https://en.wikipedia.org/wiki/FermentationPhylogenetic
tree of bacteria and archaea, highlighting those that carry out
fermentation. Their end products are also highlighted. Figure modified
from Hackmann (2024).
Because oxygen is not required, it is an alternative to aerobic respiration. Over 25% of bacteria and archaea carry out fermentation. They live in the gut, sediments, food, and other environments. Eukaryotes, including humans and other animals, also carry out fermentation.
Fermentation is important in several areas of human society. Humans have used fermentation in production of food for 13,000 years. Humans and their livestock have microbes in the gut that carry out fermentation, releasing products used by the host for energy. Fermentation is used at an industrial level to produce commodity chemicals, such as ethanol and lactate. In total, fermentation forms more than 50 metabolic end products. This process highlights the power of microbial activity.
Definition
The definition of fermentation has evolved over the years. The most modern definition is catabolism, where organic compounds are both the electron donor and acceptor. A common electron donor is glucose, and pyruvate is a common electron acceptor. This definition distinguishes fermentation from aerobic respiration, where oxygen is the acceptor and types of anaerobic respiration, where an inorganic species is the acceptor.
Fermentation had been defined differently in the past. In 1876, Louis Pasteur described it as "la vie sans air" (life without air).
This definition came before the discovery of anaerobic respiration.
Later, it had been defined as catabolism that forms ATP through only substrate-level phosphorylation. However, several pathways of fermentation have been discovered to form ATP through an electron transport chain and ATP synthase, also.
Some sources define fermentation loosely as any large-scale biological manufacturing process. See Industrial fermentation. This definition focuses on the process of manufacturing rather than metabolic details.
Biological role and prevalence
Fermentation is used by organisms to generate ATP energy for metabolism.
One advantage is that it requires no oxygen or other external electron
acceptors, and thus it can be carried out when those electron acceptors
are absent. A disadvantage is that it produces relatively little ATP,
yielding only between 2 and 4.5 per glucose compared to 32 for aerobic respiration.
Over 25% of bacteria and archaea carry out fermentation. This type of metabolism is most common in the phylum Bacillota, and it is least common in Actinomycetota. Their most common habitat is host-associated ones, such as the gut.
Animals, including humans, also carry out fermentation. The product of fermentation in humans is lactate, and it is formed during anaerobic exercise or in cancerous cells. No animal is known to survive on fermentation alone, even as one parasitic animal (Henneguya zschokkei) is known to survive without oxygen.
Substrates and products of fermentation
The most common substrates and products of fermentation. Figure modified from Hackmann (2024).
Fermentation uses a range of substrates and forms a variety of
metabolic end products. Of the 55 end products formed, the most common
are acetate and lactate. Of the 46 chemically-defined substrates that have been reported, the most common are glucose and other sugars.
Biochemical overview
Overview of the biochemical pathways for fermentation of glucose. Figure modified from Hackmann (2024).
When an organic compound is fermented, it is broken down to a simpler
molecule and releases electrons. The electrons are transferred to a
redox cofactor, which in turn transfers them to an organic compound. ATP is generated in the process, and it can be formed by substrate-level phosphorylation or by ATP synthase.
When glucose is fermented, it enters glycolysis or the pentose phosphate pathway and is converted to pyruvate.
From pyruvate, pathways branch out to form a number of end products
(e.g. lactate). At several points, electrons are released and accepted
by redox cofactors (NAD and ferredoxin).
At later points, these cofactors donate electrons to their final
acceptor and become oxidized. ATP is also formed at several points in
the pathway.
The biochemical pathways of fermentation of glucose in poster format. Figure modified from Hackmann (2024).
While fermentation is simple in overview, its details are more
complex. Across organisms, fermentation of glucose involves over 120
different biochemical reactions.
Further, multiple pathways can be responsible for forming the same
product. For forming acetate from its immediate precursor (pyruvate or acetyl-CoA), six separate pathways have been found.
