DNA repair is a collection of processes by which acell identifies and corrects damage to theDNA molecules that encode itsgenome.[1] In human cells, both normalmetabolic activities and environmental factors such asradiation can cause DNA damage, resulting in tens of thousands of individualmolecular lesions per cell per day.[2] Many of these lesions cause structural damage to the DNA molecule and can alter or eliminate the cell's ability totranscribe thegene that the affected DNA encodes. Other lesions induce potentially harmfulmutations in the cell's genome, which affect the survival of its daughter cells after it undergoesmitosis. 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 cellularapoptosis does not occur, irreparable DNA damage may occur. This can eventually lead to malignant tumors, orcancer as per thetwo-hit hypothesis.
The rate of DNA repair depends on various 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 can no longer effectively repair its DNA may enter one of three possible states:
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 influencelife span have turned out to be involved in DNA damage repair and protection.[3]
The 2015Nobel Prize in Chemistry was awarded toTomas Lindahl,Paul Modrich, andAziz Sancar for their work on the molecular mechanisms of DNA repair processes.[4][5]
DNA damage, due to environmental factors and normalmetabolic processes inside the cell, occurs at a rate of 10,000 to 1,000,000 molecular lesions per cell per day.[2] While this constitutes at most only 0.0003125% of the human genome's approximately 3.2 billion bases, unrepaired lesions in critical genes (such astumor suppressor genes) can impede a cell's ability to carry out its function and appreciably increase the likelihood oftumor formation and contribute totumor heterogeneity.
The vast majority of DNA damage affects theprimary 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 bulkyadducts that do not fit in the standard double helix. Unlikeproteins andRNA, DNA usually lackstertiary structure and therefore damage or disturbance does not occur at that level. DNA is, however,supercoiled and wound around "packaging" proteins calledhistones (in eukaryotes), and both superstructures are vulnerable to the effects of DNA damage.
DNA damage can be subdivided into two main types:
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 aback mutation, for example, throughgene conversion).
There are several types of damage to DNA due to endogenous cellular processes:
Damage caused by exogenous agents comes in many forms. Some examples are:
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, sugarring 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.[13]
Ineukaryotic cells, DNA is found in two cellular locations – inside thenucleus and inside themitochondria.Nuclear DNA (nDNA) exists aschromatin during non-replicative stages of thecell cycle and is condensed into aggregate structures known aschromosomes duringcell division. In either state the DNA is highly compacted and wound up around bead-like proteins calledhistones. Whenever a cell needs to express the genetic information encoded in its nDNA the required chromosomal region is unraveled, genes located therein are expressed, and then the region is condensed back to its resting conformation.Mitochondrial DNA (mtDNA) is located inside mitochondriaorganelles, 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), orfree radicals, byproducts of the constant production ofadenosine triphosphate (ATP) viaoxidative phosphorylation, create a highly oxidative environment that is known to damage mtDNA. A critical enzyme in counteracting thetoxicity of these species issuperoxide dismutase, which is present in both the mitochondria andcytoplasm of eukaryotic cells.
Senescence, an irreversible process in which the cell no longerdivides, is a protective response to the shortening of the chromosome ends, calledtelomeres. The telomeres are long regions of repetitivenoncoding DNA that cap chromosomes and undergo partial degradation each time a cell undergoes division (seeHayflick limit).[14] In contrast,quiescence is a reversible state of cellular dormancy that is unrelated to genome damage (seecell 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,[15] which serves as a "last resort" mechanism to prevent a cell with damaged DNA from replicating inappropriately in the absence of pro-growthcellular signaling. Unregulated cell division can lead to the formation of a tumor (seecancer), 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.[16]
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.[17]
Cells cannot function if DNA damage corrupts the integrity and accessibility of essential information in thegenome (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 sisterchromatid as a template to recover the original information. Without access to a template, cells use an error-prone recovery mechanism known astranslesion 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.
