound to expose two single-stranded DNA templates for replication. DNA helicases are responsible for unwinding the double-stranded DNA during chromosome replication. Helicases in eukaryotic cells are remarkably complex. The catalytic core of the helicase is composed of six minichromosome maintenance (Mcm2-7) proteins, forming a hexameric ring. Away from DNA, the Mcm2-7 proteins form a single heterohexamer and are loaded in an inactive form at origins of DNA replication as a head-to-head double hexamers around double-stranded DNA. The Mcm proteins are recruited to replication origins then redistributed throughout the genomic DNA during S phase, indicative of their localization to the replication fork.

Loading of Mcm proteins can only occur during the G₁ of the cell cycle, and the loaded complex is then activated during S phase by recruitment of the Cdc45 protein and the GINS complex to form the active Cdc45–Mcm–GINS (CMG) helicase at DNA replication forks. Mcm activity is required throughout the S phase for DNA replication. A variety of regulatory factors assemble around the CMG helicase to produce the ‘Replisome Progression Complex’ which associates with DNA polymerases to form the eukaryotic replisome, the structure of which is still quite poorly defined in comparison with its bacterial counterpart.

The isolated CMG helicase and Replisome Progression Complex contain a single Mcm protein ring complex suggesting that the loaded double hexamer of the Mcm proteins at origins might be broken into two single hexameric rings as part of the initiation process, with each Mcm protein complex ring forming the core of a CMG helicase at the two replication forks established from each origin.The full CMG complex is required for DNA unwinding, and the complex of CDC45-Mcm-GINS is the functional DNA helicase in eukaryotic cells.

Ctf4 and And1 proteins

The CMG complex interacts with the replisome through the interaction with Ctf4 and And1 proteins. Ctf4/And1 proteins interact with both the CMG complex and DNA polymerase α. Ctf4 is a polymerase α accessory factor, which is required for the recruitment of polymerase α to replication origins.

Mrc1 and Claspin proteins

Mrc1/Claspin proteins couple leading-strand synthesis with the CMG complex helicase activity. Mrc1 interacts with polymerase ε as well as Mcm proteins. The importance of this direct link between the helicase and the leading-strand polymerase is underscored by results in cultured human cells, where Mrc1/Claspin is required for efficient replication fork progression. These results suggest that efficient DNA replication also requires the coupling of helicases and leading-strand synthesis...

Proliferating cell nuclear antigen

DNA polymerases require additional factors to support DNA replication. DNA polymerases have a semiclosed 'hand' structure, which allows the polymerase to load onto the DNA and begin translocating. This structure permits DNA polymerase to hold the single-stranded DNA template, incorporate dNTPs at the active site, and release the newly formed double-stranded DNA. However, the structure of DNA polymerases does not allow a continuous stable interaction with the template DNA.

To strengthen the interaction between the polymerase and the template DNA, DNA sliding clamps associate with the polymerase to promote the processivity of the replicative polymerase. In eukaryotes, the sliding clamp is a homotrimer ring structure known as the proliferating cell nuclear antigen (PCNA). The PCNA ring has polarity with surfaces that interact with DNA polymerases and tethers them securely to the DNA template. PCNA-dependent stabilization of DNA polymerases has a significant effect on DNA replication because PCNAs are able to enhance the polymerase processivity up to 1,000-fold. PCNA is an essential cofactor and has the distinction of being one of the most common interaction platforms in the replisome to accommodate multiple processes at the replication fork, and so PCNA is also viewed as a regulatory cofactor for DNA polymerases.

Replication factor C

PCNA fully encircles the DNA template strand and must be loaded onto DNA at the replication fork. At the leading strand, loading of the PCNA is an infrequent process, because DNA replication on the leading strand is continuous until replication is terminated. However, at the lagging strand, DNA polymerase δ needs to be continually loaded at the start of each Okazaki fragment. This constant initiation of Okazaki fragment synthesis requires repeated PCNA loading for efficient DNA replication.

PCNA loading is accomplished by the replication factor C (RFC) complex. The RFC complex is composed of five ATPases: Rfc1, Rfc2, Rfc3, Rfc4 and Rfc5. RFC recognizes primer-template junctions and loads PCNA at these sites. The PCNA homotrimer is opened by RFC by ATP hydrolysis and is then loaded onto DNA in the proper orientation to facilitate its association with the polymerase.Clamp loaders can also unload PCNA from DNA; a mechanism needed when replication must be terminated.

Stalled replication fork

DNA replication at the replication fork can be halted by a shortage of deoxynucleotide triphosphates (dNTPs) or by DNA damage, resulting in replication stress. This halting of replication is described as a stalled replication fork. A fork protection complex of proteins stabilizes the replication fork until DNA damage or other replication problems can be fixed. Prolonged replication fork stalling can lead to further DNA damage. Stalling signals are deactivated if the problems causing the replication fork are resolved.

Termination

Termination of eukaryotic DNA replication requires different processes depending on whether the chromosomes are circular or linear. Unlike linear molecules, circular chromosomes are able to replicate the entire molecule. However, the two DNA molecules will remain linked together. This issue is handled by decatenation of the two DNA molecules by a type II topoisomerase. Type II topoisomerases are also used to separate linear strands as they are intricately folded into a nucleosome within the cell.

As previously mentioned, linear chromosomes face another issue that is not seen in circular DNA replication. Due to the fact that an RNA primer is required for initiation of DNA synthesis, the lagging strand is at a disadvantage in replicating the entire chromosome. While the leading strand can use a single RNA primer to extend the 5' terminus of the replicating DNA strand, multiple RNA primers are responsible for lagging strand synthesis, creating Okazaki fragments. This leads to an issue due to the fact that DNA polymerase is only able to add to the 3' end of the DNA strand. The 3'-5' action of DNA polymerase along the parent strand leaves a short single-stranded DNA (ssDNA) region at the 3' end of the parent strand when the Okazaki fragments have been repaired. Since replication occurs in opposite directions at opposite ends of parent chromosomes, each strand is a lagging strand at one end. Over time this would result in progressive shortening of both daughter chromosomes. This is known as the end replication problem.

The end replication problem is handled in eukaryotic cells by telomere regions and telomerase. Telomeres extend the 3' end of the parental chromosome beyond the 5' end of the daughter strand. This single-stranded DNA structure can act as an origin of replication that recruits telomerase. Telomerase is a specialized DNA polymerase that consists of multiple protein subunits and an RNA component. The RNA component of telomerase anneals to the single stranded 3' end of the template DNA and contains 1.5 copies of the telomeric sequence. Telomerase contains a protein subunit that is a reverse transcriptase called telomerase reverse transcriptase or TERT. TERT synthesizes DNA until the end of the template telomerase RNA and then disengages. This process can be repeated as many times as needed with the extension of the 3' end of the parental DNA molecule. This 3' addition provides a template for extension of the 5' end of the daughter strand by lagging strand DNA synthesis. Regulation of telomerase activity is handled by telomere-binding proteins.

Replication fork barriers

Prokaryotic DNA replication is bidirectional; within a replicative origin, replisome complexes are created at each end of the replication origin and replisomes move away from each other from the initial starting point. In prokaryotes, bidirectional replication initiates at one replicative origin on the circular chromosome and terminates at a site opposed from the initial start of the origin. These termination regions have DNA sequences known as Ter sites. These Ter sites are bound by the Tus protein. The Ter-Tus complex is able to stop helicase activity, terminating replication.

In eukaryotic cells, termination of replication usually occurs through the collision of the two replicative forks between two active replication origins. The location of the collision varies on the timing of origin firing. In this way, if a replication fork becomes stalled or collapses at a certain site, replication of the site can be rescued when a replisome traveling in the opposite direction completes copying the region. There are programmed replication fork barriers (RFBs) bound by RFB proteins in various locations, throughout the genome, which are able to terminate or pause replication forks, stopping progression of the replisome.

Replication factories

It has been found that replication happens in a localised way in the cell nucleus. Contrary to the traditional view of moving replication forks along stagnant DNA, a concept of replication factories emerged, which means replication forks are concentrated towards some immobilised 'factory' regions through which the template DNA strands pass like conveyor belts.

Cell cycle regulation

DNA replication is a tightly orchestrated process that is controlled within the context of the cell cycle. Progress through the cell cycle and in turn DNA replication is tightly regulated by the formation and activation of pre-replicative complexes (pre-RCs) which is achieved through the activation and inactivation of cyclin-dependent kinases (Cdks, CDKs). Specifically it is the interactions of cyclins and cyclin dependent kinases that are responsible for the transition from G₁ into S-phase.

