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DNA Repair

  1. Simon Huw Reed,
  2. Raymond Waters

Published Online: 23 SEP 2005

DOI: 10.1038/npg.els.0005284

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How to Cite

Reed, S. H. and Waters, R. 2005. DNA Repair. eLS. .

Author Information

  1. University of Wales College of Medicine, Cardiff, UK

Publication History

  1. Published Online: 23 SEP 2005

Introduction

  1. Top of page
  2. Introduction
  3. Nucleotide Excision Repair
  4. Base Excision Repair
  5. Mismatch Repair
  6. Recombinational Repair
  7. See also
  8. References
  9. Further Reading
  10. Web Links

The genetic material stored within our cells contains the encrypted information necessary for coordinating cellular function and organization of the intact organism. The macromolecule deoxyribonucleic acid (DNA) is the repository for this information. It might be expected that such a vital structure would be extremely stable. Surprisingly this is not the case. DNA is exposed constantly to the damaging effects of normal metabolic processes and the effects of genotoxic agents from the environment (Friedberg and Walkers, 1995). About 10000 lesions are induced in the DNA in each of our cells every day. Short-term implications of DNA damage include effects on gene expression. Some DNA lesions prevent ribonucleic acid (RNA) polymerase from completing the message during transcription. Long-term effects are caused after replication of damaged DNA. Base misincorporation during this process causes mutations that alter the genetic information. The effects of mutation include cancer initiation, inborn defects, aging and general cellular malfunction. See also Cancer Cytogenetics, DNA Replication, DNA Structure, Genetic Disease: Prevalence, and Telomeres and Telomerase in Ageing and Cancer

DNA repair operates as a component of the overall DNA damage response (see Liu and Kulesz-Martin, 2001). This process is relevant to several human diseases. Cells sense DNA damage and transiently block cell-cycle progression by arresting DNA replication and chromosome segregation. This provides the cell with time to recognize and repair DNA damage before it is converted and fixed into either mutations after DNA replication or chromosomal aberrations after cell division. When DNA damage is too extreme, cells commit themselves to a programed cell death known as apoptosis. This is the cell's fail-safe mechanism to prevent the initiation of cancer. Individual cells sacrifice themselves for the good of the whole organism (Hoeijmakers, 2001). See also Apoptosis and the Cell Cycle in Human Disease, Apoptosis: Inherited Disorders, and DNA Damage Response: From Tumourigenesis to Therapy

A structured network of DNA surveillance and repair exists to rid the cell of the damage burden. Four main pathways enable the cell to achieve this: nucleotide excision repair (NER), base excision repair (BER), mismatch repair (MMR) and recombinational (or double-strand break; DSB) repair. These processes each recognize a characteristic set of DNA lesions; together, they have a role in preventing mutagenesis and cellular toxicity. The double-stranded structure of the DNA molecule provides built-in redundancy in the genetic information. All four principal repair pathways use this redundancy to carry out their repair reactions. Some scientists think that the necessity for DNA repair was a major contributing factor during the evolution of the double helix (Hanawalt, 2001). See also DNA Repair: Evolution, and Mismatch Repair Genes

Many different human genetic diseases are caused by mutations in DNA repair genes. In addition to maintaining genome stability, DNA repair pathways also contribute to complex developmental processes including aging and development of the immune system. The best characterized of these repair deficiency syndromes is xeroderma pigmentosum (XP), a rare autosomal recessive disease that is caused by mutations in genes involved in the NER pathway. The striking sun sensitivity observed in the cells of individuals with XP is correlated with a severe defect in their ability to remove ultraviolet (UV)-induced DNA damage. DNA repair mechanisms developed early in evolution, and have been highly conserved throughout it. Much of our understanding of DNA repair derives from the study of model systems, including yeast, mouse and hamster. These models provide certain experimental advantages that are not available when examining humans or their cells. See also DNA Repair: Disorders, Mouse as a Model for Human Diseases, and Yeast as a Model for Human Diseases

Nucleotide Excision Repair

  1. Top of page
  2. Introduction
  3. Nucleotide Excision Repair
  4. Base Excision Repair
  5. Mismatch Repair
  6. Recombinational Repair
  7. See also
  8. References
  9. Further Reading
  10. Web Links

Nucleotide excision repair repairs a wide spectrum of DNA lesions and has the most complex biochemical mechanism, with over 30 polypeptides involved. DNA damage is caused by exposure to chemicals or electromagnetic radiation from the environment. The damage is removed as an oligonucleotide of about 30 nucleotides. The complexity of repairing UV damage in human cells was recognized early on: cell fusion studies on XP cells showed that it was possible to complement their inability to repair DNA damage. Cells from affected individuals fall into at least eight complementation groups designated XP-A to XP-G and XP-V.