In ethanol fermentation, one glucose molecule is converted into two ethanol molecules and two carbon dioxide (CO2) molecules.It is used to make bread dough rise: the carbon dioxide forms bubbles, expanding the dough into a foam. The ethanol is the intoxicating agent in alcoholic beverages such as wine, beer and liquor. Fermentation of feedstocks, including sugarcane, maize, and sugar beets, produces ethanol that is added to gasoline. In some species of fish, including goldfish and carp, it provides energy when oxygen is scarce (along with lactic acid fermentation).
Before fermentation, a glucose molecule breaks down into two pyruvate molecules (glycolysis). The energy from this exothermic reaction is used to bind inorganic phosphates to ADP, which converts it to ATP, and convert NAD+ to NADH. The pyruvates break down into two acetaldehyde
molecules and give off two carbon dioxide molecules as waste products.
The acetaldehyde is reduced into ethanol using the energy and hydrogen
from NADH, and the NADH is oxidized into NAD+ so that the cycle may repeat. The reaction is catalyzed by the enzymes pyruvate decarboxylase and alcohol dehydrogenase.
History of bioethanol fermentation
The history of ethanol as a fuel spans several centuries and is marked by a series of significant milestones. Samuel Morey, an American inventor, was the first to produce ethanol by fermenting corn in 1826. However, it was not until the California Gold Rush in the 1850s that ethanol was first used as a fuel in the United States. Rudolf Diesel
demonstrated his engine, which could run on vegetable oils and ethanol,
in 1895, but the widespread use of petroleum-based diesel engines made
ethanol less popular as a fuel. In the 1970s, the oil crisis reignited
interest in ethanol, and Brazil became a leader in ethanol production
and use. The United States began producing ethanol on a large scale in
the 1980s and 1990s as a fuel additive to gasoline, due to government
regulations. Today, ethanol continues to be explored as a sustainable
and renewable fuel source, with researchers developing new technologies
and biomass sources for its production.
1826: Samuel Morey,
an American inventor, was the first to produce ethanol by fermenting
corn. However, ethanol was not widely used as a fuel until many years
later. (1)
1850s: Ethanol was first used as a fuel in the United States during the California gold rush. Miners used ethanol as a fuel for lamps and stoves because it was cheaper than whale oil. (2)
1895: German engineer Rudolf Diesel
demonstrated his engine, which was designed to run on vegetable oils,
including ethanol. However, the widespread use of diesel engines fueled
by petroleum made ethanol less popular as a fuel. (3)
1970s: The oil crisis of the 1970s led to renewed interest in
ethanol as a fuel. Brazil became a leader in ethanol production and use,
due in part to government policies that encouraged the use of biofuels.
(4)
1980s–1990s: The United States began to produce ethanol on a large
scale as a fuel additive to gasoline. This was due to the passage of the
Clean Air Act in 1990, which required the use of oxygenates, such as ethanol, to reduce emissions. (5)
2000s–present: There has been continued interest in ethanol as a
renewable and sustainable fuel. Researchers are exploring new sources of
biomass for ethanol production, such as switchgrass and algae, and developing new technologies to improve the efficiency of the fermentation process. (6)
Homolactic fermentation (producing only lactic acid) is the simplest type of fermentation. Pyruvate from glycolysis undergoes a simple redox reaction, forming lactic acid. Overall, one molecule of glucose (or any six-carbon sugar) is converted to two molecules of lactic acid:
C6H12O6 → 2 CH3CHOHCOOH
It occurs in the muscles of animals when they need energy faster than the blood can supply oxygen. It also occurs in some kinds of bacteria (such as lactobacilli) and some fungi. It is the type of bacteria that convert lactose into lactic acid in yogurt, giving it its sour taste. These lactic acid bacteria can carry out either homolactic fermentation, where the end-product is mostly lactic acid, or heterolactic fermentation, where some lactate is further metabolized to ethanol and carbon dioxide (via the phosphoketolase pathway), acetate, or other metabolic products, e.g.:
C6H12O6 → CH3CHOHCOOH + C2H5OH + CO2
If lactose is fermented (as in yogurts and cheeses), it is first
converted into glucose and galactose (both six-carbon sugars with the
same atomic formula):
C12H22O11 + H2O → 2 C6H12O6
Heterolactic fermentation is in a sense intermediate between lactic acid fermentation and other types, e.g. alcoholic fermentation. Reasons to go further and convert lactic acid into something else include:
The acidity of lactic acid impedes biological processes. This
can be beneficial to the fermenting organism as it drives out
competitors that are unadapted to the acidity. As a result, the food
will have a longer shelf life (one reason foods are purposely fermented
in the first place); however, beyond a certain point, the acidity starts
affecting the organism that produces it.