During the G₁ phase of the cell cycle there are low levels of CDK activity. This low level of CDK activity allows for the formation of new pre-RC complexes but is not sufficient for DNA replication to be initiated by the newly formed pre-RCs. During the remaining phases of the cell cycle there are elevated levels of CDK activity. This high level of CDK activity is responsible for initiating DNA replication as well as inhibiting new pre-RC complex formation. Once DNA replication has been initiated the pre-RC complex is broken down. Due to the fact that CDK levels remain high during the S phase, G₂, and M phases of the cell cycle no new pre-RC complexes can be formed. This all helps to ensure that no initiation can occur until the cell division is complete.

In addition to cyclin dependent kinases a new round of replication is thought to be prevented through the downregulation of Cdt1. This is achieved via degradation of Cdt1 as well as through the inhibitory actions of a protein known as geminin. Geminin binds tightly to Cdt1 and is thought to be the major inhibitor of re-replication.^[2] Geminin first appears in S-phase and is degraded at the metaphase-anaphase transition, possibly through ubiquination by anaphase promoting complex (APC).

Various cell cycle checkpoints are present throughout the course of the cell cycle that determine whether a cell will progress through division entirely. Importantly in replication the G₁, or restriction, checkpoint makes the determination of whether or not initiation of replication will begin or whether the cell will be placed in a resting stage known as G₀. Cells in the G₀ stage of the cell cycle are prevented from initiating a round of replication because the minichromosome maintenance proteins are not expressed. Transition into the S-phase indicates replication has begun.

Replication checkpoint proteins

In order to preserve genetic information during cell division, DNA replication must be completed with high fidelity. In order to achieve this task, eukaryotic cells have proteins in place during certain points in the replication process that are able to detect any errors during DNA replication and are able to preserve genomic integrity. These checkpoint proteins are able to stop the cell cycle from entering mitosis in order to allow time for DNA repair. Checkpoint proteins are also involved in some DNA repair pathways, while they stabilize the structure of the replication fork to prevent further damage. These checkpoint proteins are essential to avoid passing down mutations or other chromosomal aberrations to offspring.

Eukaryotic checkpoint proteins are well conserved and involve two phosphatidylinositol 3-kinase-related kinases (PIKKs), ATR and ATM. Both ATR and ATM share a target phosphorylation sequence, the SQ/TQ motif, but their individual roles in cells differ.

ATR is involved in arresting the cell cycle in response to DNA double-stranded breaks. ATR has an obligate checkpoint partner, ATR-interacting-protein (ATRIP), and together these two proteins are responsive to stretches of single-stranded DNA that are coated by replication protein A (RPA). The formation of single-stranded DNA occurs frequently, more often during replication stress. ATR-ATRIP is able to arrest the cell cycle to preserve genome integrity. ATR is found on chromatin during S phase, similar to RPA and claspin.

The generation of single-stranded DNA tracts is important in initiating the checkpoint pathways downstream of replication damage. Once single-stranded DNA becomes sufficiently long, single-stranded DNA coated with RPA are able to recruit ATR-ATRIP. In order to become fully active, the ATR kinase rely on sensor proteins that sense whether the checkpoint proteins are localized to a valid site of DNA replication stress. The RAD9-HUS1-Rad1 (9-1-1) heterotrimeric clamp and its clamp loader RFC^Rad17 are able to recognize gapped or nicked DNA. The RFC^Rad17 clamp loader loads 9-1-1 onto the damaged DNA. The presence of 9-1-1 on DNA is enough to facilitate the interaction between ATR-ATRIP and a group of proteins termed checkpoint mediators, such as TOPBP1 and Mrc1/claspin. TOPBP1 interacts with and recruits the phosphorylated Rad9 component of 9-1-1 and binds ATR-ATRIP, which phosphorylates Chk1. Mrc1/Claspin is also required for the complete activation of ATR-ATRIP that phosphorylates Chk1, the major downstream checkpoint effector kinase. Claspin is a component of the replisome and contains a domain for docking with Chk1, revealing a specific function of Claspin during DNA replication: the promotion of checkpoint signaling at the replisome.

Chk1 signaling is vital for arresting the cell cycle and preventing cells from entering mitosis with incomplete DNA replication or DNA damage. The Chk1-dependent Cdk inhibition is important for the function of the ATR-Chk1 checkpoint and to arrest the cell cycle and allow sufficient time for completion of DNA repair mechanisms, which in turn prevents the inheritance of damaged DNA. In addition, Chk1-dependent Cdk inhibition plays a critical role in inhibiting origin firing during S phase. This mechanism prevents continued DNA synthesis and is required for the protection of the genome in the presence of replication stress and potential genotoxic conditions. Thus, ATR-Chk1 activity further prevents potential replication problems at the level of single replication origins by inhibiting initiation of replication throughout the genome, until the signaling cascade maintaining cell-cycle arrest is turned off.

Replication through nucleosomes

Eukaryotic DNA must be tightly compacted in order to fit within the confined space of the nucleus. Chromosomes are packaged by wrapping 147 nucleotides around an octamer of histone proteins, forming a nucleosome. The nucleosome octamer includes two copies of each histone H2A, H2B, H3, and H4. Due to the tight association of histone proteins to DNA, eukaryotic cells have proteins that are designed to remodel histones ahead of the replication fork, in order to allow smooth progression of the replisome. There are also proteins involved in reassembling histones behind the replication fork to reestablish the nucleosome conformation.

There are several histone chaperones that are known to be involved in nucleosome assembly after replication. The FACT complex has been found to interact with DNA polymerase α-primase complex, and the subunits of the FACT complex interacted genetically with replication factors. The FACT complex is a heterodimer that does not hydrolyze ATP, but is able to facilitate "loosening" of histones in nucleosomes, but how the FACT complex is able to relieve the tight association of histones for DNA removal remains unanswered.

Another histone chaperone that associates with the replisome is Asf1, which interacts with the Mcm complex dependent on histone dimers H3-H4. Asf1 is able to pass newly synthesized H3-H4 dimer to deposition factors behind the replication fork and this activity makes the H3-H4 histone dimers available at the site of histone deposition just after replication. Asf1 (and its partner Rtt109) has also been implicated in inhibiting gene expression from replicated genes during S-phase.

The heterotrimeric chaperone chromatin assembly factor 1 (CAF-1) is a chromatin formation protein that is involved in depositing histones onto both newly replicated DNA strands to form chromatin. CAF-1 contains a PCNA-binding motif, called a PIP-box, that allows CAF-1 to associate with the replisome through PCNA and is able to deposit histone H3-H4 dimers onto newly synthesized DNA. The Rtt106 chaperone is also involved in this process, and associated with CAF-1 and H3-H4 dimers during chromatin formation. These processes load newly synthesized histones onto DNA.

After the deposition of histones H3-H4, nucleosomes form by the association of histone H2A-H2B. This process is thought to occur through the FACT complex, since it already associated with the replisome and is able to bind free H2A-H2B, or there is the possibility of another H2A-H2B chaperone, Nap1. Electron microscopy studies show that this occurs very quickly, as nucleosomes can be observed forming just a few hundred base pairs after the replication fork. Therefore, the entire process of forming new nucleosomes takes place just after replication due to the coupling of histone chaperones to the replisome.

Mitotic DNA Synthesis

Mitotic DNA synthesis (MiDAS) is a process of irregular DNA replication where DNA synthesis, naturally occurring in the S phase, takes place in the M phase of the cell cycle. Mitotic DNA synthesis is known to occur when cells are experiencing stress related to DNA replication. Certain loci in the genome, considered common fragile sites (CFS) or ALT-associated replication defects can induce replication stress that may lead to MiDAS. Mitotic DNA synthesis is enabled by a protein known as RAD52, which then recruits enzymes, including MUS81 and POLD3. These enzymes work to promote MiDAS, operating outside of ATR, BRCA2, and RAD51 which are necessary to prevent replication stress at CFS loci throughout S phase. MiDAS has been recorded in mammals and yeast, however, its occurrence in other eukaryotic organisms is yet to be discovered.

Comparisons between prokaryotic and eukaryotic DNA replication

When compared to prokaryotic DNA replication, namely in bacteria, the completion of eukaryotic DNA replication is more complex and involves multiple origins of replication and replicative proteins to accomplish. Prokaryotic DNA is arranged in a circular shape, and has only one replication origin when replication starts. By contrast, eukaryotic DNA is linear. When replicated, there are as many as one thousand origins of replication.