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 ofpyrimidine dimers upon irradiation with UV light results in an abnormal covalent bond between adjacent pyrimidine bases. Thephotoreactivation process directly reverses this damage by the action of the enzymephotolyase, whose activation is obligately dependent on energy absorbed fromblue/UV light (300–500 nmwavelength) to promote catalysis.[18] Photolyase, an old enzyme present inbacteria,fungi, and mostanimals no longer functions in humans,[19] who instead usenucleotide excision repair to repair damage from UV irradiation. Another type of damage, methylation of guanine bases, is directly reversed by the enzyme methyl guanine methyl transferase (MGMT), the bacterial equivalent of which is calledogt. This is an expensive process because each MGMT molecule can be used only once; that is, the reaction isstoichiometric rather thancatalytic.[20] A generalized response to methylating agents in bacteria is known as theadaptive response and confers a level of resistance to alkylating agents upon sustained exposure by upregulation of alkylation repair enzymes.[21] The third type of DNA damage reversed by cells is certain methylation of the bases cytosine and adenine.
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 ofexcision repair mechanisms that remove the damaged nucleotide and replace it with an undamaged nucleotide complementary to that found in the undamaged DNA strand.[20]
Double-strand breaks, in which both strands in the double helix are severed, are particularly hazardous to the cell because they can lead togenome 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.[26][27] Three mechanisms exist to repair double-strand breaks (DSBs):non-homologous end joining (NHEJ),microhomology-mediated end joining (MMEJ), andhomologous recombination (HR):[20][28]
In anin vitro system, MMEJ occurred in mammalian cells at the levels of 10–20% of HR when both HR and NHEJ mechanisms were also available.[36]
TheextremophileDeinococcus radiodurans has a remarkable ability to survive DNA damage fromionizing radiation and other sources. At least two copies of the genome, with random DNA breaks, can form DNA fragments throughannealing. Partially overlapping fragments are then used for synthesis ofhomologous regions through a movingD-loop that can continue extension until complementary partner strands are found. In the final step, there iscrossover by means ofRecA-dependenthomologous recombination.[39]
Topoisomerases introduce both single- and double-strand breaks in the course of changing the DNA's state ofsupercoiling, 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 is not yet known[40]
Translesion synthesis (TLS) is a DNA damage tolerance process that allows theDNA replication machinery to replicate past DNA lesions such asthymine dimers orAP sites.[41] It involves switching out regularDNA 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 replicationprocessivity factorPCNA. 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 byUV irradiation, whereasPol ι introduces mutations at these sites. Pol η is known to add the first adenine across theT^T photodimer usingWatson-Crick base pairing and the second adenine will be added in its syn conformation usingHoogsteen base pairing. From a cellular perspective, risking the introduction ofpoint 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 specializedpolymerases 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.[42] Paromita Raychaudhury and Ashis Basu[43] studied the toxicity and mutagenesis of the same lesion inEscherichia coli by replicating a G[8,5-Me]T-modified plasmid inE. 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 byProliferating cell nuclear antigen (PCNA). Under normal circumstances, PCNA bound to polymerases replicates the DNA. At a site oflesion, PCNA is ubiquitinated, or modified, by the RAD6/RAD18proteins to provide a platform for the specialized polymerases to bypass the lesion and resume DNA replication.[44][45] 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.
Cells exposed toionizing 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 otherbiomolecules such asproteins,carbohydrates,lipids, andRNA. The accumulation of damage, to be specific, double-strand breaks or adducts stalling thereplication forks, are among known stimulation signals for a global response to DNA damage.[46] The global response to damage is an act directed toward the cells' own preservation and triggers multiple pathways of macromolecular repair, lesion bypass, tolerance, orapoptosis. The common features of global response are induction of multiplegenes,cell cycle arrest, and inhibition ofcell division.