The high concentration of lactic acid (the final product of fermentation) drives the equilibrium backwards (Le Chatelier's principle), decreasing the rate at which fermentation can occur and slowing down growth.
Ethanol, into which lactic acid can be easily converted, is volatile
and will readily escape, allowing the reaction to proceed easily. CO2 is also produced, but it is only weakly acidic and even more volatile than ethanol.
Acetic acid (another conversion product) is acidic and not as
volatile as ethanol; however, in the presence of limited oxygen, its
creation from lactic acid releases additional energy. It is a lighter
molecule than lactic acid, forming fewer hydrogen bonds with its
surroundings (due to having fewer groups that can form such bonds), thus
is more volatile and will also allow the reaction to proceed more
quickly.
If propionic acid, butyric acid,
and longer monocarboxylic acids are produced, the amount of acidity
produced per glucose consumed will decrease, as with ethanol, allowing
faster growth.
Hydrogen gas is produced in many types of fermentation as a way to regenerate NAD+ from NADH. Electrons are transferred to ferredoxin, which in turn is oxidized by hydrogenase, producing H2. Hydrogen gas is a substrate for methanogens and sulfate reducers, which keep the concentration of hydrogen low and favor the production of such an energy-rich compound, but hydrogen gas at a fairly high concentration can nevertheless be formed, as in flatus.
In
food and industrial contexts, any chemical modification performed by a
living being in a controlled container can be termed "fermentation". The
following do not fall into the biochemical sense, but are called
fermentation in the larger sense:
Fermentation can be used to make alternative protein sources. It is
commonly used to modify existing protein foods, including plant-based
ones such as soy, into more flavorful forms such as tempeh and fermented tofu.
Recombinant whey protein for dairy replacement (Perfect Day)
Recombinant casein protein for dairy replacements (Those Vegan Cowboys)
Recombinant egg white (EVERY)
Heme proteins such as myoglobin and hemoglobin
give meat its characteristic texture, flavor, color, and aroma. The
myoglobin and leghemoglobin ingredients can be used to replicate this
property, despite them coming from a vat instead of meat.
Enzymes
Industrial fermentation
can be used for enzyme production, where proteins with catalytic
activity are produced and secreted by microorganisms. The development of
fermentation processes, microbial strain engineering and recombinant
gene technologies has enabled the commercialization of a wide range of
enzymes. Enzymes
are used in all kinds of industrial segments, such as food (lactose
removal, cheese flavor), beverage (juice treatment), baking (bread
softness, dough conditioning), animal feed, detergents (protein, starch
and lipid stain removal), textile, personal care and pulp and paper
industries.
Modes of industrial operation
Most industrial fermentation
uses batch or fed-batch procedures, although continuous fermentation
can be more economical if various challenges, particularly the
difficulty of maintaining sterility, can be met.
Batch
In
a batch process, all the ingredients are combined and the reactions
proceed without any further input. Batch fermentation has been used for
millennia to make bread and alcoholic beverages, and it is still a
common method, especially when the process is not well understood. However, it can be expensive because the fermentor must be sterilized using high pressure steam between batches. Strictly speaking, there is often addition of small quantities of chemicals to control the pH or suppress foaming.
Batch fermentation goes through a series of phases. There is a
lag phase in which cells adjust to their environment; then a phase in
which exponential growth occurs. Once many of the nutrients have been
consumed, the growth slows and becomes non-exponential, but production
of secondary metabolites (including commercially important
antibiotics and enzymes) accelerates. This continues through a
stationary phase after most of the nutrients have been consumed, and
then the cells die.