Eukaryotic DNA is bidirectional. Here the meaning of the word bidirectional is different. Eukaryotic linear DNA has many origins (called O) and termini (called T). "T" is present to the right of "O". One "O" and one "T" together form one replicon. After the formation of pre-initiation complex, when one replicon starts elongation, initiation starts in second replicon. Now, if the first replicon moves in clockwise direction, the second replicon moves in anticlockwise direction, until "T" of first replicon is reached. At "T", both the replicons merge to complete the process of replication. Meanwhile, the second replicon is moving in forward direction also, to meet with the third replicon. This clockwise and counter-clockwise movement of two replicons is termed as bidirectional replication.

Eukaryotic DNA replication requires precise coordination of all DNA polymerases and associated proteins to replicate the entire genome each time a cell divides. This process is achieved through a series of steps of protein assemblies at origins of replication, mainly focusing the regulation of DNA replication on the association of the MCM helicase with the DNA. These origins of replication direct the number of protein complexes that will form to initiate replication. In bacterial DNA replication, regulation focuses on the binding of the DnaA initiator protein to the DNA, with initiation of replication occurring multiple times during one cell cycle. Both prokaryotic and eukaryotic DNA use ATP binding and hydrolysis to direct helicase loading and in both cases the helicase is loaded in the inactive form. However, eukaryotic helicases are double hexamers that are loaded onto double stranded DNA whereas bacterial helicases are single hexamers loaded onto single stranded DNA.

Segregation of chromosomes is another difference between prokaryotic and eukaryotic cells. Rapidly dividing cells, such as bacteria, will often begin to segregate chromosomes that are still in the process of replication. In eukaryotic cells chromosome segregation into the daughter cells is not initiated until replication is complete in all chromosomes. Despite these differences, however, the underlying process of replication is similar for both prokaryotic and eukaryotic DNA.

Alternative medicine

From Wikipedia, the free encyclopedia

https://en.wikipedia.org/wiki/Alternative_medicine

Alternative medicine
AM, complementary and alternative medicine (CAM), complementary medicine, heterodox medicine, integrative medicine (IM), complementary and integrative medicine (CIM), functional medicine, new-age medicine, pseudomedicine, unconventional medicine, unorthodox medicine, altmed

Alternative medicine is any practice that aims to achieve the healing effects of medicine despite lacking biological plausibility, testability, repeatability or evidence of effectiveness. Unlike modern medicine, which employs the scientific method to test plausible therapies by way of responsible and ethical clinical trials, producing repeatable evidence of either effect or of no effect, alternative therapies reside outside of mainstream medicine and do not originate from using the scientific method, but instead rely on testimonials, anecdotes, religion, tradition, superstition, belief in supernatural "energies", pseudoscience, errors in reasoning, propaganda, fraud, or other unscientific sources. Frequently used terms for relevant practices are New Age medicine, pseudo-medicine, unorthodox medicine, holistic medicine, fringe medicine, and unconventional medicine, with little distinction from quackery.

Some alternative practices are based on theories that contradict the established science of how the human body works; others appeal to the supernatural or superstitious to explain their effect or lack thereof. In others, the practice has plausibility but lacks a positive risk–benefit outcome probability. Research into alternative therapies often fails to follow proper research protocols (such as placebo-controlled trials, blind experiments and calculation of prior probability), providing invalid results. History has shown that if a method is proven to work, it eventually ceases to be alternative and becomes mainstream medicine.

Much of the perceived effect of an alternative practice arises from a belief that it will be effective, the placebo effect, or from the treated condition resolving on its own (the natural course of disease). This is further exacerbated by the tendency to turn to alternative therapies upon the failure of medicine, at which point the condition will be at its worst and most likely to spontaneously improve. In the absence of this bias, especially for diseases that are not expected to get better by themselves such as cancer or HIV infection, multiple studies have shown significantly worse outcomes if patients turn to alternative therapies. While this may be because these patients avoid effective treatment, some alternative therapies are actively harmful (e.g. cyanide poisoning from amygdalin, or the intentional ingestion of hydrogen peroxide) or actively interfere with effective treatments.

The alternative medicine sector is a highly profitable industry with a strong lobby, and faces far less regulation over the use and marketing of unproven treatments. Complementary medicine (CM), complementary and alternative medicine (CAM), integrated medicine or integrative medicine (IM), and holistic medicine attempt to combine alternative practices with those of mainstream medicine. Traditional medicine practices become "alternative" when used outside their original settings and without proper scientific explanation and evidence. Alternative methods are often marketed as more "natural" or "holistic" than methods offered by medical science, that is sometimes derogatorily called "Big Pharma" by supporters of alternative medicine. Billions of dollars have been spent studying alternative medicine, with few or no positive results and many methods thoroughly disproven.

Definitions and terminology

Marcia Angell: "There cannot be two kinds of medicine – conventional and alternative."

The terms alternative medicine, complementary medicine, integrative medicine, holistic medicine, natural medicine, unorthodox medicine, fringe medicine, unconventional medicine, and new age medicine are used interchangeably as having the same meaning and are almost synonymous in most contexts. Terminology has shifted over time, reflecting the preferred branding of practitioners. For example, the United States National Institutes of Health department studying alternative medicine, currently named the National Center for Complementary and Integrative Health (NCCIH), was established as the Office of Alternative Medicine (OAM) and was renamed the National Center for Complementary and Alternative Medicine (NCCAM) before obtaining its current name. Therapies are often framed as "natural" or "holistic", implicitly and intentionally suggesting that conventional medicine is "artificial" and "narrow in scope".

The meaning of the term "alternative" in the expression "alternative medicine", is not that it is an effective alternative to medical science (though some alternative medicine promoters may use the loose terminology to give the appearance of effectiveness).Loose terminology may also be used to suggest meaning that a dichotomy exists when it does not (e.g., the use of the expressions "Western medicine" and "Eastern medicine" to suggest that the difference is a cultural difference between the Asian east and the European west, rather than that the difference is between evidence-based medicine and treatments that do not work).

Alternative medicine

Alternative medicine is defined loosely as a set of products, practices, and theories that are believed or perceived by their users to have the healing effects of medicine, but whose effectiveness has not been established using scientific methods, or whose theory and practice is not part of biomedicine, or whose theories or practices are directly contradicted by scientific evidence or scientific principles used in biomedicine. "Biomedicine" or "medicine" is that part of medical science that applies principles of biology, physiology, molecular biology, biophysics, and other natural sciences to clinical practice, using scientific methods to establish the effectiveness of that practice. Unlike medicine, an alternative product or practice does not originate from using scientific methods, but may instead be based on hearsay, religion, tradition, superstition, belief in supernatural energies, pseudoscience, errors in reasoning, propaganda, fraud, or other unscientific sources.

Some other definitions seek to specify alternative medicine in terms of its social and political marginality to mainstream healthcare. This can refer to the lack of support that alternative therapies receive from medical scientists regarding access to research funding, sympathetic coverage in the medical press, or inclusion in the standard medical curriculum. For example, a widely used definition devised by the US NCCIH calls it "a group of diverse medical and health care systems, practices, and products that are not generally considered part of conventional medicine". However, these descriptive definitions are inadequate in the present-day when some conventional doctors offer alternative medical treatments and introductory courses or modules can be offered as part of standard undergraduate medical training; alternative medicine is taught in more than half of US medical schools and US health insurers are increasingly willing to provide reimbursement for alternative therapies.

Complementary or integrative medicine

Complementary medicine (CM) or integrative medicine (IM) is when alternative medicine is used together with mainstream medical treatment in a belief that it improves the effect of treatments. For example, acupuncture (piercing the body with needles to influence the flow of a supernatural energy) might be believed to increase the effectiveness or "complement" science-based medicine when used at the same time.Significant drug interactions caused by alternative therapies may make treatments less effective, notably in cancer therapy.

Several medical organizations differentiate between complementary and alternative medicine including the UK National Health Service (NHS), Cancer Research UK, and the US Center for Disease Control and Prevention (CDC), the latter of which states that "Complementary medicine is used in addition to standard treatments" whereas "Alternative medicine is used instead of standard treatments."

Complementary and integrative interventions are used to improve fatigue in adult cancer patients.

David Gorski has described integrative medicine as an attempt to bring pseudoscience into academic science-based medicine with skeptics such as Gorski and David Colquhoun referring to this with the pejorative term "quackademia". Robert Todd Carroll described Integrative medicine as "a synonym for 'alternative' medicine that, at its worst, integrates sense with nonsense. At its best, integrative medicine supports both consensus treatments of science-based medicine and treatments that the science, while promising perhaps, does not justify" Rose Shapiro has criticized the field of alternative medicine for rebranding the same practices as integrative medicine.

CAM is an abbreviation of the phrase complementary and alternative medicine. The 2019 World Health Organization (WHO) Global Report on Traditional and Complementary Medicine states that the terms complementary and alternative medicine "refer to a broad set of health care practices that are not part of that country's own traditional or conventional medicine and are not fully integrated into the dominant health care system. They are used interchangeably with traditional medicine in some countries."