The seven genes affected in XP that encode proteins that complement the NER activity missing in each of the XP-A to XP-G groups have been identified. XP-V cells are not NER defective and are only moderately UV-sensitive. The gene affected in these cells, polymerase (DNA directed), eta (POLH), encodes the human DNA polymerase η protein that is involved in aberrant but nonmutagenic translesion synthesis, which is part of the DNA damage tolerance mechanism. See also DNA Replication, and DNA Replication Fidelity

There are at least three human disorders that are associated with defective NER: XP, Cockayne syndrome (CS) and trichothiodystrophy (TTD). Determining the NER mechanism has explained the molecular bases of these diseases. In vitro biochemical assays have provided a detailed knowledge of NER (Wood and Robins, 1988), although whether NER is mediated through a preformed complex called the ‘repairosome’, or by the sequential assembly of NER proteins remains controversial. The sequential assembly model is described here for simplicity and comprises six steps: (1) damage recognition, (2) DNA unwinding, (3) dual incision of the damaged strand, (4) excision of the damage containing oligonucleotide, (5) DNA repair synthesis and (6) ligation of the newly synthesized strand. See also DNA Repair: Disorders

Damage recognition is the least understood step. NER selectively repairs actively transcribing regions of the genome more efficiently than nontranscribed regions. Rapid removal of DNA damage from expressed genes allows the timely resumption of RNA synthesis, which is inhibited after DNA damage. Lesions that block transcription of a gene will prevent generation of the messenger RNA and ultimately lead to altered gene expression. Therefore, two damage-recognition pathways operate in NER: the transcription-coupled repair (TCR) pathway, which operates on the transcribed strand of active genes; and the global genome repair (GGR) pathway, which operates in nontranscribed DNA. The two pathways use the same basic set of proteins, with a subset being unique to each pathway. Damage recognition in the TCR pathway may involve the elongating RNA polymerase II (pol II) complex. Three additional proteins CSA, CSB and XAB2 are required for the proper functioning of the TCR pathway. Currently, the function of these proteins is unknown, but suggested functions include displacement of the elongating RNA polymerase during the repair reaction. The GGR pathway uses the XPC–hHR23B subcomplex during damage recognition. Elongating RNA pol II and XPC–hHR23B compete for lesions in transcribed strands; consequently, lesions that stall pol II elongation are recognized more rapidly by pol II during TCR than by XPC–hHR23B during GGR, which explains the faster removal of lesions from the transcribed strand than from the nontranscribed regions. Subsequent steps are considered common to both subpathways. See also DNA Repair: Disorders, and Transcription-coupled DNA Repair

After damage recognition, the transcription factor complex TFIIH unwinds the DNA duplex around the site of damage. The involvement of this protein complex during NER explains the several distinct human diseases that are associated with defects in this pathway, because TFIIH is also involved in transcription initiation. The complex has many enzymatic activities, including the DNA-dependent ATPase and 3′–5′ and 5′–3′ directional helicase functions of the XPB and XPD subunits respectively. TFIIH facilitates DNA melting at the site of damage to form a bubble structure (this function also operates in transcription initiation). The XPA protein binds to the damaged site while it is maintained in an open single-stranded conformation and is crucial in the assembly of the other NER components at the site of damage. The heterotrimeric single-stranded binding protein RPA, in association with XPA, stabilizes the opened DNA complex. This positions the XPG and ERCC1–XPF endonucleases that cut the DNA backbone either side of the damage. The 5′ incision leaves a free 3′ OH group, which is a primer for DNA repair synthesis. It is not known how the damaged oligonucleotide is excised. RPA protects the template strand from nuclease attack and facilitates DNA synthesis. DNA synthesis uses either DNA polymerase δ or DNA polymerase ε, which require proliferating cell nuclear antigen (PCNA) and RF-C for their activity. Finally, the 5′ end of the newly incorporated DNA is ligated by DNA ligase I. Our current understanding of the GGR and TCR pathways is represented Figure 1.