The packaging of eukaryotic DNA intochromatin 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 beremodeled. In eukaryotes,ATP dependentchromatin remodeling complexes andhistone-modifying enzymes are two predominant factors employed to accomplish this remodeling process.[47]
Chromatin relaxation occurs rapidly at the site of a DNA damage.[48][49] In one of the earliest steps, the stress-activated protein kinase,c-Jun N-terminal kinase (JNK), phosphorylatesSIRT6 on serine 10 in response to double-strand breaks or other DNA damage.[50] Thispost-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.[50]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.[51] PARP1 synthesizespolymericadenosine diphosphate ribose (poly (ADP-ribose) or PAR) chains on itself. Next the chromatin remodelerALC1 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.[49] About half of the maximum chromatin relaxation, presumably due to action of ALC1, occurs by 10 seconds.[49] This then allows recruitment of the DNA repair enzymeMRE11, to initiate DNA repair, within 13 seconds.[51]
γH2AX, the phosphorylated form ofH2AX is also involved in the early steps leading to chromatin decondensation after DNA double-strand breaks. Thehistone variant H2AX constitutes about 10% of the H2A histones in human chromatin.[52] 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.[52] The extent of chromatin with phosphorylated γH2AX is about two million base pairs at the site of a DNA double-strand break.[52] γH2AX does not, itself, cause chromatin decondensation, but within 30 seconds of irradiation,RNF8 protein can be detected in association with γH2AX.[53] RNF8 mediates extensive chromatin decondensation, through its subsequent interaction withCHD4,[54] a component of the nucleosome remodeling and deacetylase complexNuRD.
DDB2 occurs in a heterodimeric complex withDDB1. This complex further complexes with theubiquitin ligase proteinCUL4A[55] and withPARP1.[56] This larger complex rapidly associates with UV-induced damage within chromatin, with half-maximum association completed in 40 seconds.[55] The PARP1 protein, attached to both DDB1 and DDB2, thenPARylates (creates a poly-ADP ribose chain) on DDB2 that attracts the DNA remodeling proteinALC1.[56] Action of ALC1 relaxes the chromatin at the site of UV damage to DNA. This relaxation allows other proteins in thenucleotide excision repair pathway to enter the chromatin and repair UV-inducedcyclobutane pyrimidine dimer damages.
After rapidchromatin remodeling,cell cyclecheckpoints are activated to allow DNA repair to occur before the cell cycle progresses. First, twokinases,ATM andATR are activated within 5 or 6 minutes after DNA is damaged. This is followed by phosphorylation of the cell cycle checkpoint proteinChk1, initiating its function, about 10 minutes after DNA is damaged.[57]
After DNA damage,cell cyclecheckpoints 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 theG1/S andG2/M boundaries. An intra-S checkpoint also exists. Checkpoint activation is controlled by two masterkinases,ATM andATR. ATM responds to DNA double-strand breaks and disruptions in chromatin structure,[58] whereas ATR primarily responds to stalledreplication forks. These kinasesphosphorylate downstream targets in asignal transduction cascade, eventually leading to cell cycle arrest. A class of checkpoint mediator proteins includingBRCA1,MDC1, and53BP1 has also been identified.[59] These proteins seem to be required for transmitting the checkpoint activation signal to downstream proteins.
DNA damage checkpoint is asignal transduction pathway that blockscell cycle progression in G1, G2 andmetaphase and slows down the rate of S phase progression whenDNA 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)-likeprotein 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 thechromosomes 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 isp53, as it is required for inducingapoptosis following DNA damage.[60] Thecyclin-dependent kinase inhibitorp21 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 deactivatingcyclin/cyclin-dependent kinase complexes.[61]
TheSOS response is the changes ingene expression inEscherichia coli and other bacteria in response to extensive DNA damage. Theprokaryotic SOS system is regulated by two key proteins:LexA andRecA. The LexAhomodimer is atranscriptionalrepressor that binds tooperator sequences commonly referred to as SOS boxes. InEscherichia coli it is known that LexA regulates transcription of approximately 48 genes including the lexA and recA genes.[62] The SOS response is known to be widespread in the Bacteria domain, but it is mostly absent in some bacterial phyla, like theSpirochetes.[63]The most common cellular signals activating the SOS response are regions of single-stranded DNA (ssDNA), arising from stalledreplication forks or double-strand breaks, which are processed byDNA helicase to separate the two DNA strands.[46] In the initiation step, RecA protein binds to ssDNA in anATP 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.