In the 1990s, integrative medicine started to be marketed by a new term, functional medicine.

The Integrative Medicine Exam by the American Board of Physician Specialties includes the following subjects: Manual Therapies, Biofield Therapies, Acupuncture, Movement Therapies, Expressive Arts, Traditional Chinese Medicine, Ayurveda, Indigenous Medical Systems, Homeopathic Medicine, Naturopathic Medicine, Osteopathic Medicine, Chiropractic, and Functional Medicine.

Other terms

Traditional medicine (TM) refers to certain practices within a culture which have existed since before the advent of medical science, Many TM practices are based on "holistic" approaches to disease and health, versus the scientific evidence-based methods in conventional medicine. The 2019 WHO report defines traditional medicine as "the sum total of the knowledge, skill and practices based on the theories, beliefs and experiences indigenous to different cultures, whether explicable or not, used in the maintenance of health as well as in the prevention, diagnosis, improvement or treatment of physical and mental illness." When used outside the original setting and in the absence of scientific evidence, TM practices are typically referred to as "alternative medicine".

Holistic medicine is another rebranding of alternative medicine. In this case, the words balance and holism are often used alongside complementary or integrative, claiming to take into fuller account the "whole" person, in contrast to the supposed reductionism of medicine.

Challenges in defining alternative medicine

Prominent members of the science and biomedical science community say that it is not meaningful to define an alternative medicine that is separate from a conventional medicine because the expressions "conventional medicine", "alternative medicine", "complementary medicine", "integrative medicine", and "holistic medicine" do not refer to any medicine at all. Others say that alternative medicine cannot be precisely defined because of the diversity of theories and practices it includes, and because the boundaries between alternative and conventional medicine overlap, are porous, and change. Healthcare practices categorized as alternative may differ in their historical origin, theoretical basis, diagnostic technique, therapeutic practice and in their relationship to the medical mainstream. Under a definition of alternative medicine as "non-mainstream", treatments considered alternative in one location may be considered conventional in another.

Critics say the expression is deceptive because it implies there is an effective alternative to science-based medicine, and that complementary is deceptive because it implies that the treatment increases the effectiveness of (complements) science-based medicine, while alternative medicines that have been tested nearly always have no measurable positive effect compared to a placebo.Journalist John Diamond wrote that "there is really no such thing as alternative medicine, just medicine that works and medicine that doesn't", a notion later echoed by Paul Offit: "The truth is there's no such thing as conventional or alternative or complementary or integrative or holistic medicine. There's only medicine that works and medicine that doesn't. And the best way to sort it out is by carefully evaluating scientific studies—not by visiting Internet chat rooms, reading magazine articles, or talking to friends."

Types

Alternative medicine consists of a wide range of health care practices, products, and therapies. The shared feature is a claim to heal that is not based on the scientific method. Alternative medicine practices are diverse in their foundations and methodologies. Alternative medicine practices may be classified by their cultural origins or by the types of beliefs upon which they are based.Methods may incorporate or be based on traditional medicinal practices of a particular culture, folk knowledge, superstition, spiritual beliefs, belief in supernatural energies (antiscience), pseudoscience, errors in reasoning, propaganda, fraud, new or different concepts of health and disease, and any bases other than being proven by scientific methods. Different cultures may have their own unique traditional or belief based practices developed recently or over thousands of years, and specific practices or entire systems of practices.

Unscientific belief systems

Alternative medicine, such as using naturopathy or homeopathy in place of conventional medicine, is based on belief systems not grounded in science.

	Proposed mechanism	Issues
Naturopathy	Naturopathic medicine is based on a belief that the body heals itself using a supernatural vital energy that guides bodily processes.	In conflict with the paradigm of evidence-based medicine. Many naturopaths have opposed vaccination, and "scientific evidence does not support claims that naturopathic medicine can cure cancer or any other disease".
Homeopathy	A belief that a substance that causes the symptoms of a disease in healthy people cures similar symptoms in sick people.	Developed before knowledge of atoms and molecules, or of basic chemistry, which shows that repeated dilution as practiced in homeopathy produces only water, and that homeopathy is not scientifically valid.

Traditional ethnic systems

Acupuncture involves insertion of needles in the body.

Alternative medical systems may be based on traditional medicine practices, such as traditional Chinese medicine (TCM), Ayurveda in India, or practices of other cultures around the world. Some useful applications of traditional medicines have been researched and accepted within ordinary medicine, however the underlying belief systems are seldom scientific and are not accepted.

Traditional medicine is considered alternative when it is used outside its home region; or when it is used together with or instead of known functional treatment; or when it can be reasonably expected that the patient or practitioner knows or should know that it will not work – such as knowing that the practice is based on superstition.

	Claims	Issues
Traditional Chinese medicine	Traditional practices and beliefs from China, together with modifications made by the Communist party make up TCM. Common practices include herbal medicine, acupuncture (insertion of needles in the body at specified points), massage (Tui na), exercise (qigong), and dietary therapy.	The practices are based on belief in a supernatural energy called qi, considerations of Chinese astrology and Chinese numerology, traditional use of herbs and other substances found in China, a belief that the tongue contains a map of the body that reflects changes in the body, and an incorrect model of the anatomy and physiology of internal organs.
Ayurveda	Traditional medicine of India. Ayurveda believes in the existence of three elemental substances, the doshas (called Vata, Pitta and Kapha), and states that a balance of the doshas results in health, while imbalance results in disease. Such disease-inducing imbalances can be adjusted and balanced using traditional herbs, minerals and heavy metals. Ayurveda stresses the use of plant-based medicines and treatments, with some animal products, and added minerals, including sulfur, arsenic, lead and copper(II) sulfate.	Safety concerns have been raised about Ayurveda, with two U.S. studies finding about 20 percent of Ayurvedic Indian-manufactured patent medicines contained toxic levels of heavy metals such as lead, mercury and arsenic. A 2015 study of users in the United States also found elevated blood lead levels in 40 percent of those tested. Other concerns include the use of herbs containing toxic compounds and the lack of quality control in Ayurvedic facilities. Incidents of heavy metal poisoning have been attributed to the use of these compounds in the United States.

Supernatural energies

Bases of belief may include belief in existence of supernatural energies undetected by the science of physics, as in biofields, or in belief in properties of the energies of physics that are inconsistent with the laws of physics, as in energy medicine.

	Claims	Issues
Biofield therapy	Intended to influence energy fields that, it is purported, surround and penetrate the body.	Advocates of scientific skepticism such as Carl Sagan have criticized the lack of empirical evidence to support the existence of the putative energy fields on which these therapies are predicated.
Bioelectromagnetic therapy	Use verifiable electromagnetic fields, such as pulsed fields, alternating-current, or direct-current fields in an unconventional manner.	Asserts that magnets can be used to defy the laws of physics to influence health and disease.
Chiropractic	Spinal manipulation aims to treat "vertebral subluxations" which are claimed to put pressure on nerves.	Chiropractic was based on the belief that manipulating the spine unblocks the flow of a supernatural vital energy called Innate Intelligence, thereby affecting health and disease. Vertebral subluxation is a pseudoscientific entity not proven to exist.
Reiki	Practitioners place their palms on the patient near Chakras that they believe are centers of supernatural energies in the belief that these supernatural energies can transfer from the practitioner's palms to heal the patient.	Lacks credible scientific evidence.

Herbal remedies and other substances

Substance based practices use substances found in nature such as herbs, foods, non-vitamin supplements and megavitamins, animal and fungal products, and minerals, including use of these products in traditional medical practices that may also incorporate other methods. Examples include healing claims for non-vitamin supplements, fish oil, Omega-3 fatty acid, glucosamine, echinacea, flaxseed oil, and ginseng. Herbal medicine, or phytotherapy, includes not just the use of plant products, but may also include the use of animal and mineral products. It is among the most commercially successful branches of alternative medicine, and includes the tablets, powders and elixirs that are sold as "nutritional supplements". Only a very small percentage of these have been shown to have any efficacy, and there is little regulation as to standards and safety of their contents.

Religion, faith healing, and prayer

	Claims	Issues
Christian faith healing	There is a divine or spiritual intervention in healing.	Lack of evidence for effectiveness. Unwanted outcomes, such as death and disability, "have occurred when faith healing was elected instead of medical care for serious injuries or illnesses". A 2001 double-blind study of 799 discharged coronary surgery patients found that "intercessory prayer had no significant effect on medical outcomes after hospitalization in a coronary care unit."

