Cell cycle

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

Life cycle of the cell

 

Onion (Allium) cells in different phases of the cell cycle. Growth in an 'organism' is carefully controlled by regulating the cell cycle.

 

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Cell cycle in Deinococcus radiodurans

The cell cycle, or cell-division cycle, is the series of events that take place in a cell that cause it to divide into two daughter cells. These events include the duplication of its DNA (DNA replication) and some of its organelles, and subsequently the partitioning of its cytoplasm and other components into two daughter cells in a process called cell division.

In cells with nuclei (eukaryotes), (i.e., animal, plant, fungal, and protist cells), the cell cycle is divided into two main stages: interphase and the mitotic (M) phase (including mitosis and cytokinesis). During interphase, the cell grows, accumulating nutrients needed for mitosis, and replicates its DNA and some of its organelles. During the mitotic phase, the replicated chromosomes, organelles, and cytoplasm separate into two new daughter cells. To ensure the proper replication of cellular components and division, there are control mechanisms known as cell cycle checkpoints after each of the key steps of the cycle that determine if the cell can progress to the next phase.

In cells without nuclei (prokaryotes), (i.e., bacteria and archaea), the cell cycle is divided into the B, C, and D periods. The B period extends from the end of cell division to the beginning of DNA replication. DNA replication occurs during the C period. The D period refers to the stage between the end of DNA replication and the splitting of the bacterial cell into two daughter cells.

The cell-division cycle is a vital process by which a single-celled fertilized egg develops into a mature organism, as well as the process by which hair, skin, blood cells, and some internal organs are renewed. After cell division, each of the daughter cells begin the interphase of a new cycle. Although the various stages of interphase are not usually morphologically distinguishable, each phase of the cell cycle has a distinct set of specialized biochemical processes that prepare the cell for initiation of the cell division.

Phases

The eukaryotic cell cycle consists of four distinct phases: G₁ phase, S phase (synthesis), G₂ phase (collectively known as interphase) and M phase (mitosis and cytokinesis). M phase is itself composed of two tightly coupled processes: mitosis, in which the cell's nucleus divides, and cytokinesis, in which the cell's cytoplasm divides forming two daughter cells. Activation of each phase is dependent on the proper progression and completion of the previous one. Cells that have temporarily or reversibly stopped dividing are said to have entered a state of quiescence called G₀ phase.

Schematic of the cell cycle. Outer ring: I = Interphase, M = Mitosis; inner ring: M = Mitosis, G₁ = Gap 1, G₂ = Gap 2, S = Synthesis; not in ring: G₀ = Gap 0/Resting

State	Phase	Abbreviation	Description
Resting	Gap 0	G0	A phase where the cell has left the cycle and has stopped dividing.
Interphase	Gap 1	G1	Cells increase in size in Gap 1. The G1 checkpoint control mechanism ensures that everything is ready for DNA synthesis.
	Synthesis	S	DNA replication occurs during this phase.
	Gap 2	G2	During the gap between DNA synthesis and mitosis, the cell will continue to grow. The G2 checkpoint control mechanism ensures that everything is ready to enter the M (mitosis) phase and divide.
Cell division	Mitosis	M	Cell growth stops at this stage and cellular energy is focused on the orderly division into two daughter cells. A checkpoint in the middle of mitosis (Metaphase Checkpoint) ensures that the cell is ready to complete cell division.

After cell division, each of the daughter cells begin the interphase of a new cycle. Although the various stages of interphase are not usually morphologically distinguishable, each phase of the cell cycle has a distinct set of specialized biochemical processes that prepare the cell for initiation of cell division.

G₀ phase (quiescence)

Plant cell cycle

 

Animal cell cycle

 

Main article: G0 phase

G₀ is a resting phase where the cell has left the cycle and has stopped dividing. The cell cycle starts with this phase. Non-proliferative (non-dividing) cells in multicellular eukaryotes generally enter the quiescent G₀ state from G₁ and may remain quiescent for long periods of time, possibly indefinitely (as is often the case for neurons). This is very common for cells that are fully differentiated. Some cells enter the G₀ phase semi-permanently and are considered post-mitotic, e.g., some liver, kidney, and stomach cells. Many cells do not enter G₀ and continue to divide throughout an organism's life, e.g., epithelial cells.

The word "post-mitotic" is sometimes used to refer to both quiescent and senescent cells. Cellular senescence occurs in response to DNA damage and external stress and usually constitutes an arrest in G₁. Cellular senescence may make a cell's progeny nonviable; it is often a biochemical alternative to the self-destruction of such a damaged cell by apoptosis.

Interphase

Main article: Interphase

Interphase represent the phase between two successive M phases. Interphase is a series of changes that takes place in a newly formed cell and its nucleus before it becomes capable of division again. It is also called preparatory phase or intermitosis. Typically interphase lasts for at least 91% of the total time required for the cell cycle.

Interphase proceeds in three stages, G₁, S, and G₂, followed by the cycle of mitosis and cytokinesis. The cell's nuclear DNA contents are duplicated during S phase.

G₁ phase (First growth phase or Post mitotic gap phase)

Main article: G1 phase

The first phase within interphase, from the end of the previous M phase until the beginning of DNA synthesis, is called G₁ (G indicating gap). It is also called the growth phase. During this phase, the biosynthetic activities of the cell, which are considerably slowed down during M phase, resume at a high rate. The duration of G₁ is highly variable, even among different cells of the same species. In this phase, the cell increases its supply of proteins, increases the number of organelles (such as mitochondria, ribosomes), and grows in size. In G₁ phase, a cell has three options.

• To continue cell cycle and enter S phase
• Stop cell cycle and enter G₀ phase for undergoing differentiation.
• Become arrested in G₁ phase hence it may enter G₀ phase or re-enter cell cycle.

The deciding point is called check point (Restriction point). This check point is called the restriction point or START and is regulated by G₁/S cyclins, which cause transition from G₁ to S phase. Passage through the G₁ check point commits the cell to division.

S phase (DNA replication)

Main article: S phase

The ensuing S phase starts when DNA synthesis commences; when it is complete, all of the chromosomes have been replicated, i.e., each chromosome consists of two sister chromatids. Thus, during this phase, the amount of DNA in the cell has doubled, though the ploidy and number of chromosomes are unchanged. Rates of RNA transcription and protein synthesis are very low during this phase. An exception to this is histone production, most of which occurs during the S phase.

G₂ phase (growth)

Main article: G2 phase

G₂ phase occurs after DNA replication and is a period of protein synthesis and rapid cell growth to prepare the cell for mitosis. During this phase microtubules begin to reorganize to form a spindle (preprophase). Before proceeding to mitotic phase, cells must be checked at the G₂ checkpoint for any DNA damage within the chromosomes. The G₂ checkpoint is mainly regulated by the tumor protein p53. If the DNA is damaged, p53 will either repair the DNA or trigger the apoptosis of the cell. If p53 is dysfunctional or mutated, cells with damaged DNA may continue through the cell cycle, leading to the development of cancer.

Mitotic phase (chromosome separation)

Main article: Mitosis

The relatively brief M phase consists of nuclear division (karyokinesis). It is a relatively short period of the cell cycle. M phase is complex and highly regulated. The sequence of events is divided into phases, corresponding to the completion of one set of activities and the start of the next. These phases are sequentially known as:

• prophase
• prometaphase
• metaphase
• anaphase
• telophase

Mitosis is the process by which a eukaryotic cell separates the chromosomes in its cell nucleus into two identical sets in two nuclei. During the process of mitosis the pairs of chromosomes condense and attach to microtubules that pull the sister chromatids to opposite sides of the cell.

Mitosis occurs exclusively in eukaryotic cells, but occurs in different ways in different species. For example, animal cells undergo an "open" mitosis, where the nuclear envelope breaks down before the chromosomes separate, while fungi such as Aspergillus nidulans and Saccharomyces cerevisiae (yeast) undergo a "closed" mitosis, where chromosomes divide within an intact cell nucleus.

Cytokinesis phase (separation of all cell components)

Main article: Cytokinesis

Mitosis is immediately followed by cytokinesis, which divides the nuclei, cytoplasm, organelles and cell membrane into two cells containing roughly equal shares of these cellular components. Mitosis and cytokinesis together define the division of the mother cell into two daughter cells, genetically identical to each other and to their parent cell. This accounts for approximately 10% of the cell cycle.

Because cytokinesis usually occurs in conjunction with mitosis, "mitosis" is often used interchangeably with "M phase". However, there are many cells where mitosis and cytokinesis occur separately, forming single cells with multiple nuclei in a process called endoreplication. This occurs most notably among the fungi and slime molds, but is found in various groups. Even in animals, cytokinesis and mitosis may occur independently, for instance during certain stages of fruit fly embryonic development. Errors in mitosis can result in cell death through apoptosis or cause mutations that may lead to cancer.

Regulation of eukaryotic cell cycle

Levels of the three major cyclin types oscillate during the cell cycle (top), providing the basis for oscillations in the cyclin–Cdk complexes that drive cell-cycle events (bottom). In general, Cdk levels are constant and in large excess over cyclin levels; thus, cyclin–Cdk complexes form in parallel with cyclin levels. The enzymatic activities of cyclin–Cdk complexes also tend to rise and fall in parallel with cyclin levels, although in some cases Cdk inhibitor proteins or phosphorylation introduce a delay between the formation and activation of cyclin–Cdk complexes. Formation of active G1/S–Cdk complexes commits the cell to a new division cycle at the Start checkpoint in late G1. G1/S–Cdks then activate the S–Cdk complexes that initiate DNA replication at the beginning of S phase. M–Cdk activation occurs after the completion of S phase, resulting in progression through the G2/M checkpoint and assembly of the mitotic spindle. APC activation then triggers sister-chromatid separation at the metaphase-to-anaphase transition. APC activity also causes the destruction of S and M cyclins and thus the inactivation of Cdks, which promotes the completion of mitosis and cytokinesis. APC activity is maintained in G1 until G1/S–Cdk activity rises again and commits the cell to the next cycle. This scheme serves only as a general guide and does not apply to all cell types.