Fed-batch fermentation is a variation of batch fermentation where
some of the ingredients are added during the fermentation. This allows
greater control over the stages of the process. In particular,
production of secondary metabolites can be increased by adding a limited
quantity of nutrients during the non-exponential growth phase.
Fed-batch operations are often sandwiched between batch operations.
Open
The
high cost of sterilizing the fermentor between batches can be avoided
using various open fermentation approaches that are able to resist
contamination. One is to use a naturally evolved mixed culture. This is
particularly favored in wastewater treatment, since mixed populations
can adapt to a wide variety of wastes. Thermophilic
bacteria can produce lactic acid at temperatures of around 50 °Celsius,
sufficient to discourage microbial contamination; and ethanol has been
produced at a temperature of 70 °C. This is just below its boiling point
(78 °C), making it easy to extract. Halophilic
bacteria can produce bioplastics in hypersaline conditions. Solid-state
fermentation adds a small amount of water to a solid substrate; it is
widely used in the food industry to produce flavors, enzymes and organic
acids.
Continuous
In continuous fermentation, substrates are added and final products removed continuously. There are three varieties: chemostats, which hold nutrient levels constant; turbidostats, which keep cell mass constant; and plug flow reactors in which the culture medium flows steadily through a tube while the cells are recycled from the outlet to the inlet.
If the process works well, there is a steady flow of feed and effluent
and the costs of repeatedly setting up a batch are avoided. Also, it can
prolong the exponential growth phase and avoid byproducts that inhibit
the reactions by continuously removing them. However, it is difficult to
maintain a steady state and avoid contamination, and the design tends
to be complex. Typically the fermentor must run for over 500 hours to be more economical than batch processors.
The use of fermentation, particularly for beverages, has existed since the Neolithic and has been documented dating from 7000 to 6600 BCE in Jiahu, China, 5000 BCE in India, Ayurveda mentions many Medicated Wines, 6000 BCE in Georgia, 3150 BCE in ancient Egypt, 3000 BCE in Babylon, 2000 BCE in pre-Hispanic Mexico, and 1500 BC in Sudan. Fermented foods have a religious significance in Judaism and Christianity. The Baltic god Rugutis was worshiped as the agent of fermentation.
In alchemy, fermentation ("putrefaction") was symbolized by Capricorn ♑︎.
Louis Pasteur in his laboratory
In 1837, Charles Cagniard de la Tour, Theodor Schwann and Friedrich Traugott Kützing
independently published papers concluding, as a result of microscopic
investigations, that yeast is a living organism that reproduces by budding.
Schwann boiled grape juice to kill the yeast and found that no
fermentation would occur until new yeast was added. However, a lot of
chemists, including Antoine Lavoisier,
continued to view fermentation as a simple chemical reaction and
rejected the notion that living organisms could be involved. This was
seen as a reversion to vitalism and was lampooned in an anonymous publication by Justus von Liebig and Friedrich Wöhler.
The turning point came when Louis Pasteur
(1822–1895), during the 1850s and 1860s, repeated Schwann's experiments
and showed fermentation is initiated by living organisms in a series of
investigations. In 1857, Pasteur showed lactic acid fermentation is caused by living organisms. In 1860, he demonstrated how bacteria cause souring
in milk, a process formerly thought to be merely a chemical change. His
work in identifying the role of microorganisms in food spoilage led to
the process of pasteurization.
In 1877, working to improve the French brewing industry, Pasteur published his famous paper on fermentation, "Etudes sur la Bière", which was translated into English in 1879 as "Studies on fermentation". He defined fermentation (incorrectly) as "Life without air", yet he correctly showed how specific types of microorganisms cause specific types of fermentations and specific end-products.
Although showing fermentation resulted from the action of living
microorganisms was a breakthrough, it did not explain the basic nature
of fermentation; nor did it prove it is caused by microorganisms which
appear to be always present. Many scientists, including Pasteur, had
unsuccessfully attempted to extract the fermentation enzyme from yeast.