NCCIH classification

The United States agency National Center for Complementary and Integrative Health (NCCIH) has created a classification system for branches of complementary and alternative medicine that divides them into five major groups. These groups have some overlap, and distinguish two types of energy medicine: veritable which involves scientifically observable energy (including magnet therapy, colorpuncture and light therapy) and putative, which invokes physically undetectable or unverifiable energy. None of these energies have any evidence to support that they affect the body in any positive or health promoting way.

Whole medical systems: Cut across more than one of the other groups; examples include traditional Chinese medicine, naturopathy, homeopathy, and ayurveda.
Mind-body interventions: Explore the interconnection between the mind, body, and spirit, under the premise that they affect "bodily functions and symptoms". A connection between mind and body is conventional medical fact, and this classification does not include therapies with proven function such as cognitive behavioral therapy.
"Biology"-based practices: Use substances found in nature such as herbs, foods, vitamins, and other natural substances. (As used here, "biology" does not refer to the science of biology, but is a usage newly coined by NCCIH in the primary source used for this article. "Biology-based" as coined by NCCIH may refer to chemicals from a nonbiological source, such as use of the poison lead in traditional Chinese medicine, and to other nonbiological substances.)
Manipulative and body-based practices: feature manipulation or movement of body parts, such as is done in bodywork, chiropractic, and osteopathic manipulation.
Energy medicine: is a domain that deals with putative and verifiable energy fields:
- Biofield therapies are intended to influence energy fields that are purported to surround and penetrate the body. The existence of such energy fields have been disproven.
- Bioelectromagnetic-based therapies use verifiable electromagnetic fields, such as pulsed fields, alternating-current, or direct-current fields in a non-scientific manner.

History

The history of alternative medicine may refer to the history of a group of diverse medical practices that were collectively promoted as "alternative medicine" beginning in the 1970s, to the collection of individual histories of members of that group, or to the history of western medical practices that were labeled "irregular practices" by the western medical establishment. It includes the histories of complementary medicine and of integrative medicine. Before the 1970s, western practitioners that were not part of the increasingly science-based medical establishment were referred to "irregular practitioners", and were dismissed by the medical establishment as unscientific and as practicing quackery. Until the 1970s, irregular practice became increasingly marginalized as quackery and fraud, as western medicine increasingly incorporated scientific methods and discoveries, and had a corresponding increase in success of its treatments. In the 1970s, irregular practices were grouped with traditional practices of nonwestern cultures and with other unproven or disproven practices that were not part of biomedicine, with the entire group collectively marketed and promoted under the single expression "alternative medicine".

Use of alternative medicine in the west began to rise following the counterculture movement of the 1960s, as part of the rising new age movement of the 1970s. This was due to misleading mass marketing of "alternative medicine" being an effective "alternative" to biomedicine, changing social attitudes about not using chemicals and challenging the establishment and authority of any kind, sensitivity to giving equal measure to beliefs and practices of other cultures (cultural relativism), and growing frustration and desperation by patients about limitations and side effects of science-based medicine. At the same time, in 1975, the American Medical Association, which played the central role in fighting quackery in the United States, abolished its quackery committee and closed down its Department of Investigation. By the early to mid 1970s the expression "alternative medicine" came into widespread use, and the expression became mass marketed as a collection of "natural" and effective treatment "alternatives" to science-based biomedicine.By 1983, mass marketing of "alternative medicine" was so pervasive that the British Medical Journal (BMJ) pointed to "an apparently endless stream of books, articles, and radio and television programmes urge on the public the virtues of (alternative medicine) treatments ranging from meditation to drilling a hole in the skull to let in more oxygen".

An analysis of trends in the criticism of complementary and alternative medicine (CAM) in five prestigious American medical journals during the period of reorganization within medicine (1965–1999) was reported as showing that the medical profession had responded to the growth of CAM in three phases, and that in each phase, changes in the medical marketplace had influenced the type of response in the journals. Changes included relaxed medical licensing, the development of managed care, rising consumerism, and the establishment of the USA Office of Alternative Medicine (later National Center for Complementary and Alternative Medicine, currently National Center for Complementary and Integrative Health).

Medical education

Mainly as a result of reforms following the Flexner Report of 1910 medical education in established medical schools in the US has generally not included alternative medicine as a teaching topic. Typically, their teaching is based on current practice and scientific knowledge about: anatomy, physiology, histology, embryology, neuroanatomy, pathology, pharmacology, microbiology and immunology. Medical schools' teaching includes such topics as doctor-patient communication, ethics, the art of medicine, and engaging in complex clinical reasoning (medical decision-making). Writing in 2002, Snyderman and Weil remarked that by the early twentieth century the Flexner model had helped to create the 20th-century academic health center, in which education, research, and practice were inseparable. While this had much improved medical practice by defining with increasing certainty the pathophysiological basis of disease, a single-minded focus on the pathophysiological had diverted much of mainstream American medicine from clinical conditions that were not well understood in mechanistic terms, and were not effectively treated by conventional therapies.

By 2001 some form of CAM training was being offered by at least 75 out of 125 medical schools in the US. Exceptionally, the School of Medicine of the University of Maryland, Baltimore, includes a research institute for integrative medicine (a member entity of the Cochrane Collaboration).Medical schools are responsible for conferring medical degrees, but a physician typically may not legally practice medicine until licensed by the local government authority. Licensed physicians in the US who have attended one of the established medical schools there have usually graduated Doctor of Medicine (MD). All states require that applicants for MD licensure be graduates of an approved medical school and complete the United States Medical Licensing Examination (USMLE).

Efficacy

There is a general scientific consensus that alternative therapies lack the requisite scientific validation, and their effectiveness is either unproved or disproved. Many of the claims regarding the efficacy of alternative medicines are controversial, since research on them is frequently of low quality and methodologically flawed. Selective publication bias, marked differences in product quality and standardisation, and some companies making unsubstantiated claims call into question the claims of efficacy of isolated examples where there is evidence for alternative therapies.

The Scientific Review of Alternative Medicine points to confusions in the general population – a person may attribute symptomatic relief to an otherwise-ineffective therapy just because they are taking something (the placebo effect); the natural recovery from or the cyclical nature of an illness (the regression fallacy) gets misattributed to an alternative medicine being taken; a person not diagnosed with science-based medicine may never originally have had a true illness diagnosed as an alternative disease category.

Edzard Ernst, the first university professor of Complementary and Alternative Medicine, characterized the evidence for many alternative techniques as weak, nonexistent, or negative and in 2011 published his estimate that about 7.4% were based on "sound evidence", although he believes that may be an overestimate. Ernst has concluded that 95% of the alternative therapies he and his team studied, including acupuncture, herbal medicine, homeopathy, and reflexology, are "statistically indistinguishable from placebo treatments", but he also believes there is something that conventional doctors can usefully learn from the chiropractors and homeopath: this is the therapeutic value of the placebo effect, one of the strangest phenomena in medicine.

In 2003, a project funded by the CDC identified 208 condition-treatment pairs, of which 58% had been studied by at least one randomized controlled trial (RCT), and 23% had been assessed with a meta-analysis. According to a 2005 book by a US Institute of Medicine panel, the number of RCTs focused on CAM has risen dramatically.

As of 2005, the Cochrane Library had 145 CAM-related Cochrane systematic reviews and 340 non-Cochrane systematic reviews. An analysis of the conclusions of only the 145 Cochrane reviews was done by two readers. In 83% of the cases, the readers agreed. In the 17% in which they disagreed, a third reader agreed with one of the initial readers to set a rating. These studies found that, for CAM, 38.4% concluded positive effect or possibly positive (12.4%), 4.8% concluded no effect, 0.7% concluded harmful effect, and 56.6% concluded insufficient evidence. An assessment of conventional treatments found that 41.3% concluded positive or possibly positive effect, 20% concluded no effect, 8.1% concluded net harmful effects, and 21.3% concluded insufficient evidence. However, the CAM review used the more developed 2004 Cochrane database, while the conventional review used the initial 1998 Cochrane database.

Alternative therapies do not "complement" (improve the effect of, or mitigate the side effects of) functional medical treatment.Significant drug interactions caused by alternative therapies may instead negatively impact functional treatment by making prescription drugs less effective, such as interference by herbal preparations with warfarin.

In the same way as for conventional therapies, drugs, and interventions, it can be difficult to test the efficacy of alternative medicine in clinical trials. In instances where an established, effective, treatment for a condition is already available, the Helsinki Declaration states that withholding such treatment is unethical in most circumstances. Use of standard-of-care treatment in addition to an alternative technique being tested may produce confounded or difficult-to-interpret results.

Cancer researcher Andrew J. Vickers has stated:

Contrary to much popular and scientific writing, many alternative cancer treatments have been investigated in good-quality clinical trials, and they have been shown to be ineffective. The label "unproven" is inappropriate for such therapies; it is time to assert that many alternative cancer therapies have been "disproven".