Regulation of the cell cycle involves processes crucial to the survival of a cell, including the detection and repair of genetic damage as well as the prevention of uncontrolled cell division. The molecular events that control the cell cycle are ordered and directional; that is, each process occurs in a sequential fashion and it is impossible to "reverse" the cycle.

Role of cyclins and CDKs

Nobel Laureate Paul Nurse

Nobel Laureate Tim Hunt

Two key classes of regulatory molecules, cyclins and cyclin-dependent kinases (CDKs), determine a cell's progress through the cell cycle. Leland H. Hartwell, R. Timothy Hunt, and Paul M. Nurse won the 2001 Nobel Prize in Physiology or Medicine for their discovery of these central molecules. Many of the genes encoding cyclins and CDKs are conserved among all eukaryotes, but in general, more complex organisms have more elaborate cell cycle control systems that incorporate more individual components. Many of the relevant genes were first identified by studying yeast, especially Saccharomyces cerevisiae; genetic nomenclature in yeast dubs many of these genes cdc (for "cell division cycle") followed by an identifying number, e.g. cdc25 or cdc20.

Cyclins form the regulatory subunits and CDKs the catalytic subunits of an activated heterodimer; cyclins have no catalytic activity and CDKs are inactive in the absence of a partner cyclin. When activated by a bound cyclin, CDKs perform a common biochemical reaction called phosphorylation that activates or inactivates target proteins to orchestrate coordinated entry into the next phase of the cell cycle. Different cyclin-CDK combinations determine the downstream proteins targeted. CDKs are constitutively expressed in cells whereas cyclins are synthesised at specific stages of the cell cycle, in response to various molecular signals.

General mechanism of cyclin-CDK interaction

Upon receiving a pro-mitotic extracellular signal, G₁ cyclin-CDK complexes become active to prepare the cell for S phase, promoting the expression of transcription factors that in turn promote the expression of S cyclins and of enzymes required for DNA replication. The G₁ cyclin-CDK complexes also promote the degradation of molecules that function as S phase inhibitors by targeting them for ubiquitination. Once a protein has been ubiquitinated, it is targeted for proteolytic degradation by the proteasome. However, results from a recent study of E2F transcriptional dynamics at the single-cell level argue that the role of G1 cyclin-CDK activities, in particular cyclin D-CDK4/6, is to tune the timing rather than the commitment of cell cycle entry.

Active S cyclin-CDK complexes phosphorylate proteins that make up the pre-replication complexes assembled during G₁ phase on DNA replication origins. The phosphorylation serves two purposes: to activate each already-assembled pre-replication complex, and to prevent new complexes from forming. This ensures that every portion of the cell's genome will be replicated once and only once. The reason for prevention of gaps in replication is fairly clear, because daughter cells that are missing all or part of crucial genes will die. However, for reasons related to gene copy number effects, possession of extra copies of certain genes is also deleterious to the daughter cells.

Mitotic cyclin-CDK complexes, which are synthesized but inactivated during S and G₂ phases, promote the initiation of mitosis by stimulating downstream proteins involved in chromosome condensation and mitotic spindle assembly. A critical complex activated during this process is a ubiquitin ligase known as the anaphase-promoting complex (APC), which promotes degradation of structural proteins associated with the chromosomal kinetochore. APC also targets the mitotic cyclins for degradation, ensuring that telophase and cytokinesis can proceed.

Specific action of cyclin-CDK complexes

Cyclin D is the first cyclin produced in the cells that enter the cell cycle, in response to extracellular signals (e.g. growth factors). Cyclin D levels stay low in resting cells that are not proliferating. Additionally, CDK4/6 and CDK2 are also inactive because CDK4/6 are bound by INK4 family members (e.g., p16), limiting kinase activity. Meanwhile, CDK2 complexes are inhibited by the CIP/KIP proteins such as p21 and p27, When it is time for a cell to enter the cell cycle, which is triggered by a mitogenic stimuli, levels of cyclin D increase. In response to this trigger, cyclin D binds to existing CDK4/6, forming the active cyclin D-CDK4/6 complex. Cyclin D-CDK4/6 complexes in turn mono-phosphorylates the retinoblastoma susceptibility protein (Rb) to pRb. The un-phosphorylated Rb tumour suppressor functions in inducing cell cycle exit and maintaining G0 arrest (senescence).

In the last few decades, a model has been widely accepted whereby pRB proteins are inactivated by cyclin D-Cdk4/6-mediated phosphorylation. Rb has 14+ potential phosphorylation sites. Cyclin D-Cdk 4/6 progressively phosphorylates Rb to hyperphosphorylated state, which triggers dissociation of pRB–E2F complexes, thereby inducing G1/S cell cycle gene expression and progression into S phase.

However, scientific observations from a recent study show that Rb is present in three types of isoforms: (1) un-phosphorylated Rb in G0 state; (2) mono-phosphorylated Rb, also referred to as "hypo-phosphorylated' or 'partially' phosphorylated Rb in early G1 state; and (3) inactive hyper-phosphorylated Rb in late G1 state. In early G1 cells, mono-phosphorylated Rb exits as 14 different isoforms, one of each has distinct E2F binding affinity. Rb has been found to associate with hundreds of different proteins and the idea that different mono-phosphorylated Rb isoforms have different protein partners was very appealing. A recent report confirmed that mono-phosphorylation controls Rb's association with other proteins and generates functional distinct forms of Rb. All different mono-phosphorylated Rb isoforms inhibit E2F transcriptional program and are able to arrest cells in G1-phase. Importantly, different mono-phosphorylated forms of RB have distinct transcriptional outputs that are extended beyond E2F regulation.

In general, the binding of pRb to E2F inhibits the E2F target gene expression of certain G1/S and S transition genes including E-type cyclins. The partial phosphorylation of RB de-represses the Rb-mediated suppression of E2F target gene expression, begins the expression of cyclin E. The molecular mechanism that causes the cell switched to cyclin E activation is currently not known, but as cyclin E levels rise, the active cyclin E-CDK2 complex is formed, bringing Rb to be inactivated by hyper-phosphorylation. Hyperphosphorylated Rb is completely dissociated from E2F, enabling further expression of a wide range of E2F target genes are required for driving cells to proceed into S phase. Recently, it has been identified that cyclin D-Cdk4/6 binds to a C-terminal alpha-helix region of Rb that is only distinguishable to cyclin D rather than other cyclins, cyclin E, A and B. This observation based on the structural analysis of Rb phosphorylation supports that Rb is phosphorylated in a different level through multiple Cyclin-Cdk complexes. This also makes feasible the current model of a simultaneous switch-like inactivation of all mono-phosphorylated Rb isoforms through one type of Rb hyper-phosphorylation mechanism. In addition, mutational analysis of the cyclin D- Cdk 4/6 specific Rb C-terminal helix shows that disruptions of cyclin D-Cdk 4/6 binding to Rb prevents Rb phosphorylation, arrests cells in G1, and bolsters Rb's functions in tumor suppressor. This cyclin-Cdk driven cell cycle transitional mechanism governs a cell committed to the cell cycle that allows cell proliferation. A cancerous cell growth often accompanies with deregulation of Cyclin D-Cdk 4/6 activity.

The hyperphosphorylated Rb dissociates from the E2F/DP1/Rb complex (which was bound to the E2F responsive genes, effectively "blocking" them from transcription), activating E2F. Activation of E2F results in transcription of various genes like cyclin E, cyclin A, DNA polymerase, thymidine kinase, etc. Cyclin E thus produced binds to CDK2, forming the cyclin E-CDK2 complex, which pushes the cell from G₁ to S phase (G₁/S, which initiates the G₂/M transition). Cyclin B-cdk1 complex activation causes breakdown of nuclear envelope and initiation of prophase, and subsequently, its deactivation causes the cell to exit mitosis. A quantitative study of E2F transcriptional dynamics at the single-cell level by using engineered fluorescent reporter cells provided a quantitative framework for understanding the control logic of cell cycle entry, challenging the canonical textbook model. Genes that regulate the amplitude of E2F accumulation, such as Myc, determine the commitment in cell cycle and S phase entry. G1 cyclin-CDK activities are not the driver of cell cycle entry. Instead, they primarily tune the timing of E2F increase, thereby modulating the pace of cell cycle progression.

Inhibitors

Endogenous

Overview of signal transduction pathways involved in apoptosis, also known as "programmed cell death"

Two families of genes, the cip/kip (CDK interacting protein/Kinase inhibitory protein) family and the INK4a/ARF (Inhibitor of Kinase 4/Alternative Reading Frame) family, prevent the progression of the cell cycle. Because these genes are instrumental in prevention of tumor formation, they are known as tumor suppressors.

The cip/kip family includes the genes p21, p27 and p57. They halt the cell cycle in G₁ phase by binding to and inactivating cyclin-CDK complexes. p21 is activated by p53 (which, in turn, is triggered by DNA damage e.g. due to radiation). p27 is activated by Transforming Growth Factor β (TGF β), a growth inhibitor.

The INK4a/ARF family includes p16^INK4a, which binds to CDK4 and arrests the cell cycle in G₁ phase, and p14^ARF which prevents p53 degradation.

Synthetic

Synthetic inhibitors of Cdc25 could also be useful for the arrest of cell cycle and therefore be useful as antineoplastic and anticancer agents.