InEscherichia coli, SOS boxes are 20-nucleotide long sequences near promoters withpalindromic 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.[63] 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.[64] 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 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 ofyeastSaccharomyces cerevisiae to DNA damaging agents results in overlapping but distinct transcriptional profiles. Similarities to environmentalshock 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 theheterogeneity ofmammalian cells. In an animal different types of cells are distributed among different organs that have evolved different sensitivities to DNA damage.[65]
In general global response to DNA damage involves expression of multiple genes responsible forpostreplication 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 DNApolymerases present during global response to DNA damage and are responsible for enhanced mutagenesis during a global response to DNA damage in eukaryotes.[46]
Experimental animals with genetic deficiencies in DNA repair often show decreased life span and increased cancer incidence.[17] For example, mice deficient in the dominant NHEJ pathway and in telomere maintenance mechanisms getlymphoma and infections more often, and, as a consequence, have shorter lifespans than wild-type mice.[66] 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.[67] 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.[68]
Themaximum life spans ofmice,naked mole-rats andhumans are respectively ~3, ~30 and ~129 years.[69] Of these, the shortest lived species, mouse, expresses DNA repair genes, including core genes in several DNA repair pathways, at a lower level than do humans and naked mole rats.[69] Furthermore several DNA repair pathways in humans and naked mole-rats are up-regulated compared to mouse. These observations suggest that elevated DNA repair facilitates greaterlongevity.[69]
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,[17] increased sensitivity to carcinogens and correspondingly increased cancer risk (seebelow). On the other hand, organisms with enhanced DNA repair systems, such asDeinococcus radiodurans, the most radiation-resistant known organism, exhibit remarkable resistance to the double-strand break-inducing effects ofradioactivity, likely due to enhanced efficiency of DNA repair and especially NHEJ.[70]
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 vianutrient sensing pathways and decreasedmetabolic rate. The molecular mechanisms by which such restriction results in lengthened lifespan are as yet unclear (see[71] 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 ofmTOR signaling, an evidence of reduction ofmetabolic activity, and concurrently to reduce constitutive level ofDNA damage induced by endogenously generated reactive oxygen species.[72]
For example, increasing thegene dosage of the gene SIR-2, which regulates DNA packaging in the nematode wormCaenorhabditis elegans, can significantly extend lifespan.[73] 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.[74] Caloric restriction has been closely linked to the rate of base excision repair in the nuclear DNA of rodents,[75] although similar effects have not been observed in mitochondrial DNA.[76]
TheC. 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.[77] This observation supports thepleiotropy theory of thebiological 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.
Defects in the NER mechanism are responsible for several genetic disorders, including:
Mental retardation often accompanies the latter two disorders, suggesting increased vulnerability of developmental neurons.
Other DNA repair disorders include:
All of the above diseases are often called "segmentalprogerias" ("accelerated aging diseases") because those affected appear elderly and experience 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 includeFanconi anemia, hereditarybreast cancer and hereditarycolon cancer.
Because of inherent limitations in the DNA repair mechanisms, if humans lived long enough, they would all eventually develop cancer.[78][79] There are at least 34Inherited 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 andBRCA2, two important genes whose mutations confer a hugely increased risk of breast cancer on carriers,[80] are both associated with a large number of DNA repair pathways, especially NHEJ and homologous recombination.