Perceived mechanism of effect

Anything classified as alternative medicine by definition does not have a proven healing or medical effect. However, there are different mechanisms through which it can be perceived to "work". The common denominator of these mechanisms is that effects are mis-attributed to the alternative treatment.

Placebo effect

A placebo is a treatment with no intended therapeutic value. An example of a placebo is an inert pill, but it can include more dramatic interventions like sham surgery. The placebo effect is the concept that patients will perceive an improvement after being treated with an inert treatment. The opposite of the placebo effect is the nocebo effect, when patients who expect a treatment to be harmful will perceive harmful effects after taking it.

Placebos do not have a physical effect on diseases or improve overall outcomes, but patients may report improvements in subjective outcomes such as pain and nausea. A 1955 study suggested that a substantial part of a medicine's impact was due to the placebo effect. However, reassessments found the study to have flawed methodology. This and other modern reviews suggest that other factors like natural recovery and reporting bias should also be considered.

All of these are reasons why alternative therapies may be credited for improving a patient's condition even though the objective effect is non-existent, or even harmful. David Gorski argues that alternative treatments should be treated as a placebo, rather than as medicine. Almost none have performed significantly better than a placebo in clinical trials. Furthermore, distrust of conventional medicine may lead to patients experiencing the nocebo effect when taking effective medication.

Regression to the mean

A patient who receives an inert treatment may report improvements afterwards that it did not cause. Assuming it was the cause without evidence is an example of the regression fallacy. This may be due to a natural recovery from the illness, or a fluctuation in the symptoms of a long-term condition. The concept of regression toward the mean implies that an extreme result is more likely to be followed by a less extreme result.

Other factors

There are also reasons why a placebo treatment group may outperform a "no-treatment" group in a test which are not related to a patient's experience. These include patients reporting more favourable results than they really felt due to politeness or "experimental subordination", observer bias, and misleading wording of questions. In their 2010 systematic review of studies into placebos, Asbjørn Hróbjartsson and Peter C. Gøtzsche write that "even if there were no true effect of placebo, one would expect to record differences between placebo and no-treatment groups due to bias associated with lack of blinding." Alternative therapies may also be credited for perceived improvement through decreased use or effect of medical treatment, and therefore either decreased side effects or nocebo effects towards standard treatment.

Use and regulation

Appeal

Practitioners of complementary medicine usually discuss and advise patients as to available alternative therapies. Patients often express interest in mind-body complementary therapies because they offer a non-drug approach to treating some health conditions.

In addition to the social-cultural underpinnings of the popularity of alternative medicine, there are several psychological issues that are critical to its growth, notably psychological effects, such as the will to believe, cognitive biases that help maintain self-esteem and promote harmonious social functioning, and the post hoc, ergo propter hoc fallacy.

In a 2018 interview with The BMJ, Edzard Ernst stated: "The present popularity of complementary and alternative medicine is also inviting criticism of what we are doing in mainstream medicine. It shows that we aren't fulfilling a certain need-we are not giving patients enough time, compassion, or empathy. These are things that complementary practitioners are very good at. Mainstream medicine could learn something from complementary medicine."

Marketing

Alternative medicine is a profitable industry with large media advertising expenditures. Accordingly, alternative practices are often portrayed positively and compared favorably to "big pharma".

The popularity of complementary & alternative medicine (CAM) may be related to other factors that Ernst mentioned in a 2008 interview in The Independent:

Why is it so popular, then? Ernst blames the providers, customers and the doctors whose neglect, he says, has created the opening into which alternative therapists have stepped. "People are told lies. There are 40 million websites and 39.9 million tell lies, sometimes outrageous lies. They mislead cancer patients, who are encouraged not only to pay their last penny but to be treated with something that shortens their lives." At the same time, people are gullible. It needs gullibility for the industry to succeed. It doesn't make me popular with the public, but it's the truth.

Paul Offit proposed that "alternative medicine becomes quackery" in four ways: by recommending against conventional therapies that are helpful, promoting potentially harmful therapies without adequate warning, draining patients' bank accounts, or by promoting "magical thinking". Promoting alternative medicine has been called dangerous and unethical.

Friendly and colorful images of herbal treatments may look less threatening or dangerous when compared to conventional medicine. This is an intentional marketing strategy.

Social factors

Authors have speculated on the socio-cultural and psychological reasons for the appeal of alternative medicines among the minority using them in lieu of conventional medicine. There are several socio-cultural reasons for the interest in these treatments centered on the low level of scientific literacy among the public at large and a concomitant increase in antiscientific attitudes and new age mysticism. Related to this are vigorous marketing of extravagant claims by the alternative medical community combined with inadequate media scrutiny and attacks on critics. Alternative medicine is criticized for taking advantage of the least fortunate members of society.

There is also an increase in conspiracy theories toward conventional medicine and pharmaceutical companies, mistrust of traditional authority figures, such as the physician, and a dislike of the current delivery methods of scientific biomedicine, all of which have led patients to seek out alternative medicine to treat a variety of ailments. Many patients lack access to contemporary medicine, due to a lack of private or public health insurance, which leads them to seek out lower-cost alternative medicine. Medical doctors are also aggressively marketing alternative medicine to profit from this market.

Patients can be averse to the painful, unpleasant, and sometimes-dangerous side effects of biomedical treatments. Treatments for severe diseases such as cancer and HIV infection have well-known, significant side-effects. Even low-risk medications such as antibiotics can have potential to cause life-threatening anaphylactic reactions in a very few individuals. Many medications may cause minor but bothersome symptoms such as cough or upset stomach. In all of these cases, patients may be seeking out alternative therapies to avoid the adverse effects of conventional treatments.

Prevalence of use

According to research published in 2015, the increasing popularity of CAM needs to be explained by moral convictions or lifestyle choices rather than by economic reasoning.

In developing nations, access to essential medicines is severely restricted by lack of resources and poverty. Traditional remedies, often closely resembling or forming the basis for alternative remedies, may comprise primary healthcare or be integrated into the healthcare system. In Africa, traditional medicine is used for 80% of primary healthcare, and in developing nations as a whole over one-third of the population lack access to essential medicines.

In Latin America, inequities against BIPOC communities keep them tied to their traditional practices and therefore, it is often these communities that constitute the majority of users of alternative medicine. Racist attitudes towards certain communities disable them from accessing more urbanized modes of care. In a study that assessed access to care in rural communities of Latin America, it was found that discrimination is a huge barrier to the ability of citizens to access care; more specifically, women of Indigenous and African descent, and lower-income families were especially hurt. Such exclusion exacerbates the inequities that minorities in Latin America already face. Consistently excluded from many systems of westernized care for socioeconomic and other reasons, low-income communities of color often turn to traditional medicine for care as it has proved reliable to them across generations.

Commentators including David Horrobin have proposed adopting a prize system to reward medical research. This stands in opposition to the current mechanism for funding research proposals in most countries around the world. In the US, the NCCIH provides public research funding for alternative medicine. The NCCIH has spent more than US$2.5 billion on such research since 1992 and this research has not demonstrated the efficacy of alternative therapies. As of 2011, the NCCIH's sister organization in the NIC Office of Cancer Complementary and Alternative Medicine had given out grants of around $105 million each year for several years. Testing alternative medicine that has no scientific basis (as in the aforementioned grants) has been called a waste of scarce research resources.

That alternative medicine has been on the rise "in countries where Western science and scientific method generally are accepted as the major foundations for healthcare, and 'evidence-based' practice is the dominant paradigm" was described as an "enigma" in the Medical Journal of Australia. A 15-year systematic review published in 2022 on the global acceptance and use of CAM among medical specialists found the overall acceptance of CAM at 52% and the overall use at 45%.

In the United States

In the United States, the 1974 Child Abuse Prevention and Treatment Act (CAPTA) required that for states to receive federal money, they had to grant religious exemptions to child neglect and abuse laws regarding religion-based healing practices. Thirty-one states have child-abuse religious exemptions.

The use of alternative medicine in the US has increased, with a 50 percent increase in expenditures and a 25 percent increase in the use of alternative therapies between 1990 and 1997 in America. According to a national survey conducted in 2002, "36 percent of U.S. adults aged 18 years and over use some form of complementary and alternative medicine." Americans spend many billions on the therapies annually. Most Americans used CAM to treat and/or prevent musculoskeletal conditions or other conditions associated with chronic or recurring pain. In America, women were more likely than men to use CAM, with the biggest difference in use of mind-body therapies including prayer specifically for health reasons". In 2008, more than 37% of American hospitals offered alternative therapies, up from 27 percent in 2005, and 25% in 2004. More than 70% of the hospitals offering CAM were in urban areas.