Success came in 1897 when the German chemist Eduard Buechner
ground up yeast, extracted a juice from them, then found to his
amazement this "dead" liquid would ferment a sugar solution, forming
carbon dioxide and alcohol much like living yeasts.
Buechner's results are considered to mark the birth of
biochemistry. The "unorganized ferments" behaved just like the organized
ones. From that time on, the term enzyme came to be applied to all
ferments. It was then understood fermentation is caused by enzymes
produced by microorganisms. In 1907, Buechner won the Nobel Prize in chemistry for his work.
Advances in microbiology and fermentation technology have
continued steadily up until the present. For example, in the 1930s, it
was discovered microorganisms could be mutated
with physical and chemical treatments to be higher-yielding,
faster-growing, tolerant of less oxygen, and able to use a more
concentrated medium. Strain selection and hybridization developed as well, affecting most modern food fermentations.
Post 1930s
The
field of fermentation has been critical to producing a wide range of
consumer goods, from food and drink to industrial chemicals and
pharmaceuticals. Since its early beginnings in ancient civilizations,
fermentation has continued to evolve and expand, with new techniques and
technologies driving advances in product quality, yield, and
efficiency. The period from the 1930s onward saw a number of significant
advancements in fermentation technology, including the development of
new processes for producing high-value products like antibiotics and enzymes,
the increasing importance of fermentation in the production of bulk
chemicals, and a growing interest in the use of fermentation for the
production of functional foods and nutraceuticals.
The 1950s and 1960s saw the development of new fermentation
technologies, such as immobilized cells and enzymes, which allowed for
more precise control over fermentation processes and increased the
production of high-value products like antibiotics and enzymes. In the
1970s and 1980s, fermentation became increasingly important in producing
bulk chemicals like ethanol, lactic acid, and citric acid.
This led to developing new fermentation techniques and genetically
engineered microorganisms to improve yields and reduce production costs.
In the 1990s and 2000s, there was a growing interest in fermentation to
produce functional foods and nutraceuticals, which have potential
health benefits beyond basic nutrition. This led to new fermentation
processes, probiotics, and other functional ingredients.
Overall, the period from 1930 onward saw significant advancements
in the use of fermentation for industrial purposes, leading to the
production of a wide range of fermented products that are now consumed
worldwide.
The frontal lobe is the largest of the four major lobes of the brain in mammals, and is located at the front of each cerebral hemisphere (in front of the parietal lobe and the temporal lobe). It is parted from the parietal lobe by a groove between tissues called the central sulcus and from the temporal lobe by a deeper groove called the lateral sulcus
(Sylvian fissure). The most anterior rounded part of the frontal lobe
(though not well-defined) is known as the frontal pole, one of the three
poles of the cerebrum.
The frontal lobe is the largest lobe of the brain and makes up about a third of the surface area of each hemisphere. On the lateral surface of each hemisphere, the central sulcus separates the frontal lobe from the parietal lobe. The lateral sulcus separates the frontal lobe from the temporal lobe.
The frontal lobe can be divided into a lateral, polar, orbital (above the orbit; also called basal or ventral), and medial part. Each of these parts consists of a particular gyrus:
The gyri are separated by sulci. E.g., the precentral gyrus is in front of the central sulcus, and behind the precentral sulcus. The superior and middle frontal gyri are divided by the superior frontal sulcus. The middle and inferior frontal gyri are divided by the inferior frontal sulcus.
In humans the frontal lobe reaches full maturity only after the
20s—the prefrontal cortex, in particular, continues in maturing 'til the
second and third decades of life—which, thereafter, marks the cognitive maturity associated with adulthood. A small amount of atrophy,
however, is normal in the aging person's frontal lobe. Fjell, in 2009,
studied atrophy of the brain in people aged 60–91 years. The 142 healthy
participants were scanned using MRI. Their results were compared to those of 122 participants with Alzheimer's disease. A follow-up
one year later showed there to have been a marked volumetric decline in
those with Alzheimer's and a much smaller decline (averaging 0.5%) in
the healthy group.