Many human cancers possess the hyper-activated Cdk 4/6 activities. Given the observations of cyclin D-Cdk 4/6 functions, inhibition of Cdk 4/6 should result in preventing a malignant tumor from proliferating. Consequently, scientists have tried to invent the synthetic Cdk4/6 inhibitor as Cdk4/6 has been characterized to be a therapeutic target for anti-tumor effectiveness. Three Cdk4/6 inhibitors - palbociclib, ribociclib, and abemaciclib - currently received FDA approval for clinical use to treat advanced-stage or metastatic, hormone-receptor-positive (HR-positive, HR+), HER2-negative (HER2-) breast cancer. For example, palbociclib is an orally active CDK4/6 inhibitor which has demonstrated improved outcomes for ER-positive/HER2-negative advanced breast cancer. The main side effect is neutropenia which can be managed by dose reduction.

Cdk4/6 targeted therapy will only treat cancer types where Rb is expressed. Cancer cells with loss of Rb have primary resistance to Cdk4/6 inhibitors.

Transcriptional regulatory network

Current evidence suggests that a semi-autonomous transcriptional network acts in concert with the CDK-cyclin machinery to regulate the cell cycle. Several gene expression studies in Saccharomyces cerevisiae have identified 800–1200 genes that change expression over the course of the cell cycle. They are transcribed at high levels at specific points in the cell cycle, and remain at lower levels throughout the rest of the cycle. While the set of identified genes differs between studies due to the computational methods and criteria used to identify them, each study indicates that a large portion of yeast genes are temporally regulated.

Many periodically expressed genes are driven by transcription factors that are also periodically expressed. One screen of single-gene knockouts identified 48 transcription factors (about 20% of all non-essential transcription factors) that show cell cycle progression defects. Genome-wide studies using high throughput technologies have identified the transcription factors that bind to the promoters of yeast genes, and correlating these findings with temporal expression patterns have allowed the identification of transcription factors that drive phase-specific gene expression. The expression profiles of these transcription factors are driven by the transcription factors that peak in the prior phase, and computational models have shown that a CDK-autonomous network of these transcription factors is sufficient to produce steady-state oscillations in gene expression).

Experimental evidence also suggests that gene expression can oscillate with the period seen in dividing wild-type cells independently of the CDK machinery. Orlando et al. used microarrays to measure the expression of a set of 1,271 genes that they identified as periodic in both wild type cells and cells lacking all S-phase and mitotic cyclins (clb1,2,3,4,5,6). Of the 1,271 genes assayed, 882 continued to be expressed in the cyclin-deficient cells at the same time as in the wild type cells, despite the fact that the cyclin-deficient cells arrest at the border between G₁ and S phase. However, 833 of the genes assayed changed behavior between the wild type and mutant cells, indicating that these genes are likely directly or indirectly regulated by the CDK-cyclin machinery. Some genes that continued to be expressed on time in the mutant cells were also expressed at different levels in the mutant and wild type cells. These findings suggest that while the transcriptional network may oscillate independently of the CDK-cyclin oscillator, they are coupled in a manner that requires both to ensure the proper timing of cell cycle events. Other work indicates that phosphorylation, a post-translational modification, of cell cycle transcription factors by Cdk1 may alter the localization or activity of the transcription factors in order to tightly control timing of target genes.

While oscillatory transcription plays a key role in the progression of the yeast cell cycle, the CDK-cyclin machinery operates independently in the early embryonic cell cycle. Before the midblastula transition, zygotic transcription does not occur and all needed proteins, such as the B-type cyclins, are translated from maternally loaded mRNA.

DNA replication and DNA replication origin activity

Analyses of synchronized cultures of Saccharomyces cerevisiae under conditions that prevent DNA replication initiation without delaying cell cycle progression showed that origin licensing decreases the expression of genes with origins near their 3' ends, revealing that downstream origins can regulate the expression of upstream genes. This confirms previous predictions from mathematical modeling of a global causal coordination between DNA replication origin activity and mRNA expression, and shows that mathematical modeling of DNA microarray data can be used to correctly predict previously unknown biological modes of regulation.

Checkpoints

Main article: Cell cycle checkpoint

Cell cycle checkpoints are used by the cell to monitor and regulate the progress of the cell cycle. Checkpoints prevent cell cycle progression at specific points, allowing verification of necessary phase processes and repair of DNA damage. The cell cannot proceed to the next phase until checkpoint requirements have been met. Checkpoints typically consist of a network of regulatory proteins that monitor and dictate the progression of the cell through the different stages of the cell cycle.

It is estimated that in normal human cells about 1% of single-strand DNA damages are converted to about 50 endogenous DNA double-strand breaks per cell per cell cycle. Although such double-strand breaks are usually repaired with high fidelity, errors in their repair are considered to contribute significantly to the rate of cancer in humans.

There are several checkpoints to ensure that damaged or incomplete DNA is not passed on to daughter cells. Three main checkpoints exist: the G₁/S checkpoint, the G₂/M checkpoint and the metaphase (mitotic) checkpoint. Another checkpoint is the Go checkpoint, in which the cells are checked for maturity. If the cells fail to pass this checkpoint by not being ready yet, they will be discarded from dividing.

G₁/S transition is a rate-limiting step in the cell cycle and is also known as restriction point. This is where the cell checks whether it has enough raw materials to fully replicate its DNA (nucleotide bases, DNA synthase, chromatin, etc.). An unhealthy or malnourished cell will get stuck at this checkpoint.

The G₂/M checkpoint is where the cell ensures that it has enough cytoplasm and phospholipids for two daughter cells. But sometimes more importantly, it checks to see if it is the right time to replicate. There are some situations where many cells need to all replicate simultaneously (for example, a growing embryo should have a symmetric cell distribution until it reaches the mid-blastula transition). This is done by controlling the G₂/M checkpoint.

The metaphase checkpoint is a fairly minor checkpoint, in that once a cell is in metaphase, it has committed to undergoing mitosis. However that's not to say it isn't important. In this checkpoint, the cell checks to ensure that the spindle has formed and that all of the chromosomes are aligned at the spindle equator before anaphase begins.

While these are the three "main" checkpoints, not all cells have to pass through each of these checkpoints in this order to replicate. Many types of cancer are caused by mutations that allow the cells to speed through the various checkpoints or even skip them altogether. Going from S to M to S phase almost consecutively. Because these cells have lost their checkpoints, any DNA mutations that may have occurred are disregarded and passed on to the daughter cells. This is one reason why cancer cells have a tendency to exponentially accrue mutations. Aside from cancer cells, many fully differentiated cell types no longer replicate so they leave the cell cycle and stay in G₀ until their death. Thus removing the need for cellular checkpoints. An alternative model of the cell cycle response to DNA damage has also been proposed, known as the postreplication checkpoint.

Checkpoint regulation plays an important role in an organism's development. In sexual reproduction, when egg fertilization occurs, when the sperm binds to the egg, it releases signalling factors that notify the egg that it has been fertilized. Among other things, this induces the now fertilized oocyte to return from its previously dormant, G₀, state back into the cell cycle and on to mitotic replication and division.

p53 plays an important role in triggering the control mechanisms at both G₁/S and G₂/M checkpoints. In addition to p53, checkpoint regulators are being heavily researched for their roles in cancer growth and proliferation.

Fluorescence imaging of the cell cycle

Fluorescent proteins visualize the cell cycle progression. IFP2.0-hGem(1/110) fluorescence is shown in green and highlights the S/G₂/M phases. smURFP-hCdtI(30/120) fluorescence is shown in red and highlights the G₀/G₁ phases.

Pioneering work by Atsushi Miyawaki and coworkers developed the fluorescent ubiquitination-based cell cycle indicator (FUCCI), which enables fluorescence imaging of the cell cycle. Originally, a green fluorescent protein, mAG, was fused to hGem(1/110) and an orange fluorescent protein (mKO₂) was fused to hCdt1(30/120). Note, these fusions are fragments that contain a nuclear localization signal and ubiquitination sites for degradation, but are not functional proteins. The green fluorescent protein is made during the S, G₂, or M phase and degraded during the G₀ or G₁ phase, while the orange fluorescent protein is made during the G₀ or G₁ phase and destroyed during the S, G₂, or M phase. A far-red and near-infrared FUCCI was developed using a cyanobacteria-derived fluorescent protein (smURFP) and a bacteriophytochrome-derived fluorescent protein (movie found at this link).

Role in tumor formation

A disregulation of the cell cycle components may lead to tumor formation. As mentioned above, when some genes like the cell cycle inhibitors, RB, p53 etc. mutate, they may cause the cell to multiply uncontrollably, forming a tumor. Although the duration of cell cycle in tumor cells is equal to or longer than that of normal cell cycle, the proportion of cells that are in active cell division (versus quiescent cells in G₀ phase) in tumors is much higher than that in normal tissue. Thus there is a net increase in cell number as the number of cells that die by apoptosis or senescence remains the same.

The cells which are actively undergoing cell cycle are targeted in cancer therapy as the DNA is relatively exposed during cell division and hence susceptible to damage by drugs or radiation. This fact is made use of in cancer treatment; by a process known as debulking, a significant mass of the tumor is removed which pushes a significant number of the remaining tumor cells from G₀ to G₁ phase (due to increased availability of nutrients, oxygen, growth factors etc.). Radiation or chemotherapy following the debulking procedure kills these cells which have newly entered the cell cycle.

The fastest cycling mammalian cells in culture, crypt cells in the intestinal epithelium, have a cycle time as short as 9 to 10 hours. Stem cells in resting mouse skin may have a cycle time of more than 200 hours. Most of this difference is due to the varying length of G₁, the most variable phase of the cycle. M and S do not vary much.

In general, cells are most radiosensitive in late M and G₂ phases and most resistant in late S phase.

For cells with a longer cell cycle time and a significantly long G₁ phase, there is a second peak of resistance late in G₁.