A survey of Americans found that 88 percent thought that "there are some good ways of treating sickness that medical science does not recognize". Use of magnets was the most common tool in energy medicine in America, and among users of it, 58 percent described it as at least "sort of scientific", when it is not at all scientific. In 2002, at least 60 percent of US medical schools have at least some class time spent teaching alternative therapies. "Therapeutic touch" was taught at more than 100 colleges and universities in 75 countries before the practice was debunked by a nine-year-old child for a school science project.

Prevalence of use of specific therapies

The most common CAM therapies used in the US in 2002 were prayer (45%), herbalism (19%), breathing meditation (12%), meditation (8%), chiropractic medicine (8%), yoga (5–6%), body work (5%), diet-based therapy (4%), progressive relaxation (3%), mega-vitamin therapy (3%) and visualization (2%).

In Britain, the most often used alternative therapies were Alexander technique, aromatherapy, Bach and other flower remedies, body work therapies including massage, Counseling stress therapies, hypnotherapy, meditation, reflexology, Shiatsu, Ayurvedic medicine, nutritional medicine, and yoga. Ayurvedic medicine remedies are mainly plant based with some use of animal materials. Safety concerns include the use of herbs containing toxic compounds and the lack of quality control in Ayurvedic facilities.

According to the National Health Service (England), the most commonly used complementary and alternative medicines (CAM) supported by the NHS in the UK are: acupuncture, aromatherapy, chiropractic, homeopathy, massage, osteopathy and clinical hypnotherapy.

In palliative care

Complementary therapies are often used in palliative care or by practitioners attempting to manage chronic pain in patients. Integrative medicine is considered more acceptable in the interdisciplinary approach used in palliative care than in other areas of medicine. "From its early experiences of care for the dying, palliative care took for granted the necessity of placing patient values and lifestyle habits at the core of any design and delivery of quality care at the end of life. If the patient desired complementary therapies, and as long as such treatments provided additional support and did not endanger the patient, they were considered acceptable." The non-pharmacologic interventions of complementary medicine can employ mind-body interventions designed to "reduce pain and concomitant mood disturbance and increase quality of life."

Regulation

The alternative medicine lobby has successfully pushed for alternative therapies to be subject to far less regulation than conventional medicine. Some professions of complementary/traditional/alternative medicine, such as chiropractic, have achieved full regulation in North America and other parts of the world and are regulated in a manner similar to that governing science-based medicine. In contrast, other approaches may be partially recognized and others have no regulation at all. In some cases, promotion of alternative therapies is allowed when there is demonstrably no effect, only a tradition of use. Despite laws making it illegal to market or promote alternative therapies for use in cancer treatment, many practitioners promote them.

Regulation and licensing of alternative medicine ranges widely from country to country, and state to state. In Austria and Germany complementary and alternative medicine is mainly in the hands of doctors with MDs, and half or more of the American alternative practitioners are licensed MDs. In Germany herbs are tightly regulated: half are prescribed by doctors and covered by health insurance.

Government bodies in the US and elsewhere have published information or guidance about alternative medicine. The U.S. Food and Drug Administration (FDA), has issued online warnings for consumers about medication health fraud. This includes a section on Alternative Medicine Fraud, such as a warning that Ayurvedic products generally have not been approved by the FDA before marketing.

Risks and problems

The National Science Foundation has studied the problematic side of the public's attitudes and understandings of science fiction, pseudoscience, and belief in alternative medicine. They use a quote from Robert L. Park to describe some issues with alternative medicine:

Alternative medicine is another concern. As used here, alternative medicine refers to all treatments that have not been proven effective using scientific methods. A scientist's view of the situation appeared in a recent book (Park 2000b)":
Between homeopathy and herbal therapy lies a bewildering array of untested and unregulated treatments, all labeled alternative by their proponents. Alternative seems to define a culture rather than a field of medicine—a culture that is not scientifically demanding. It is a culture in which ancient traditions are given more weight than biological science, and anecdotes are preferred over clinical trials. Alternative therapies steadfastly resist change, often for centuries or even millennia, unaffected by scientific advances in the understanding of physiology or disease. Incredible explanations invoking modern physics are sometimes offered for how alternative therapies might work, but there seems to be little interest in testing these speculations scientifically.

Negative outcomes

According to the Institute of Medicine, use of alternative medical techniques may result in several types of harm:

"Direct harm, which results in adverse patient outcome."
"Economic harm, which results in monetary loss but presents no health hazard;"
"Indirect harm, which results in a delay of appropriate treatment, or in unreasonable expectations that discourage patients and their families from accepting and dealing effectively with their medical conditions;"

Interactions with conventional pharmaceuticals

Forms of alternative medicine that are biologically active can be dangerous even when used in conjunction with conventional medicine. Examples include immuno-augmentation therapy, shark cartilage, bioresonance therapy, oxygen and ozone therapies, and insulin potentiation therapy. Some herbal remedies can cause dangerous interactions with chemotherapy drugs, radiation therapy, or anesthetics during surgery, among other problems. An example of these dangers was reported by Associate Professor Alastair MacLennan of Adelaide University, Australia regarding a patient who almost bled to death on the operating table after neglecting to mention that she had been taking "natural" potions to "build up her strength" before the operation, including a powerful anticoagulant that nearly caused her death.

To ABC Online, MacLennan also gives another possible mechanism:

And lastly there's the cynicism and disappointment and depression that some patients get from going on from one alternative medicine to the next, and they find after three months the placebo effect wears off, and they're disappointed and they move on to the next one, and they're disappointed and disillusioned, and that can create depression and make the eventual treatment of the patient with anything effective difficult, because you may not get compliance, because they've seen the failure so often in the past.

Side-effects

Conventional treatments are subjected to testing for undesired side-effects, whereas alternative therapies, in general, are not subjected to such testing at all. Any treatment – whether conventional or alternative – that has a biological or psychological effect on a patient may also have potential to possess dangerous biological or psychological side-effects. Attempts to refute this fact with regard to alternative therapies sometimes use the appeal to nature fallacy, i.e., "That which is natural cannot be harmful." Specific groups of patients such as patients with impaired hepatic or renal function are more susceptible to side effects of alternative remedies.

An exception to the normal thinking regarding side-effects is homeopathy. Since 1938, the FDA has regulated homeopathic products in "several significantly different ways from other drugs." Homeopathic preparations, termed "remedies", are extremely dilute, often far beyond the point where a single molecule of the original active (and possibly toxic) ingredient is likely to remain. They are, thus, considered safe on that count, but "their products are exempt from good manufacturing practice requirements related to expiration dating and from finished product testing for identity and strength", and their alcohol concentration may be much higher than allowed in conventional drugs.

Treatment delay

Alternative medicine may discourage people from getting the best possible treatment. Those having experienced or perceived success with one alternative therapy for a minor ailment may be convinced of its efficacy and persuaded to extrapolate that success to some other alternative therapy for a more serious, possibly life-threatening illness. For this reason, critics argue that therapies that rely on the placebo effect to define success are very dangerous. According to mental health journalist Scott Lilienfeld in 2002, "unvalidated or scientifically unsupported mental health practices can lead individuals to forgo effective treatments" and refers to this as opportunity cost. Individuals who spend large amounts of time and money on ineffective treatments may be left with precious little of either, and may forfeit the opportunity to obtain treatments that could be more helpful. In short, even innocuous treatments can indirectly produce negative outcomes. Between 2001 and 2003, four children died in Australia because their parents chose ineffective naturopathic, homeopathic, or other alternative medicines and diets rather than conventional therapies.

Unconventional cancer "cures"

There have always been "many therapies offered outside of conventional cancer treatment centers and based on theories not found in biomedicine. These alternative cancer cures have often been described as 'unproven,' suggesting that appropriate clinical trials have not been conducted and that the therapeutic value of the treatment is unknown." However, "many alternative cancer treatments have been investigated in good-quality clinical trials, and they have been shown to be ineffective.... The label 'unproven' is inappropriate for such therapies; it is time to assert that many alternative cancer therapies have been 'disproven'."

Edzard Ernst has stated:

any alternative cancer cure is bogus by definition. There will never be an alternative cancer cure. Why? Because if something looked halfway promising, then mainstream oncology would scrutinize it, and if there is anything to it, it would become mainstream almost automatically and very quickly. All curative "alternative cancer cures" are based on false claims, are bogus, and, I would say, even criminal.