Cancer therapy procedures such aschemotherapy andradiotherapy 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'.[81]
Perhaps the most well-known of these 'synthetic lethality' drugs is the poly(ADP-ribose) polymerase 1 (PARP1) inhibitorolaparib, 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.[82]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.[83]
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.[84] Previous studies have shown an elevated DNA damage response in cell-culture models with oncogene activation[85] and preneoplastic colon adenomas.[86] 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).[87]
Replication stress, along with the selection for inactivating mutations in DNA damage response genes in the evolution of the tumor,[88] 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 ashort hairpin RNA (shRNA) to inhibit the double-strand break response kinase ataxia telangiectasia (ATM), leading to increased tumor size and invasiveness.[86] Humans born with inherited defects in DNA repair mechanisms (for example,Li-Fraumeni syndrome) have a higher cancer risk.[89]
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.[84] 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).[90] As well as mutations to DNA damage repair genes, mutations also arise in the genes responsible for arresting thecell 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.[91]
| HR[a] | NHEJ[b] | SSA[c] | FA[d] | BER[e] | NER[f] | MMR[g] | |
|---|---|---|---|---|---|---|---|
| ATM |  | | | ||||
| ATR | | | | ||||
| PAXIP | | | |||||
| RPA | | | | ||||
| BRCA1 | | | |||||
| BRCA2 | | | |||||
| RAD51 | | | |||||
| RFC | | | | ||||
| XRCC1 | | | |||||
| PCNA | | | | ||||
| PARP1 | | | |||||
| ERCC1 | | | | | |||
| MSH3 | | | | ||||
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 byepigenetic alterations.[92]
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 inDNA methylation (hypermethylation and hypomethylation) andhistone modification,[93] changes in chromosomal architecture (caused by inappropriate expression of proteins such asHMGA2 orHMGA1)[94] and changes caused bymicroRNAs. Each of these epigenetic alterations serves to regulate gene expression without altering the underlyingDNA sequence. These changes usually remain throughcell 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 thegenetic instability characteristic of cancers.[95][96][97]
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 inDNA mismatch repair[98][99] or inhomologous recombinational repair (HRR).[100] Chromosomal rearrangements and aneuploidy also increase in HRR defective cells.[101]
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.[102][103]
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 articleDNA 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).[104] However, suchgermline mutations (which cause highly penetrant cancer syndromes) are the cause of only about 1 percent of cancers.[105]
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 amissense mutation in the DNA repair geneMGMT, while the majority had reduced MGMT expression due to methylation of the MGMT promoter region (an epigenetic alteration).[106] Five different studies found that between 40% and 90% of colorectal cancers have reduced MGMT expression due to methylation of the MGMT promoter region.[107][108][109][110][111]
Similarly, out of 119 cases of mismatch repair-deficient colorectal cancers that lacked DNA repair genePMS2 expression, PMS2 was deficient in 6 due to mutations in the PMS2 gene, while in 103 cases PMS2 expression was deficient because its pairing partnerMLH1 was repressed due to promoter methylation (PMS2 protein is unstable in the absence of MLH1).[112] In the other 10 cases, loss of PMS2 expression was likely due to epigenetic overexpression of themicroRNA,miR-155, which down-regulates MLH1.[113]
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 ofERCC1,XPF or PMS2 occur simultaneously in the majority of 49 colon cancers evaluated by Facista et al.[114]
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.[115] Of these, 83 are directly employed in repairing the 5 types of DNA damages illustrated in the chart.[citation needed]
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,[116] and broad experimental survey articles[117][118] 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 (seetranslesion synthesis) can lead to increased mutations and, ultimately, cancer. Epigenetic repression of DNA repair genes inaccurate DNA repair pathways appear to be central tocarcinogenesis.
The two gray-highlighted genesRAD51 andBRCA2, are required forhomologous recombinational repair. They are sometimes epigenetically over-expressed and sometimes under-expressed in certain cancers. As indicated in the Wikipedia articles onRAD51 andBRCA2, such cancers ordinarily have epigenetic deficiencies in other DNA repair genes. These repair deficiencies would likely cause increased unrepaired DNA damages. The over-expression ofRAD51 andBRCA2 seen in these cancers may reflect selective pressures for compensatoryRAD51 orBRCA2 over-expression and increased homologous recombinational repair to at least partially deal with such excess DNA damages. In those cases whereRAD51 orBRCA2 are under-expressed, this would itself lead to increased unrepaired DNA damages. Replication errors past these damages (seetranslesion synthesis) could cause increased mutations and cancer, so that under-expression ofRAD51 orBRCA2 would be carcinogenic in itself.
Cyan-highlighted genes are in themicrohomology-mediated end joining (MMEJ) pathway and are up-regulated in cancer. MMEJ is an additional error-proneinaccurate 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.[28]FEN1, the flap endonuclease in MMEJ, is epigenetically increased by promoter hypomethylation and is over-expressed in the majority of cancers of the breast,[119] prostate,[120] stomach,[121][122] neuroblastomas,[123] pancreas,[124] and lung.[125] PARP1 is also over-expressed when its promoter regionETS site isepigenetically hypomethylated, and this contributes to progression to endometrial cancer[126] and BRCA-mutated serous ovarian cancer.[127] Other genes in theMMEJ pathway are also over-expressed in a number of cancers (seeMMEJ for summary), and are also shown in cyan.