The pattern of resistance and sensitivity correlates with the level of sulfhydryl compounds in the cell. Sulfhydryls are natural substances that protect cells from radiation damage and tend to be at their highest levels in S and at their lowest near mitosis.

Homologous recombination (HR) is an accurate process for repairing DNA double-strand breaks. HR is nearly absent in G1 phase, is most active in S phase, and declines in G₂/M. Non-homologous end joining, a less accurate and more mutagenic process for repairing double strand breaks, is active throughout the cell cycle.

DNA repair

From Wikipedia, the free encyclopedia

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

DNA damage resulting in multiple broken chromosomes

DNA repair is a collection of processes by which a cell identifies and corrects damage to the DNA molecules that encode its genome. In human cells, both normal metabolic activities and environmental factors such as radiation can cause DNA damage, resulting in tens of thousands of individual molecular lesions per cell per day. Many of these lesions cause structural damage to the DNA molecule and can alter or eliminate the cell's ability to transcribe the gene that the affected DNA encodes. Other lesions induce potentially harmful mutations in the cell's genome, which affect the survival of its daughter cells after it undergoes mitosis. As a consequence, the DNA repair process is constantly active as it responds to damage in the DNA structure. When normal repair processes fail, and when cellular apoptosis does not occur, irreparable DNA damage may occur, including double-strand breaks and DNA crosslinkages (interstrand crosslinks or ICLs). This can eventually lead to malignant tumors, or cancer as per the two hit hypothesis.

The rate of DNA repair is dependent on many factors, including the cell type, the age of the cell, and the extracellular environment. A cell that has accumulated a large amount of DNA damage, or one that no longer effectively repairs damage incurred to its DNA, can enter one of three possible states:

1. an irreversible state of dormancy, known as senescence
2. cell suicide, also known as apoptosis or programmed cell death
3. unregulated cell division, which can lead to the formation of a tumor that is cancerous

The DNA repair ability of a cell is vital to the integrity of its genome and thus to the normal functionality of that organism. Many genes that were initially shown to influence life span have turned out to be involved in DNA damage repair and protection.

Play media

Paul Modrich talks about himself and his work in DNA repair.

The 2015 Nobel Prize in Chemistry was awarded to Tomas Lindahl, Paul Modrich, and Aziz Sancar for their work on the molecular mechanisms of DNA repair processes.

DNA damage

Further information: DNA damage (naturally occurring) and Free radical damage to DNA

DNA damage, due to environmental factors and normal metabolic processes inside the cell, occurs at a rate of 10,000 to 1,000,000 molecular lesions per cell per day. While this constitutes only 0.000165% of the human genome's approximately 6 billion bases, unrepaired lesions in critical genes (such as tumor suppressor genes) can impede a cell's ability to carry out its function and appreciably increase the likelihood of tumor formation and contribute to tumour heterogeneity.

The vast majority of DNA damage affects the primary structure of the double helix; that is, the bases themselves are chemically modified. These modifications can in turn disrupt the molecules' regular helical structure by introducing non-native chemical bonds or bulky adducts that do not fit in the standard double helix. Unlike proteins and RNA, DNA usually lacks tertiary structure and therefore damage or disturbance does not occur at that level. DNA is, however, supercoiled and wound around "packaging" proteins called histones (in eukaryotes), and both superstructures are vulnerable to the effects of DNA damage.

Sources

DNA damage can be subdivided into two main types:

1. endogenous damage such as attack by reactive oxygen species produced from normal metabolic byproducts (spontaneous mutation), especially the process of oxidative deamination
   1. also includes replication errors
2. exogenous damage caused by external agents such as
   1. ultraviolet [UV 200–400 nm] radiation from the sun or other artificial light sources
   2. other radiation frequencies, including x-rays and gamma rays
   3. hydrolysis or thermal disruption
   4. certain plant toxins
   5. human-made mutagenic chemicals, especially aromatic compounds that act as DNA intercalating agents
   6. viruses

The replication of damaged DNA before cell division can lead to the incorporation of wrong bases opposite damaged ones. Daughter cells that inherit these wrong bases carry mutations from which the original DNA sequence is unrecoverable (except in the rare case of a back mutation, for example, through gene conversion).

Types

There are several types of damage to DNA due to endogenous cellular processes:

1. oxidation of bases [e.g. 8-oxo-7,8-dihydroguanine (8-oxoG)] and generation of DNA strand interruptions from reactive oxygen species,
2. alkylation of bases (usually methylation), such as formation of 7-methylguanosine, 1-methyladenine, 6-O-Methylguanine
3. hydrolysis of bases, such as deamination, depurination, and depyrimidination.
4. "bulky adduct formation" (e.g., benzo[a]pyrene diol epoxide-dG adduct, aristolactam I-dA adduct)
5. mismatch of bases, due to errors in DNA replication, in which the wrong DNA base is stitched into place in a newly forming DNA strand, or a DNA base is skipped over or mistakenly inserted.
6. Monoadduct damage cause by change in single nitrogenous base of DNA
7. Diadduct damage

Damage caused by exogenous agents comes in many forms. Some examples are:

1. UV-B light causes crosslinking between adjacent cytosine and thymine bases creating pyrimidine dimers. This is called direct DNA damage.
2. UV-A light creates mostly free radicals. The damage caused by free radicals is called indirect DNA damage.
3. Ionizing radiation such as that created by radioactive decay or in cosmic rays causes breaks in DNA strands. Intermediate-level ionizing radiation may induce irreparable DNA damage (leading to replicational and transcriptional errors needed for neoplasia or may trigger viral interactions) leading to pre-mature aging and cancer.
4. Thermal disruption at elevated temperature increases the rate of depurination (loss of purine bases from the DNA backbone) and single-strand breaks. For example, hydrolytic depurination is seen in the thermophilic bacteria, which grow in hot springs at 40–80 °C. The rate of depurination (300 purine residues per genome per generation) is too high in these species to be repaired by normal repair machinery, hence a possibility of an adaptive response cannot be ruled out.
5. Industrial chemicals such as vinyl chloride and hydrogen peroxide, and environmental chemicals such as polycyclic aromatic hydrocarbons found in smoke, soot and tar create a huge diversity of DNA adducts- ethenobases, oxidized bases, alkylated phosphotriesters and crosslinking of DNA, just to name a few.

UV damage, alkylation/methylation, X-ray damage and oxidative damage are examples of induced damage. Spontaneous damage can include the loss of a base, deamination, sugar ring puckering and tautomeric shift. Constitutive (spontaneous) DNA damage caused by endogenous oxidants can be detected as a low level of histone H2AX phosphorylation in untreated cells.

Nuclear versus mitochondrial

In human cells, and eukaryotic cells in general, DNA is found in two cellular locations – inside the nucleus and inside the mitochondria. Nuclear DNA (nDNA) exists as chromatin during non-replicative stages of the cell cycle and is condensed into aggregate structures known as chromosomes during cell division. In either state the DNA is highly compacted and wound up around bead-like proteins called histones. Whenever a cell needs to express the genetic information encoded in its nDNA the required chromosomal region is unravelled, genes located therein are expressed, and then the region is condensed back to its resting conformation. Mitochondrial DNA (mtDNA) is located inside mitochondria organelles, exists in multiple copies, and is also tightly associated with a number of proteins to form a complex known as the nucleoid. Inside mitochondria, reactive oxygen species (ROS), or free radicals, byproducts of the constant production of adenosine triphosphate (ATP) via oxidative phosphorylation, create a highly oxidative environment that is known to damage mtDNA. A critical enzyme in counteracting the toxicity of these species is superoxide dismutase, which is present in both the mitochondria and cytoplasm of eukaryotic cells.

Senescence and apoptosis

Senescence, an irreversible process in which the cell no longer divides, is a protective response to the shortening of the chromosome ends, called telomeres. The telomeres are long regions of repetitive noncoding DNA that cap chromosomes and undergo partial degradation each time a cell undergoes division (see Hayflick limit). In contrast, quiescence is a reversible state of cellular dormancy that is unrelated to genome damage (see cell cycle). Senescence in cells may serve as a functional alternative to apoptosis in cases where the physical presence of a cell for spatial reasons is required by the organism, which serves as a "last resort" mechanism to prevent a cell with damaged DNA from replicating inappropriately in the absence of pro-growth cellular signaling. Unregulated cell division can lead to the formation of a tumor (see cancer), which is potentially lethal to an organism. Therefore, the induction of senescence and apoptosis is considered to be part of a strategy of protection against cancer.

Mutation

It is important to distinguish between DNA damage and mutation, the two major types of error in DNA. DNA damage and mutation are fundamentally different. Damage results in physical abnormalities in the DNA, such as single- and double-strand breaks, 8-hydroxydeoxyguanosine residues, and polycyclic aromatic hydrocarbon adducts. DNA damage can be recognized by enzymes, and thus can be correctly repaired if redundant information, such as the undamaged sequence in the complementary DNA strand or in a homologous chromosome, is available for copying. If a cell retains DNA damage, transcription of a gene can be prevented, and thus translation into a protein will also be blocked. Replication may also be blocked or the cell may die.

In contrast to DNA damage, a mutation is a change in the base sequence of the DNA. A mutation cannot be recognized by enzymes once the base change is present in both DNA strands, and thus a mutation cannot be repaired. At the cellular level, mutations can cause alterations in protein function and regulation. Mutations are replicated when the cell replicates. In a population of cells, mutant cells will increase or decrease in frequency according to the effects of the mutation on the ability of the cell to survive and reproduce.

Although distinctly different from each other, DNA damage and mutation are related because DNA damage often causes errors of DNA synthesis during replication or repair; these errors are a major source of mutation.