Rejection of science

There is no alternative medicine. There is only scientifically proven,
evidence-based medicine supported by solid data
or unproven medicine, for which scientific evidence is lacking.
— P.B. Fontanarosa, JAMA (1998)

Complementary and alternative medicine (CAM) is not as well researched as conventional medicine, which undergoes intense research before release to the public. Practitioners of science-based medicine also discard practices and treatments when they are shown ineffective, while alternative practitioners do not. Funding for research is also sparse making it difficult to do further research for effectiveness of CAM. Most funding for CAM is funded by government agencies. Proposed research for CAM are rejected by most private funding agencies because the results of research are not reliable. The research for CAM has to meet certain standards from research ethics committees, which most CAM researchers find almost impossible to meet. Even with the little research done on it, CAM has not been proven to be effective. Studies that have been done will be cited by CAM practitioners in an attempt to claim a basis in science. These studies tend to have a variety of problems, such as small samples, various biases, poor research design, lack of controls, negative results, etc. Even those with positive results can be better explained as resulting in false positives due to bias and noisy data.

Alternative medicine may lead to a false understanding of the body and of the process of science. Steven Novella, a neurologist at Yale School of Medicine, wrote that government-funded studies of integrating alternative medicine techniques into the mainstream are "used to lend an appearance of legitimacy to treatments that are not legitimate." Marcia Angell considered that critics felt that healthcare practices should be classified based solely on scientific evidence, and if a treatment had been rigorously tested and found safe and effective, science-based medicine will adopt it regardless of whether it was considered "alternative" to begin with. It is possible for a method to change categories (proven vs. unproven), based on increased knowledge of its effectiveness or lack thereof. Prominent supporters of this position are George D. Lundberg, former editor of the Journal of the American Medical Association (JAMA) and the journal's interim editor-in-chief Phil Fontanarosa.

Writing in 1999 in CA: A Cancer Journal for Clinicians Barrie R. Cassileth mentioned a 1997 letter to the United States Senate's Subcommittee on Public Health and Safety, which had deplored the lack of critical thinking and scientific rigor in OAM-supported research, had been signed by four Nobel Laureates and other prominent scientists. (This was supported by the National Institutes of Health (NIH).)

In March 2009, a staff writer for The Washington Post reported that the impending national discussion about broadening access to health care, improving medical practice and saving money was giving a group of scientists an opening to propose shutting down the National Center for Complementary and Alternative Medicine. They quoted one of these scientists, Steven Salzberg, a genome researcher and computational biologist at the University of Maryland, as saying "One of our concerns is that NIH is funding pseudoscience." They noted that the vast majority of studies were based on fundamental misunderstandings of physiology and disease, and had shown little or no effect.

Writers such as Carl Sagan, a noted astrophysicist, advocate of scientific skepticism and the author of The Demon-Haunted World: Science as a Candle in the Dark (1996), have lambasted the lack of empirical evidence to support the existence of the putative energy fields on which these therapies are predicated.

Sampson has also pointed out that CAM tolerated contradiction without thorough reason and experiment. Barrett has pointed out that there is a policy at the NIH of never saying something does not work, only that a different version or dose might give different results. Barrett also expressed concern that, just because some "alternatives" have merit, there is the impression that the rest deserve equal consideration and respect even though most are worthless, since they are all classified under the one heading of alternative medicine.

Some critics of alternative medicine are focused upon health fraud, misinformation, and quackery as public health problems, notably Wallace Sampson and Paul Kurtz founders of Scientific Review of Alternative Medicine and Stephen Barrett, co-founder of The National Council Against Health Fraud and webmaster of Quackwatch. Grounds for opposing alternative medicine include that:

Alternative therapies typically lack any scientific validation, and their effectiveness either is unproven or has been disproved.
It is usually based on religion, tradition, superstition, belief in supernatural energies, pseudoscience, errors in reasoning, propaganda, or fraud.
Methods may incorporate or base themselves on traditional medicine, folk knowledge, spiritual beliefs, ignorance or misunderstanding of scientific principles, errors in reasoning, or newly conceived approaches claiming to heal.
Research on alternative medicine is frequently of low quality and methodologically flawed.
Treatments are not part of the conventional, science-based healthcare system.
Where alternative therapies have replaced conventional science-based medicine, even with the safest alternative medicines, failure to use or delay in using conventional science-based medicine has caused deaths.

Many alternative medical treatments are not patentable, which may lead to less research funding from the private sector. In addition, in most countries, alternative therapies (in contrast to pharmaceuticals) can be marketed without any proof of efficacy – also a disincentive for manufacturers to fund scientific research.

English evolutionary biologist Richard Dawkins, in his 2003 book A Devil's Chaplain, defined alternative medicine as a "set of practices that cannot be tested, refuse to be tested, or consistently fail tests." Dawkins argued that if a technique is demonstrated effective in properly performed trials then it ceases to be alternative and simply becomes medicine.

CAM is also often less regulated than conventional medicine. There are ethical concerns about whether people who perform CAM have the proper knowledge to treat patients. CAM is often done by non-physicians who do not operate with the same medical licensing laws which govern conventional medicine, and it is often described as an issue of non-maleficence.

According to two writers, Wallace Sampson and K. Butler, marketing is part of the training required in alternative medicine, and propaganda methods in alternative medicine have been traced back to those used by Hitler and Goebels in their promotion of pseudoscience in medicine.

In November 2011 Edzard Ernst stated that the "level of misinformation about alternative medicine has now reached the point where it has become dangerous and unethical. So far, alternative medicine has remained an ethics-free zone. It is time to change this."

Harriet Hall criticized the low standard of evidence accepted by the alternative medicine community:

Science-based medicine has one rigorous standard of evidence, the kind [used for pharmaceuticals] .... CAM has a double standard. They gladly accept a lower standard of evidence for treatments they believe in. However, I suspect they would reject a pharmaceutical if it were approved for marketing on the kind of evidence they accept for CAM.

Conflicts of interest

Some commentators have said that special consideration must be given to the issue of conflicts of interest in alternative medicine. Edzard Ernst has said that most researchers into alternative medicine are at risk of "unidirectional bias" because of a generally uncritical belief in their chosen subject.Ernst cites as evidence the phenomenon whereby 100% of a sample of acupuncture trials originating in China had positive conclusions. David Gorski contrasts evidence-based medicine, in which researchers try to disprove hyphotheses, with what he says is the frequent practice in pseudoscience-based research, of striving to confirm pre-existing notions. Harriet Hall writes that there is a contrast between the circumstances of alternative medicine practitioners and disinterested scientists: in the case of acupuncture, for example, an acupuncturist would have "a great deal to lose" if acupuncture were rejected by research; but the disinterested skeptic would not lose anything if its effects were confirmed; rather their change of mind would enhance their skeptical credentials.

Use of health and research resources

Research into alternative therapies has been criticized for "diverting research time, money, and other resources from more fruitful lines of investigation in order to pursue a theory that has no basis in biology." Research methods expert and author of Snake Oil Science, R. Barker Bausell, has stated that "it's become politically correct to investigate nonsense." A commonly cited statistic is that the US National Institute of Health had spent $2.5 billion on investigating alternative therapies prior to 2009, with none being found to be effective.

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Tuesday, April 15, 2025

Eukaryotic DNA replication

Initiation

Origin of replication

Pre-replicative complex

Origin recognition complex

Cdc6 protein

Cdt1 protein

Minichromosome maintenance protein complex

Initiation complex

Cdc45 protein

GINS

Mcm10

DDK and CDK kinases

Dpb11, Sld3, and Sld2 proteins

Elongation

Replication fork

Leading strand

Lagging strand

Replicative DNA polymerases

Cdc45–Mcm–GINS helicase complex

Ctf4 and And1 proteins

Mrc1 and Claspin proteins

Proliferating cell nuclear antigen

Replication factor C

Stalled replication fork

Termination

Replication fork barriers

Replication factories

Cell cycle regulation

Replication checkpoint proteins

Replication through nucleosomes

Mitotic DNA Synthesis

Comparisons between prokaryotic and eukaryotic DNA replication

Alternative medicine

Definitions and terminology

Alternative medicine

Complementary or integrative medicine

Other terms

Challenges in defining alternative medicine

Types

Unscientific belief systems

Traditional ethnic systems

Supernatural energies

Herbal remedies and other substances

Religion, faith healing, and prayer

NCCIH classification

History

Medical education

Efficacy

Perceived mechanism of effect

Placebo effect

Regression to the mean

Other factors

Use and regulation

Appeal

Marketing

Social factors

Prevalence of use

In the United States

Prevalence of use of specific therapies

In palliative care

Regulation

Risks and problems

Negative outcomes

Interactions with conventional pharmaceuticals

Side-effects

Treatment delay

Unconventional cancer "cures"

Rejection of science

Conflicts of interest

Use of health and research resources

Human extinction