Differential activity of DNA repair pathways across various regions of the human genome causes mutations to be very unevenly distributed within tumor genomes.[128][129] In particular, the gene-rich, early-replicating regions of the human genome exhibit lower mutation frequencies than the gene-poor, late-replicatingheterochromatin. One mechanism underlying this involves thehistone modificationH3K36me3, which can recruitmismatch repair proteins,[130] thereby lowering mutation rates inH3K36me3-marked regions.[131] Another important mechanism concernsnucleotide excision repair, which can be recruited by the transcription machinery, lowering somatic mutation rates in active genes[129] and other open chromatin regions.[132]
Damage to DNA is very common and is constantly being repaired. Epigenetic alterations can accompany DNA repair of oxidative damage or double-strand breaks. In human cells, oxidative DNA damage occurs about 10,000 times a day and DNA double-strand breaks occur about 10 to 50 times a cell cycle in somatic replicating cells (seeDNA damage (naturally occurring)). The selective advantage of DNA repair is to allow the cell to survive in the face of DNA damage. The selective advantage of epigenetic alterations that occur with DNA repair is not clear.[citation needed]
In the steady state (with endogenous damages occurring and being repaired), there are about 2,400 oxidatively damaged guanines that form8-oxo-2'-deoxyguanosine (8-OHdG) in the average mammalian cell DNA.[133] 8-OHdG constitutes about 5% of the oxidative damages commonly present in DNA.[134] The oxidized guanines do not occur randomly among all guanines in DNA. There is a sequence preference for the guanine at amethylatedCpG site (a cytosine followed by guanine along its5' → 3' direction and where the cytosine is methylated (5-mCpG)).[135] A 5-mCpG site has the lowest ionization potential for guanine oxidation.
Oxidized guanine has mispairing potential and is mutagenic.[137]Oxoguanine glycosylase (OGG1) is the primary enzyme responsible for the excision of the oxidized guanine during DNA repair. OGG1 finds and binds to an 8-OHdG within a few seconds.[138] However, OGG1 does not immediately excise 8-OHdG. In HeLa cells half maximum removal of 8-OHdG occurs in 30 minutes,[139] and in irradiated mice, the 8-OHdGs induced in the mouse liver are removed with a half-life of 11 minutes.[134]
When OGG1 is present at an oxidized guanine within a methylatedCpG site it recruitsTET1 to the 8-OHdG lesion (see Figure). This allows TET1 to demethylate an adjacent methylated cytosine.[140] Demethylation of cytosine is an epigenetic alteration.[141]
As an example, when human mammary epithelial cells were treated with H2O2 for six hours, 8-OHdG increased about 3.5-fold in DNA and this caused about 80% demethylation of the 5-methylcytosines in the genome.[136] Demethylation of CpGs in a gene promoter byTET enzyme activity increases transcription of the gene into messenger RNA.[142] In cells treated with H2O2, one particular gene was examined,BACE1.[136] The methylation level of theBACE1CpG island was reduced (an epigenetic alteration) and this allowed about 6.5 fold increase of expression ofBACE1 messenger RNA.[citation needed]
While six-hour incubation with H2O2 causes considerable demethylation of 5-mCpG sites, shorter times of H2O2 incubation appear to promote other epigenetic alterations. Treatment of cells with H2O2 for 30 minutes causes the mismatch repair protein heterodimer MSH2-MSH6 to recruit DNA methyltransferase 1 (DNMT1) to sites of some kinds of oxidative DNA damage.[143] This could cause increased methylation of cytosines (epigenetic alterations) at these locations.