Given these properties of DNA damage and mutation, it can be seen that DNA damage is a special problem in non-dividing or slowly-dividing cells, where unrepaired damage will tend to accumulate over time. On the other hand, in rapidly dividing cells, unrepaired DNA damage that does not kill the cell by blocking replication will tend to cause replication errors and thus mutation. The great majority of mutations that are not neutral in their effect are deleterious to a cell's survival. Thus, in a population of cells composing a tissue with replicating cells, mutant cells will tend to be lost. However, infrequent mutations that provide a survival advantage will tend to clonally expand at the expense of neighboring cells in the tissue. This advantage to the cell is disadvantageous to the whole organism because such mutant cells can give rise to cancer. Thus, DNA damage in frequently dividing cells, because it gives rise to mutations, is a prominent cause of cancer. In contrast, DNA damage in infrequently-dividing cells is likely a prominent cause of aging.

Mechanisms

Main article: DNA damage (naturally occurring) § Repair of damaged DNA

Cells cannot function if DNA damage corrupts the integrity and accessibility of essential information in the genome (but cells remain superficially functional when non-essential genes are missing or damaged). Depending on the type of damage inflicted on the DNA's double helical structure, a variety of repair strategies have evolved to restore lost information. If possible, cells use the unmodified complementary strand of the DNA or the sister chromatid as a template to recover the original information. Without access to a template, cells use an error-prone recovery mechanism known as translesion synthesis as a last resort.

Damage to DNA alters the spatial configuration of the helix, and such alterations can be detected by the cell. Once damage is localized, specific DNA repair molecules bind at or near the site of damage, inducing other molecules to bind and form a complex that enables the actual repair to take place.

Direct reversal

Cells are known to eliminate three types of damage to their DNA by chemically reversing it. These mechanisms do not require a template, since the types of damage they counteract can occur in only one of the four bases. Such direct reversal mechanisms are specific to the type of damage incurred and do not involve breakage of the phosphodiester backbone. The formation of pyrimidine dimers upon irradiation with UV light results in an abnormal covalent bond between adjacent pyrimidine bases. The photoreactivation process directly reverses this damage by the action of the enzyme photolyase, whose activation is obligately dependent on energy absorbed from blue/UV light (300–500 nm wavelength) to promote catalysis. Photolyase, an old enzyme present in bacteria, fungi, and most animals no longer functions in humans, who instead use nucleotide excision repair to repair damage from UV irradiation. Another type of damage, methylation of guanine bases, is directly reversed by the protein methyl guanine methyl transferase (MGMT), the bacterial equivalent of which is called ogt. This is an expensive process because each MGMT molecule can be used only once; that is, the reaction is stoichiometric rather than catalytic. A generalized response to methylating agents in bacteria is known as the adaptive response and confers a level of resistance to alkylating agents upon sustained exposure by upregulation of alkylation repair enzymes. The third type of DNA damage reversed by cells is certain methylation of the bases cytosine and adenine.

Single-strand damage

Structure of the base-excision repair enzyme uracil-DNA glycosylase excising a hydrolytically-produced uracil residue from DNA. The uracil residue is shown in yellow.

When only one of the two strands of a double helix has a defect, the other strand can be used as a template to guide the correction of the damaged strand. In order to repair damage to one of the two paired molecules of DNA, there exist a number of excision repair mechanisms that remove the damaged nucleotide and replace it with an undamaged nucleotide complementary to that found in the undamaged DNA strand.

1. Base excision repair (BER): damaged single bases or nucleotides are most commonly repaired by removing the base or the nucleotide involved and then inserting the correct base or nucleotide. In base excision repair, a glycosylase enzyme removes the damaged base from the DNA by cleaving the bond between the base and the deoxyribose. These enzymes remove a single base to create an apurinic or apyrimidinic site (AP site). Enzymes called AP endonucleases nick the damaged DNA backbone at the AP site. DNA polymerase then removes the damaged region using its 5’ to 3’ exonuclease activity and correctly synthesizes the new strand using the complementary strand as a template. The gap is then sealed by enzyme DNA ligase.
2. Nucleotide excision repair (NER): bulky, helix-distorting damage, such as pyrimidine dimerization caused by UV light is usually repaired by a three-step process. First the damage is recognized, then 12-24 nucleotide-long strands of DNA are removed both upstream and downstream of the damage site by endonucleases, and the removed DNA region is then resynthesized. NER is a highly evolutionarily conserved repair mechanism and is used in nearly all eukaryotic and prokaryotic cells. In prokaryotes, NER is mediated by Uvr proteins. In eukaryotes, many more proteins are involved, although the general strategy is the same.
3. Mismatch repair systems are present in essentially all cells to correct errors that are not corrected by proofreading. These systems consist of at least two proteins. One detects the mismatch, and the other recruits an endonuclease that cleaves the newly synthesized DNA strand close to the region of damage. In E. coli , the proteins involved are the Mut class proteins: MutS, MutL, and MutH. In most Eukaryotes, the analog for MutS is MSH and the analog for MutL is MLH. MutH is only present in bacteria. This is followed by removal of damaged region by an exonuclease, resynthesis by DNA polymerase, and nick sealing by DNA ligase.

Double-strand breaks

Double-strand break repair pathway models

Double-strand breaks, in which both strands in the double helix are severed, are particularly hazardous to the cell because they can lead to genome rearrangements. In fact, when a double-strand break is accompanied by a cross-linkage joining the two strands at the same point, neither strand can be used as a template for the repair mechanisms, so that the cell will not be able to complete mitosis when it next divides, and will either die or, in rare cases, undergo a mutation. Three mechanisms exist to repair double-strand breaks (DSBs): non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), and homologous recombination (HR). In an in vitro system, MMEJ occurred in mammalian cells at the levels of 10–20% of HR when both HR and NHEJ mechanisms were also available.

DNA ligase, shown above repairing chromosomal damage, is an enzyme that joins broken nucleotides together by catalyzing the formation of an internucleotide ester bond between the phosphate backbone and the deoxyribose nucleotides.

In NHEJ, DNA Ligase IV, a specialized DNA ligase that forms a complex with the cofactor XRCC4, directly joins the two ends. To guide accurate repair, NHEJ relies on short homologous sequences called microhomologies present on the single-stranded tails of the DNA ends to be joined. If these overhangs are compatible, repair is usually accurate. NHEJ can also introduce mutations during repair. Loss of damaged nucleotides at the break site can lead to deletions, and joining of nonmatching termini forms insertions or translocations. NHEJ is especially important before the cell has replicated its DNA, since there is no template available for repair by homologous recombination. There are "backup" NHEJ pathways in higher eukaryotes. Besides its role as a genome caretaker, NHEJ is required for joining hairpin-capped double-strand breaks induced during V(D)J recombination, the process that generates diversity in B-cell and T-cell receptors in the vertebrate immune system.

Homologous recombination requires the presence of an identical or nearly identical sequence to be used as a template for repair of the break. The enzymatic machinery responsible for this repair process is nearly identical to the machinery responsible for chromosomal crossover during meiosis. This pathway allows a damaged chromosome to be repaired using a sister chromatid (available in G2 after DNA replication) or a homologous chromosome as a template. DSBs caused by the replication machinery attempting to synthesize across a single-strand break or unrepaired lesion cause collapse of the replication fork and are typically repaired by recombination.

MMEJ starts with short-range end resection by MRE11 nuclease on either side of a double-strand break to reveal microhomology regions. In further steps, Poly (ADP-ribose) polymerase 1 (PARP1) is required and may be an early step in MMEJ. There is pairing of microhomology regions followed by recruitment of flap structure-specific endonuclease 1 (FEN1) to remove overhanging flaps. This is followed by recruitment of XRCC1–LIG3 to the site for ligating the DNA ends, leading to an intact DNA. MMEJ is always accompanied by a deletion, so that MMEJ is a mutagenic pathway for DNA repair.

The extremophile Deinococcus radiodurans has a remarkable ability to survive DNA damage from ionizing radiation and other sources. At least two copies of the genome, with random DNA breaks, can form DNA fragments through annealing. Partially overlapping fragments are then used for synthesis of homologous regions through a moving D-loop that can continue extension until they find complementary partner strands. In the final step there is crossover by means of RecA-dependent homologous recombination.

Topoisomerases introduce both single- and double-strand breaks in the course of changing the DNA's state of supercoiling, which is especially common in regions near an open replication fork. Such breaks are not considered DNA damage because they are a natural intermediate in the topoisomerase biochemical mechanism and are immediately repaired by the enzymes that created them.

Another type of DNA double-strand breaks originates from the DNA heat-sensitive or heat-labile sites. These DNA sites are not initial DSBs. However, they convert to DSB after treating with elevated temperature. Ionizing irradiation can induces a highly complex form of DNA damage as clustered damage. It consists of different types of DNA lesions in various locations of the DNA helix. Some of these closely located lesions can probably convert to DSB by exposure to high temperatures. But the exact nature of these lesions and their interactions are not discovered yet.