These findings corroborate those of Coffey, who in 1992 indicated that
the frontal lobe decreases in volume approximately 0.5–1% per year.
Function
The entirety of the frontal cortex can be considered the "action cortex", much as the posterior cortex
is considered the "sensory cortex". It is devoted to action of one kind
or another: skeletal movement, ocular movement, speech control, and the
expression of emotions. In humans, the largest part of the frontal
cortex, the prefrontal cortex (PFC), is responsible for internal, purposeful mental action, commonly called reasoning or prefrontal synthesis.
The function of the PFC involves the ability to project future
consequences that result from current actions. PFC functions also
include override and suppression of socially unacceptable responses as
well as differentiation of tasks.
The PFC also plays an important part in integrating longer
non-task based memories stored across the brain. These are often
memories associated with emotions derived from input from the brain's limbic system. The frontal lobe modifies those emotions, generally to fit socially acceptable norms.
Damage to the frontal lobe can occur in a number of ways and result in many different consequences. Transient ischemic attacks (TIAs) also known as mini-strokes, and strokes
are common causes of frontal lobe damage in older adults (65 and over).
These strokes and mini-strokes can occur due to the blockage of blood flow to the brain or as a result of the rupturing of an aneurysm in a cerebral artery. Other ways in which injury can occur include traumatic brain injuries incurred following accidents, diagnoses such as Alzheimer's disease or Parkinson's disease (which cause dementia symptoms), and frontal lobe epilepsy (which can occur at any age). Very often, frontal lobe damage is recognized in those with prenatal alcohol exposure.
Symptoms
Common
effects of damage to the frontal lobe are varied. Patients who have
experienced frontal lobe trauma may know the appropriate response to a
situation but display inappropriate responses to those same situations
in real life .
Similarly, emotions that are felt may not be expressed in the face or
voice. For example, someone who is feeling happy would not smile, and
the voice would be devoid of emotion. Along the same lines, though, the
person may also exhibit excessive, unwarranted displays of emotion.
Depression is common in stroke patients. Also common is a loss of or
decrease in motivation. Someone might not want to carry out normal daily
activities and would not feel "up to it". Those who are close to the person who has experienced the damage may notice changes in behavior. The case of Phineas Gage was long considered exemplary of these symptoms, though more recent research
has suggested that accounts of his personality change have been poorly
evidenced. The frontal lobe is the same part of the brain that is
responsible for executive functions such as planning for the future, judgment, decision-making skills, attention span, and inhibition. These functions can decrease in someone whose frontal lobe is damaged.
Consequences that are seen less frequently are also varied. Confabulation
may be the most frequently indicated "less common" effect. In the case
of confabulation, someone gives false information while maintaining the
belief that it is the truth. In a small number of patients,
uncharacteristic cheerfulness can be noted. This effect is seen mostly
in patients with lesions to the right frontal portion of the brain.
Another infrequent effect is that of reduplicative paramnesia,
in which patients believe that the location in which they currently
reside is a replica of one located somewhere else. Similarly, those who
experience Capgras syndrome
after frontal lobe damage believe that an identical "replacement" has
taken the identity of a close friend, relative, or other person and is
posing as that person. This last effect is seen mostly in schizophrenic
patients who also have a neurological disorder in the frontal lobe.
DNA damage
In the human frontal cortex, a set of genes undergo reduced expression after age 40 and especially after age 70. This set includes genes that have key functions in synaptic plasticity important in learning and memory, vesicular transport and mitochondrial function. During aging, DNA damage is markedly increased in the promoters
of the genes displaying reduced expression in the frontal cortex. In
cultured human neurons, these promoters are selectively damaged by
oxidative stress.
Individuals with HIV associated neurocognitive disorders accumulate nuclear and mitochondrial DNA damage in the frontal cortex.