Jiang et al.[144] treatedHEK 293 cells with agents causing oxidative DNA damage, (potassium bromate (KBrO3) orpotassium chromate (K2CrO4)).Base excision repair (BER) of oxidative damage occurred with the DNA repair enzymepolymerase beta localizing to oxidized guanines. Polymerase beta is the main human polymerase in short-patch BER of oxidative DNA damage. Jiang et al.[144] also found that polymerase beta recruited theDNA methyltransferase protein DNMT3b to BER repair sites. They then evaluated the methylation pattern at the single nucleotide level in a small region of DNA including thepromoter region and the early transcription region of theBRCA1 gene. Oxidative DNA damage from bromate modulated the DNA methylation pattern (caused epigenetic alterations) at CpG sites within the region of DNA studied. In untreated cells, CpGs located at −189, −134, −29, −19, +16, and +19 of the BRCA1 gene had methylated cytosines (where numbering is from themessenger RNA transcription start site, and negative numbers indicate nucleotides in the upstreampromoter region). Bromate treatment-induced oxidation resulted in the loss of cytosine methylation at −189, −134, +16 and +19 while also leading to the formation of new methylation at the CpGs located at −80, −55, −21 and +8 after DNA repair was allowed.[citation needed]
At least four articles report the recruitment ofDNA methyltransferase 1 (DNMT1) to sites of DNA double-strand breaks.[145][146][102][147] Duringhomologous recombinational repair (HR) of the double-strand break, the involvement of DNMT1 causes the two repaired strands of DNA to have different levels of methylated cytosines. One strand becomes frequently methylated at about 21CpG sites downstream of the repaired double-strand break. The other DNA strand loses methylation at about six CpG sites that were previously methylated downstream of the double-strand break, as well as losing methylation at about five CpG sites that were previously methylated upstream of the double-strand break. When the chromosome is replicated, this gives rise to one daughter chromosome that is heavily methylated downstream of the previous break site and one that is unmethylated in the region both upstream and downstream of the previous break site. With respect to the gene that was broken by the double-strand break, half of the progeny cells express that gene at a high level and in the other half of the progeny cells expression of that gene is repressed. When clones of these cells were maintained for three years, the new methylation patterns were maintained over that time period.[148]
In mice with a CRISPR-mediated homology-directed recombination insertion in their genome there were a large number of increased methylations of CpG sites within the double-strand break-associated insertion.[149]
Non-homologous end joining (NHEJ) repair of a double-strand break can cause a small number of demethylations of pre-existing cytosine DNA methylations downstream of the repaired double-strand break.[146] Further work by Allen et al.[150] showed that NHEJ of a DNA double-strand break in a cell could give rise to some progeny cells having repressed expression of the gene harboring the initial double-strand break and some progeny having high expression of that gene due to epigenetic alterations associated with NHEJ repair. The frequency of epigenetic alterations causing repression of a gene after an NHEJ repair of a DNA double-strand break in that gene may be about 0.9%.[102]
The basic processes of DNA repair are highlyconserved among bothprokaryotes andeukaryotes and even amongbacteriophages (viruses which infectbacteria); however, more complex organisms with more complex genomes have correspondingly more complex repair mechanisms.[151] The ability of a large number of proteinstructural 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.[152]
Thefossil record indicates that single-cell life began to proliferate on the planet at some point during thePrecambrian 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 tophotosynthetic organisms, as well as the presence of potentially damagingfree radicals in the cell due tooxidative phosphorylation, necessitated the evolution of DNA repair mechanisms that act specifically to counter the types of damage induced byoxidative stress. The mechanism by which this came about, however, is unclear.[citation needed]
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 agerm line cell that will eventually produce agamete, the mutation has the potential to be passed on to the organism's offspring. The rate ofevolution 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.[153] 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.[citation needed]
A technology named clustered regularly interspaced short palindromic repeat (shortened toCRISPR-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.[154] 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.[citation needed]
{{expression of concern|...}} with{{expression of concern|...|intentional=yes}}.)If we lived long enough, sooner or later we all would get cancer.
A certain irreducible background incidence of cancer is to be expected regardless of circumstances: mutations can never be absolutely avoided, because they are an inescapable consequence of fundamental limitations on the accuracy of DNA replication, as discussed in Chapter 5. If a human could live long enough, it is inevitable that at least one of his or her cells would eventually accumulate a set of mutations sufficient for cancer to develop.
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