Translesion synthesis

Translesion synthesis (TLS) is a DNA damage tolerance process that allows the DNA replication machinery to replicate past DNA lesions such as thymine dimers or AP sites. It involves switching out regular DNA polymerases for specialized translesion polymerases (i.e. DNA polymerase IV or V, from the Y Polymerase family), often with larger active sites that can facilitate the insertion of bases opposite damaged nucleotides. The polymerase switching is thought to be mediated by, among other factors, the post-translational modification of the replication processivity factor PCNA. Translesion synthesis polymerases often have low fidelity (high propensity to insert wrong bases) on undamaged templates relative to regular polymerases. However, many are extremely efficient at inserting correct bases opposite specific types of damage. For example, Pol η mediates error-free bypass of lesions induced by UV irradiation, whereas Pol ι introduces mutations at these sites. Pol η is known to add the first adenine across the T^T photodimer using Watson-Crick base pairing and the second adenine will be added in its syn conformation using Hoogsteen base pairing. From a cellular perspective, risking the introduction of point mutations during translesion synthesis may be preferable to resorting to more drastic mechanisms of DNA repair, which may cause gross chromosomal aberrations or cell death. In short, the process involves specialized polymerases either bypassing or repairing lesions at locations of stalled DNA replication. For example, Human DNA polymerase eta can bypass complex DNA lesions like guanine-thymine intra-strand crosslink, G[8,5-Me]T, although it can cause targeted and semi-targeted mutations. Paromita Raychaudhury and Ashis Basu studied the toxicity and mutagenesis of the same lesion in Escherichia coli by replicating a G[8,5-Me]T-modified plasmid in E. coli with specific DNA polymerase knockouts. Viability was very low in a strain lacking pol II, pol IV, and pol V, the three SOS-inducible DNA polymerases, indicating that translesion synthesis is conducted primarily by these specialized DNA polymerases. A bypass platform is provided to these polymerases by Proliferating cell nuclear antigen (PCNA). Under normal circumstances, PCNA bound to polymerases replicates the DNA. At a site of lesion, PCNA is ubiquitinated, or modified, by the RAD6/RAD18 proteins to provide a platform for the specialized polymerases to bypass the lesion and resume DNA replication. After translesion synthesis, extension is required. This extension can be carried out by a replicative polymerase if the TLS is error-free, as in the case of Pol η, yet if TLS results in a mismatch, a specialized polymerase is needed to extend it; Pol ζ. Pol ζ is unique in that it can extend terminal mismatches, whereas more processive polymerases cannot. So when a lesion is encountered, the replication fork will stall, PCNA will switch from a processive polymerase to a TLS polymerase such as Pol ι to fix the lesion, then PCNA may switch to Pol ζ to extend the mismatch, and last PCNA will switch to the processive polymerase to continue replication.

Global response to DNA damage

Cells exposed to ionizing radiation, ultraviolet light or chemicals are prone to acquire multiple sites of bulky DNA lesions and double-strand breaks. Moreover, DNA damaging agents can damage other biomolecules such as proteins, carbohydrates, lipids, and RNA. The accumulation of damage, to be specific, double-strand breaks or adducts stalling the replication forks, are among known stimulation signals for a global response to DNA damage. The global response to damage is an act directed toward the cells' own preservation and triggers multiple pathways of macromolecular repair, lesion bypass, tolerance, or apoptosis. The common features of global response are induction of multiple genes, cell cycle arrest, and inhibition of cell division.

Initial steps

The packaging of eukaryotic DNA into chromatin presents a barrier to all DNA-based processes that require recruitment of enzymes to their sites of action. To allow DNA repair, the chromatin must be remodeled. In eukaryotes, ATP dependent chromatin remodeling complexes and histone-modifying enzymes are two predominant factors employed to accomplish this remodeling process.

Chromatin relaxation occurs rapidly at the site of a DNA damage. In one of the earliest steps, the stress-activated protein kinase, c-Jun N-terminal kinase (JNK), phosphorylates SIRT6 on serine 10 in response to double-strand breaks or other DNA damage. This post-translational modification facilitates the mobilization of SIRT6 to DNA damage sites, and is required for efficient recruitment of poly (ADP-ribose) polymerase 1 (PARP1) to DNA break sites and for efficient repair of DSBs. PARP1 protein starts to appear at DNA damage sites in less than a second, with half maximum accumulation within 1.6 seconds after the damage occurs. PARP1 synthesizes polymeric adenosine diphosphate ribose (poly (ADP-ribose) or PAR) chains on itself. Next the chromatin remodeler ALC1 quickly attaches to the product of PARP1 action, a poly-ADP ribose chain, and ALC1 completes arrival at the DNA damage within 10 seconds of the occurrence of the damage. About half of the maximum chromatin relaxation, presumably due to action of ALC1, occurs by 10 seconds. This then allows recruitment of the DNA repair enzyme MRE11, to initiate DNA repair, within 13 seconds.

γH2AX, the phosphorylated form of H2AX is also involved in the early steps leading to chromatin decondensation after DNA double-strand breaks. The histone variant H2AX constitutes about 10% of the H2A histones in human chromatin. γH2AX (H2AX phosphorylated on serine 139) can be detected as soon as 20 seconds after irradiation of cells (with DNA double-strand break formation), and half maximum accumulation of γH2AX occurs in one minute. The extent of chromatin with phosphorylated γH2AX is about two million base pairs at the site of a DNA double-strand break. γH2AX does not, itself, cause chromatin decondensation, but within 30 seconds of irradiation, RNF8 protein can be detected in association with γH2AX. RNF8 mediates extensive chromatin decondensation, through its subsequent interaction with CHD4, a component of the nucleosome remodeling and deacetylase complex NuRD.

DDB2 occurs in a heterodimeric complex with DDB1. This complex further complexes with the ubiquitin ligase protein CUL4A and with PARP1. This larger complex rapidly associates with UV-induced damage within chromatin, with half-maximum association completed in 40 seconds. The PARP1 protein, attached to both DDB1 and DDB2, then PARylates (creates a poly-ADP ribose chain) on DDB2 that attracts the DNA remodeling protein ALC1. Action of ALC1 relaxes the chromatin at the site of UV damage to DNA. This relaxation allows other proteins in the nucleotide excision repair pathway to enter the chromatin and repair UV-induced cyclobutane pyrimidine dimer damages.

After rapid chromatin remodeling, cell cycle checkpoints are activated to allow DNA repair to occur before the cell cycle progresses. First, two kinases, ATM and ATR are activated within 5 or 6 minutes after DNA is damaged. This is followed by phosphorylation of the cell cycle checkpoint protein Chk1, initiating its function, about 10 minutes after DNA is damaged.

DNA damage checkpoints

After DNA damage, cell cycle checkpoints are activated. Checkpoint activation pauses the cell cycle and gives the cell time to repair the damage before continuing to divide. DNA damage checkpoints occur at the G1/S and G2/M boundaries. An intra-S checkpoint also exists. Checkpoint activation is controlled by two master kinases, ATM and ATR. ATM responds to DNA double-strand breaks and disruptions in chromatin structure, whereas ATR primarily responds to stalled replication forks. These kinases phosphorylate downstream targets in a signal transduction cascade, eventually leading to cell cycle arrest. A class of checkpoint mediator proteins including BRCA1, MDC1, and 53BP1 has also been identified. These proteins seem to be required for transmitting the checkpoint activation signal to downstream proteins.

DNA damage checkpoint is a signal transduction pathway that blocks cell cycle progression in G1, G2 and metaphase and slows down the rate of S phase progression when DNA is damaged. It leads to a pause in cell cycle allowing the cell time to repair the damage before continuing to divide.

Checkpoint Proteins can be separated into four groups: phosphatidylinositol 3-kinase (PI3K)-like protein kinase, proliferating cell nuclear antigen (PCNA)-like group, two serine/threonine(S/T) kinases and their adaptors. Central to all DNA damage induced checkpoints responses is a pair of large protein kinases belonging to the first group of PI3K-like protein kinases-the ATM (Ataxia telangiectasia mutated) and ATR (Ataxia- and Rad-related) kinases, whose sequence and functions have been well conserved in evolution. All DNA damage response requires either ATM or ATR because they have the ability to bind to the chromosomes at the site of DNA damage, together with accessory proteins that are platforms on which DNA damage response components and DNA repair complexes can be assembled.

An important downstream target of ATM and ATR is p53, as it is required for inducing apoptosis following DNA damage. The cyclin-dependent kinase inhibitor p21 is induced by both p53-dependent and p53-independent mechanisms and can arrest the cell cycle at the G1/S and G2/M checkpoints by deactivating cyclin/cyclin-dependent kinase complexes.

The prokaryotic SOS response

The SOS response is the changes in gene expression in Escherichia coli and other bacteria in response to extensive DNA damage. The prokaryotic SOS system is regulated by two key proteins: LexA and RecA. The LexA homodimer is a transcriptional repressor that binds to operator sequences commonly referred to as SOS boxes. In Escherichia coli it is known that LexA regulates transcription of approximately 48 genes including the lexA and recA genes. The SOS response is known to be widespread in the Bacteria domain, but it is mostly absent in some bacterial phyla, like the Spirochetes. The most common cellular signals activating the SOS response are regions of single-stranded DNA (ssDNA), arising from stalled replication forks or double-strand breaks, which are processed by DNA helicase to separate the two DNA strands. In the initiation step, RecA protein binds to ssDNA in an ATP hydrolysis driven reaction creating RecA–ssDNA filaments. RecA–ssDNA filaments activate LexA autoprotease activity, which ultimately leads to cleavage of LexA dimer and subsequent LexA degradation. The loss of LexA repressor induces transcription of the SOS genes and allows for further signal induction, inhibition of cell division and an increase in levels of proteins responsible for damage processing.

In Escherichia coli, SOS boxes are 20-nucleotide long sequences near promoters with palindromic structure and a high degree of sequence conservation. In other classes and phyla, the sequence of SOS boxes varies considerably, with different length and composition, but it is always highly conserved and one of the strongest short signals in the genome. The high information content of SOS boxes permits differential binding of LexA to different promoters and allows for timing of the SOS response. The lesion repair genes are induced at the beginning of SOS response. The error-prone translesion polymerases, for example, UmuCD'2 (also called DNA polymerase V), are induced later on as a last resort. Once the DNA damage is repaired or bypassed using polymerases or through recombination, the amount of single-stranded DNA in cells is decreased, lowering the amounts of RecA filaments decreases cleavage activity of LexA homodimer, which then binds to the SOS boxes near promoters and restores normal gene expression.