Genetic
A report from the National Institute of Mental Health says a gene variant of (COMT) that reduces dopamine activity in the prefrontal cortex
is related to poorer performance and inefficient functioning of that
brain region during working memory, tasks, and to a slightly increased
risk for schizophrenia.
History
Psychosurgery
In the early 20th century, a medical treatment for mental illness, first developed by PortugueseneurologistEgas Moniz, involved damaging the pathways connecting the frontal lobe to the limbic system. A frontal lobotomy
(sometimes called frontal leucotomy) successfully reduced distress but
at the cost of often blunting the subject's emotions, volition and personality. The indiscriminate use of this psychosurgical procedure, combined with its severe side effects and a mortality rate of 7.4 to 17 per cent,
earned it a bad reputation. The frontal lobotomy has largely died out
as a psychiatric treatment. More precise psychosurgical procedures are
still used, although rarely. They may include anterior capsulotomy
(bilateral thermal lesions of the anterior limbs of the internal capsule) or the bilateral cingulotomy (involving lesions of the anterior cingulate gyri) and might be used to treat otherwise untreatable obsessional disorders or clinical depression.
Theories of function
Theories of frontal lobe function can be separated into four categories:
Single-process theories, which propose that "damage to a single process or system is responsible for a number of different dysexecutive symptoms"
Multi-process theories, which propose "that the frontal lobe
executive system consists of a number of components that typically work
together in everyday actions (heterogeneity of function)"
Construct-led theories, which propose that "most if not all frontal
functions can be explained by one construct (homogeneity of function)
such as working memory or inhibition"
Single-symptom theories, which propose that a specific dysexecutive
symptom (e.g., confabulation) is related to the processes and construct
of the underlying structures.
Other theories include:
Stuss (1999) suggests a differentiation into two categories according to homogeneity and heterogeneity of function.
It may be highlighted that the theories described above differ in their focus on certain processes/systems or construct-lets.
Stuss (1999) remarks that the question of homogeneity (single
construct) or heterogeneity (multiple processes/systems) of function
"may represent a problem of semantics and/or incomplete functional
analysis rather than an unresolvable dichotomy" (p. 348). However,
further research will show if a unified theory of frontal lobe function
that fully accounts for the diversity of functions will be available.
Other primates
Many
scientists had thought that the frontal lobe was disproportionately
enlarged in humans compared to other primates. This was thought to be an
important feature of human evolution and seen as the primary reason why
human cognition differs from that of other primates. However, this view
in relation to great apes has since been challenged by neuroimaging studies. Using magnetic resonance imaging to determine the volume of the frontal cortex in humans, all extant ape species, and several monkey species, it was found that the human frontal cortex was not relatively larger than the cortex of other great apes, but was relatively larger than the frontal cortex of lesser apes and the monkeys. The higher cognition of the humans is instead seen to relate to a greater connectedness given by neural tracts that do not affect the cortical volume. This is also evident in the pathways of the language network connecting the frontal and temporal lobes.
Eukaryotic DNA replication is a conserved mechanism that restricts DNA replication to once per cell cycle. Eukaryotic DNA replication of chromosomalDNA 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.
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.
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.
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.
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.
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.
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
Mcm2-7 double hexamer arranged in a head-to-head (NTD-to-NTD)
orientation. Each hexameric ring is slightly tilted, twisted and
off-centred relative to each other. Top panel, side views. Bottom panel, CTD view.
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.
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.
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
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.
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.
Eukaryotic replisome complex and associated proteins. A loop occurs in the lagging strand
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 purinenucleobases, 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.
Depiction of DNA replication at replication fork. a: template strands, b: leading strand, c: lagging strand, d: replication fork, e: RNA primer, f: Okazaki fragment
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...
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
A depiction of telomerase progressively elongating telomeric DNA.
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.
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 RFCRad17 are able to recognize gapped or nicked DNA. The RFCRad17 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.
Depiction
of replication through histones. Histones are removed from DNA by the
FACT complex and Asf1. Histones are reassembled onto newly replicated
DNA after the replication fork by CAF-1 and Rtt106.
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.
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