Eukaryotic transcriptional responses to DNA damage

Eukaryotic cells exposed to DNA damaging agents also activate important defensive pathways by inducing multiple proteins involved in DNA repair, cell cycle checkpoint control, protein trafficking and degradation. Such genome wide transcriptional response is very complex and tightly regulated, thus allowing coordinated global response to damage. Exposure of yeast Saccharomyces cerevisiae to DNA damaging agents results in overlapping but distinct transcriptional profiles. Similarities to environmental shock response indicates that a general global stress response pathway exist at the level of transcriptional activation. In contrast, different human cell types respond to damage differently indicating an absence of a common global response. The probable explanation for this difference between yeast and human cells may be in the heterogeneity of mammalian cells. In an animal different types of cells are distributed among different organs that have evolved different sensitivities to DNA damage.

In general global response to DNA damage involves expression of multiple genes responsible for postreplication repair, homologous recombination, nucleotide excision repair, DNA damage checkpoint, global transcriptional activation, genes controlling mRNA decay, and many others. A large amount of damage to a cell leaves it with an important decision: undergo apoptosis and die, or survive at the cost of living with a modified genome. An increase in tolerance to damage can lead to an increased rate of survival that will allow a greater accumulation of mutations. Yeast Rev1 and human polymerase η are members of [Y family translesion DNA polymerases present during global response to DNA damage and are responsible for enhanced mutagenesis during a global response to DNA damage in eukaryotes.

Aging

Main article: DNA damage theory of aging

Pathological effects of poor DNA repair

DNA repair rate is an important determinant of cell pathology

Experimental animals with genetic deficiencies in DNA repair often show decreased life span and increased cancer incidence. For example, mice deficient in the dominant NHEJ pathway and in telomere maintenance mechanisms get lymphoma and infections more often, and, as a consequence, have shorter lifespans than wild-type mice. In similar manner, mice deficient in a key repair and transcription protein that unwinds DNA helices have premature onset of aging-related diseases and consequent shortening of lifespan. However, not every DNA repair deficiency creates exactly the predicted effects; mice deficient in the NER pathway exhibited shortened life span without correspondingly higher rates of mutation.

If the rate of DNA damage exceeds the capacity of the cell to repair it, the accumulation of errors can overwhelm the cell and result in early senescence, apoptosis, or cancer. Inherited diseases associated with faulty DNA repair functioning result in premature aging, increased sensitivity to carcinogens and correspondingly increased cancer risk (see below). On the other hand, organisms with enhanced DNA repair systems, such as Deinococcus radiodurans, the most radiation-resistant known organism, exhibit remarkable resistance to the double-strand break-inducing effects of radioactivity, likely due to enhanced efficiency of DNA repair and especially NHEJ.

Longevity and caloric restriction

Most life span influencing genes affect the rate of DNA damage

A number of individual genes have been identified as influencing variations in life span within a population of organisms. The effects of these genes is strongly dependent on the environment, in particular, on the organism's diet. Caloric restriction reproducibly results in extended lifespan in a variety of organisms, likely via nutrient sensing pathways and decreased metabolic rate. The molecular mechanisms by which such restriction results in lengthened lifespan are as yet unclear (see for some discussion); however, the behavior of many genes known to be involved in DNA repair is altered under conditions of caloric restriction. Several agents reported to have anti-aging properties have been shown to attenuate constitutive level of mTOR signaling, an evidence of reduction of metabolic activity, and concurrently to reduce constitutive level of DNA damage induced by endogenously generated reactive oxygen species.

For example, increasing the gene dosage of the gene SIR-2, which regulates DNA packaging in the nematode worm Caenorhabditis elegans, can significantly extend lifespan. The mammalian homolog of SIR-2 is known to induce downstream DNA repair factors involved in NHEJ, an activity that is especially promoted under conditions of caloric restriction. Caloric restriction has been closely linked to the rate of base excision repair in the nuclear DNA of rodents, although similar effects have not been observed in mitochondrial DNA.

The C. elegans gene AGE-1, an upstream effector of DNA repair pathways, confers dramatically extended life span under free-feeding conditions but leads to a decrease in reproductive fitness under conditions of caloric restriction. This observation supports the pleiotropy theory of the biological origins of aging, which suggests that genes conferring a large survival advantage early in life will be selected for even if they carry a corresponding disadvantage late in life.

Medicine and DNA repair modulation

Main article: DNA repair-deficiency disorder

Hereditary DNA repair disorders

Defects in the NER mechanism are responsible for several genetic disorders, including:

• Xeroderma pigmentosum: hypersensitivity to sunlight/UV, resulting in increased skin cancer incidence and premature aging
• Cockayne syndrome: hypersensitivity to UV and chemical agents
• Trichothiodystrophy: sensitive skin, brittle hair and nails

Mental retardation often accompanies the latter two disorders, suggesting increased vulnerability of developmental neurons.

Other DNA repair disorders include:

• Werner's syndrome: premature aging and retarded growth
• Bloom's syndrome: sunlight hypersensitivity, high incidence of malignancies (especially leukemias).
• Ataxia telangiectasia: sensitivity to ionizing radiation and some chemical agents

All of the above diseases are often called "segmental progerias" ("accelerated aging diseases") because their victims appear elderly and suffer from aging-related diseases at an abnormally young age, while not manifesting all the symptoms of old age.

Other diseases associated with reduced DNA repair function include Fanconi anemia, hereditary breast cancer and hereditary colon cancer.

Cancer

Because of inherent limitations in the DNA repair mechanisms, if humans lived long enough, they would all eventually develop cancer. There are at least 34 Inherited human DNA repair gene mutations that increase cancer risk. Many of these mutations cause DNA repair to be less effective than normal. In particular, Hereditary nonpolyposis colorectal cancer (HNPCC) is strongly associated with specific mutations in the DNA mismatch repair pathway. BRCA1 and BRCA2, two important genes whose mutations confer a hugely increased risk of breast cancer on carriers, are both associated with a large number of DNA repair pathways, especially NHEJ and homologous recombination.

Cancer therapy procedures such as chemotherapy and radiotherapy work by overwhelming the capacity of the cell to repair DNA damage, resulting in cell death. Cells that are most rapidly dividing – most typically cancer cells – are preferentially affected. The side-effect is that other non-cancerous but rapidly dividing cells such as progenitor cells in the gut, skin, and hematopoietic system are also affected. Modern cancer treatments attempt to localize the DNA damage to cells and tissues only associated with cancer, either by physical means (concentrating the therapeutic agent in the region of the tumor) or by biochemical means (exploiting a feature unique to cancer cells in the body). In the context of therapies targeting DNA damage response genes, the latter approach has been termed 'synthetic lethality'.

Perhaps the most well-known of these 'synthetic lethality' drugs is the poly(ADP-ribose) polymerase 1 (PARP1) inhibitor olaparib, which was approved by the Food and Drug Administration in 2015 for the treatment in women of BRCA-defective ovarian cancer. Tumor cells with partial loss of DNA damage response (specifically, homologous recombination repair) are dependent on another mechanism – single-strand break repair – which is a mechanism consisting, in part, of the PARP1 gene product. Olaparib is combined with chemotherapeutics to inhibit single-strand break repair induced by DNA damage caused by the co-administered chemotherapy. Tumor cells relying on this residual DNA repair mechanism are unable to repair the damage and hence are not able to survive and proliferate, whereas normal cells can repair the damage with the functioning homologous recombination mechanism.

Many other drugs for use against other residual DNA repair mechanisms commonly found in cancer are currently under investigation. However, synthetic lethality therapeutic approaches have been questioned due to emerging evidence of acquired resistance, achieved through rewiring of DNA damage response pathways and reversion of previously inhibited defects.

DNA repair defects in cancer

It has become apparent over the past several years that the DNA damage response acts as a barrier to the malignant transformation of preneoplastic cells. Previous studies have shown an elevated DNA damage response in cell-culture models with oncogene activation and preneoplastic colon adenomas. DNA damage response mechanisms trigger cell-cycle arrest, and attempt to repair DNA lesions or promote cell death/senescence if repair is not possible. Replication stress is observed in preneoplastic cells due to increased proliferation signals from oncogenic mutations. Replication stress is characterized by: increased replication initiation/origin firing; increased transcription and collisions of transcription-replication complexes; nucleotide deficiency; increase in reactive oxygen species (ROS).

Replication stress, along with the selection for inactivating mutations in DNA damage response genes in the evolution of the tumor, leads to downregulation and/or loss of some DNA damage response mechanisms, and hence loss of DNA repair and/or senescence/programmed cell death. In experimental mouse models, loss of DNA damage response-mediated cell senescence was observed after using a short hairpin RNA (shRNA) to inhibit the double-strand break response kinase ataxia telangiectasia (ATM), leading to increased tumor size and invasiveness. Humans born with inherited defects in DNA repair mechanisms (for example, Li-Fraumeni syndrome) have a higher cancer risk.

The prevalence of DNA damage response mutations differs across cancer types; for example, 30% of breast invasive carcinomas have mutations in genes involved in homologous recombination. In cancer, downregulation is observed across all DNA damage response mechanisms (base excision repair (BER), nucleotide excision repair (NER), DNA mismatch repair (MMR), homologous recombination repair (HR), non-homologous end joining (NHEJ) and translesion DNA synthesis (TLS). As well as mutations to DNA damage repair genes, mutations also arise in the genes responsible for arresting the cell cycle to allow sufficient time for DNA repair to occur, and some genes are involved in both DNA damage repair and cell cycle checkpoint control, for example ATM and checkpoint kinase 2 (CHEK2) – a tumor suppressor that is often absent or downregulated in non-small cell lung cancer.

Genes involved in DNA damage response pathways and frequently mutated in cancer (HR = homologous recombination; NHEJ = non-homologous end joining; SSA = single-strand annealing; FA = fanconi anemia pathway; BER = base excision repair; NER = nucleotide excision repair; MMR = mismatch repair)

	HR	NHEJ	SSA	FA	BER	NER	MMR
ATM
ATR
PAXIP
RPA
BRCA1
BRCA2
RAD51
RFC
XRCC1
PCNA
PARP1
ERCC1
MSH3

Epigenetic DNA repair defects in cancer

Classically, cancer has been viewed as a set of diseases that are driven by progressive genetic abnormalities that include mutations in tumour-suppressor genes and oncogenes, and chromosomal aberrations. However, it has become apparent that cancer is also driven by epigenetic alterations.

Epigenetic alterations refer to functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence. Examples of such modifications are changes in DNA methylation (hypermethylation and hypomethylation) and histone modification, changes in chromosomal architecture (caused by inappropriate expression of proteins such as HMGA2 or HMGA1) and changes caused by microRNAs. Each of these epigenetic alterations serves to regulate gene expression without altering the underlying DNA sequence. These changes usually remain through cell divisions, last for multiple cell generations, and can be considered to be epimutations (equivalent to mutations).

While large numbers of epigenetic alterations are found in cancers, the epigenetic alterations in DNA repair genes, causing reduced expression of DNA repair proteins, appear to be particularly important. Such alterations are thought to occur early in progression to cancer and to be a likely cause of the genetic instability characteristic of cancers.

Reduced expression of DNA repair genes causes deficient DNA repair. When DNA repair is deficient DNA damages remain in cells at a higher than usual level and these excess damages cause increased frequencies of mutation or epimutation. Mutation rates increase substantially in cells defective in DNA mismatch repair^[93][94] or in homologous recombinational repair (HRR). Chromosomal rearrangements and aneuploidy also increase in HRR defective cells.

Higher levels of DNA damage not only cause increased mutation, but also cause increased epimutation. During repair of DNA double strand breaks, or repair of other DNA damages, incompletely cleared sites of repair can cause epigenetic gene silencing.

Deficient expression of DNA repair proteins due to an inherited mutation can cause increased risk of cancer. Individuals with an inherited impairment in any of 34 DNA repair genes (see article DNA repair-deficiency disorder) have an increased risk of cancer, with some defects causing up to a 100% lifetime chance of cancer (e.g. p53 mutations). However, such germline mutations (which cause highly penetrant cancer syndromes) are the cause of only about 1 percent of cancers.

Frequencies of epimutations in DNA repair genes

A chart of common DNA damaging agents, examples of lesions they cause in DNA, and pathways used to repair these lesions. Also shown are many of the genes in these pathways, an indication of which genes are epigenetically regulated to have reduced (or increased) expression in various cancers. It also shows genes in the error-prone microhomology-mediated end joining pathway with increased expression in various cancers.

Deficiencies in DNA repair enzymes are occasionally caused by a newly arising somatic mutation in a DNA repair gene, but are much more frequently caused by epigenetic alterations that reduce or silence expression of DNA repair genes. For example, when 113 colorectal cancers were examined in sequence, only four had a missense mutation in the DNA repair gene MGMT, while the majority had reduced MGMT expression due to methylation of the MGMT promoter region (an epigenetic alteration). Five different studies found that between 40% and 90% of colorectal cancers have reduced MGMT expression due to methylation of the MGMT promoter region.

Similarly, out of 119 cases of mismatch repair-deficient colorectal cancers that lacked DNA repair gene PMS2 expression, PMS2 was deficient in 6 due to mutations in the PMS2 gene, while in 103 cases PMS2 expression was deficient because its pairing partner MLH1 was repressed due to promoter methylation (PMS2 protein is unstable in the absence of MLH1). In the other 10 cases, loss of PMS2 expression was likely due to epigenetic overexpression of the microRNA, miR-155, which down-regulates MLH1.

In a further example, epigenetic defects were found in various cancers (e.g. breast, ovarian, colorectal and head and neck). Two or three deficiencies in the expression of ERCC1, XPF or PMS2 occur simultaneously in the majority of 49 colon cancers evaluated by Facista et al.

The chart in this section shows some frequent DNA damaging agents, examples of DNA lesions they cause, and the pathways that deal with these DNA damages. At least 169 enzymes are either directly employed in DNA repair or influence DNA repair processes. Of these, 83 are directly employed in repairing the 5 types of DNA damages illustrated in the chart.

Some of the more well studied genes central to these repair processes are shown in the chart. The gene designations shown in red, gray or cyan indicate genes frequently epigenetically altered in various types of cancers. Wikipedia articles on each of the genes highlighted by red, gray or cyan describe the epigenetic alteration(s) and the cancer(s) in which these epimutations are found. Review articles, and broad experimental survey articles also document most of these epigenetic DNA repair deficiencies in cancers.

Red-highlighted genes are frequently reduced or silenced by epigenetic mechanisms in various cancers. When these genes have low or absent expression, DNA damages can accumulate. Replication errors past these damages (see translesion synthesis) can lead to increased mutations and, ultimately, cancer. Epigenetic repression of DNA repair genes in accurate DNA repair pathways appear to be central to carcinogenesis.

The two gray-highlighted genes RAD51 and BRCA2, are required for homologous recombinational repair. They are sometimes epigenetically over-expressed and sometimes under-expressed in certain cancers. As indicated in the Wikipedia articles on RAD51 and BRCA2, such cancers ordinarily have epigenetic deficiencies in other DNA repair genes. These repair deficiencies would likely cause increased unrepaired DNA damages. The over-expression of RAD51 and BRCA2 seen in these cancers may reflect selective pressures for compensatory RAD51 or BRCA2 over-expression and increased homologous recombinational repair to at least partially deal with such excess DNA damages. In those cases where RAD51 or BRCA2 are under-expressed, this would itself lead to increased unrepaired DNA damages. Replication errors past these damages (see translesion synthesis) could cause increased mutations and cancer, so that under-expression of RAD51 or BRCA2 would be carcinogenic in itself.

Cyan-highlighted genes are in the microhomology-mediated end joining (MMEJ) pathway and are up-regulated in cancer. MMEJ is an additional error-prone inaccurate repair pathway for double-strand breaks. In MMEJ repair of a double-strand break, an homology of 5–25 complementary base pairs between both paired strands is sufficient to align the strands, but mismatched ends (flaps) are usually present. MMEJ removes the extra nucleotides (flaps) where strands are joined, and then ligates the strands to create an intact DNA double helix. MMEJ almost always involves at least a small deletion, so that it is a mutagenic pathway. FEN1, the flap endonuclease in MMEJ, is epigenetically increased by promoter hypomethylation and is over-expressed in the majority of cancers of the breast, prostate, stomach, neuroblastomas, pancreas, and lung. PARP1 is also over-expressed when its promoter region ETS site is epigenetically hypomethylated, and this contributes to progression to endometrial cancer and BRCA-mutated serous ovarian cancer. Other genes in the MMEJ pathway are also over-expressed in a number of cancers (see MMEJ for summary), and are also shown in cyan.

Genome-wide distribution of DNA repair in human somatic cells

Differential activity of DNA repair pathways across various regions of the human genome causes mutations to be very unevenly distributed within tumor genomes. In particular, the gene-rich, early-replicating regions of the human genome exhibit lower mutation frequencies than the gene-poor, late-replicating heterochromatin. One mechanism underlying this involves the histone modification H3K36me3, which can recruit mismatch repair proteins, thereby lowering mutation rates in H3K36me3-marked regions. Another important mechanism concerns nucleotide excision repair, which can be recruited by the transcription machinery, lowering somatic mutation rates in active genes and other open chromatin regions.

Evolution

The basic processes of DNA repair are highly conserved among both prokaryotes and eukaryotes and even among bacteriophages (viruses which infect bacteria); however, more complex organisms with more complex genomes have correspondingly more complex repair mechanisms. The ability of a large number of protein structural motifs to catalyze relevant chemical reactions has played a significant role in the elaboration of repair mechanisms during evolution. For an extremely detailed review of hypotheses relating to the evolution of DNA repair, see.

The fossil record indicates that single-cell life began to proliferate on the planet at some point during the Precambrian period, although exactly when recognizably modern life first emerged is unclear. Nucleic acids became the sole and universal means of encoding genetic information, requiring DNA repair mechanisms that in their basic form have been inherited by all extant life forms from their common ancestor. The emergence of Earth's oxygen-rich atmosphere (known as the "oxygen catastrophe") due to photosynthetic organisms, as well as the presence of potentially damaging free radicals in the cell due to oxidative phosphorylation, necessitated the evolution of DNA repair mechanisms that act specifically to counter the types of damage induced by oxidative stress.

Rate of evolutionary change

On some occasions, DNA damage is not repaired or is repaired by an error-prone mechanism that results in a change from the original sequence. When this occurs, mutations may propagate into the genomes of the cell's progeny. Should such an event occur in a germ line cell that will eventually produce a gamete, the mutation has the potential to be passed on to the organism's offspring. The rate of evolution in a particular species (or, in a particular gene) is a function of the rate of mutation. As a consequence, the rate and accuracy of DNA repair mechanisms have an influence over the process of evolutionary change. DNA damage protection and repair does not influence the rate of adaptation by gene regulation and by recombination and selection of alleles. On the other hand, DNA damage repair and protection does influence the rate of accumulation of irreparable, advantageous, code expanding, inheritable mutations, and slows down the evolutionary mechanism for expansion of the genome of organisms with new functionalities. The tension between evolvability and mutation repair and protection needs further investigation.

Technology

A technology named clustered regularly interspaced short palindromic repeat (shortened to CRISPR-Cas9) was discovered in 2012. The new technology allows anyone with molecular biology training to alter the genes of any species with precision, by inducing DNA damage at a specific point and then altering DNA repair mechanisms to insert new genes. It is cheaper, more efficient, and more precise than other technologies. With the help of CRISPR–Cas9, parts of a genome can be edited by scientists by removing, adding, or altering parts in a DNA sequence.