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Figure 1. Molecular model for the incision stage of nucleotide excision repair (NER). In step I, global genome repair (GGR) XPC–hHR23B (C) senses DNA helix-distorting NER lesions that lead to conformational alterations in the DNA. In transcription-coupled repair (TCR), lesions are detected by elongating RNA pol that is blocked, for example, by CPDs (NER lesions) and thymine glycols (non-NER lesion). In step II (left), XPC–hHR23B at the lesion attracts TFIIH, and possibly XPG (G; left). TFIIH creates an opened DNA complex (∼10–20 nt) around the lesion through its helicases XPB and XPD; this step requires ATP. XPC–hHR23B may be released at this or one of the subsequent stages. In step II (right) CSA, CSB, TFIIH, XPG and possibly other cofactors displace the stalled pol II from the lesion, which now becomes accessible for further repair processing; depending on the type of lesion, repair is completed by either NER or other repair pathways. In step III, XPA (A) and RPA stabilize the opening of 10–20 nt and position other factors. XPA binds to the damaged nucleotides and RPA to the undamaged DNA strand. Possibly, RPA binds 8–10 nt, and transition to its 30-nt binding mode (RPA stretching) may be important in full open complex formation. XPG stabilizes the fully opened complex. In step IV, XPG, positioned by TFIIH and RPA, makes the 3′ incision. ERCC1–XPF (F), positioned by RPA and XPA, makes the second incision 5′ of the lesion. In step V, dual incision is followed by gap-filling DNA synthesis and ligation. Drawn contacts between molecules reflect reported protein–protein interactions. (Reproduced with permission from de Laat and Jaspers 1999.)

As mentioned above, the molecular basis of NER reveals how defects in this process lead to the three distinct diseases. The complexity derives from the function of TFIIH in both repair and transcription. Mutations that affect only the repair pathway result in pure XP. This includes XP-C individuals, in which only GGR is affected. By contrast, pure CS is caused by mutations in the genes encoding CSA (Cockayne Syndrome 1; CNK1) and CSB (excision repair cross-complementing rodent repair deficiency, complementation group 6; ERCC6), which affect only the TCR pathway. Specific mutations in the genes encoding XPB (excision repair cross-complementing rodent repair deficiency, complementation group 3; ERCC3), XPD (ERCC2) and XPG (ERCC5), which affect both transcription and repair, lead to combined XP/CS. The clinical features of XP and CS are very different. Individuals affected with XP are UV-sensitive and have a higher risk of developing skin cancer. Many individuals have neurological degeneration that is related to premature aging features. Individuals affected with CS show a failure to thrive and are small. They have severe neurological abnormalities and certain features that resemble premature aging. These individuals are sunlight-sensitive but not cancer-prone. The third disease, TTD, is very similar to CS. Affected individuals have the unique features of very brittle hair, scaly skin and fragile nails. The genetic defect in TTD has been attributed to specific mutations in the TFIIH components XPB and XPD (there are currently three TTD complementation groups, A, B and D). See also DNA Repair: Disorders

Base Excision Repair

  1. Top of page
  2. Introduction
  3. Nucleotide Excision Repair
  4. Base Excision Repair
  5. Mismatch Repair
  6. Recombinational Repair
  7. See also
  8. References
  9. Further Reading
  10. Web Links

Many DNA base lesions, particularly those generated by spontaneous deamination, oxidative damage or methylation, are recognized and repaired by specific DNA N-glycosylases that remove the damaged base (Krokan and Nilsen, 2000). The glycosylase leaves an apurinic or apyrimidinic (AP) site, which in mammalian cells is processed further by removal of the sugar phosphate group, DNA gap filling and finally ligation. DNA polymerase β, XRCC1, APE1/HAP-1 and DNA ligase III are responsible for completing the major one-nucleotide removal pathway. A minor pathway that uses FEN1 and PCNA also exists.

APE1/HAP-1 is a multifunctional AP endonuclease that may be involved in brain development, and functions in the cellular stress response as well as in BER. DNA polymerase β is an error-prone polymerase, whose expression is induced by substrates of the BER pathway. The glycosylases that operate in this mode of repair can usually recognize specific types of base damage and therefore, unlike those in the NER pathway, tend to be specific. Notably, some substrate overlap exists between the BER and NER pathways. In addition, certain lesions that are substrates for the BER pathway are also repaired in a transcription-coupled fashion. The suggestion is that all RNA pol II blocking lesions can stimulate TCR of other DNA repair pathways including MMR (see below). There are no well-characterized human diseases associated with BER. Both NER and BER operate on the nuclear genome, whereas BER, but not NER operates on mitochondrial DNA. See also Mitochondrial DNA Repair in Mammals

Mismatch Repair

  1. Top of page
  2. Introduction
  3. Nucleotide Excision Repair
  4. Base Excision Repair
  5. Mismatch Repair
  6. Recombinational Repair
  7. See also
  8. References
  9. Further Reading
  10. Web Links

Postreplicative MMR removes base/base mismatches or insertion/deletion type loops created by DNA polymerase slippage during replication (Peltomaki, 2001). In humans, at least six different MMR proteins are used including the MSH proteins involved in mismatch recognition, and the MLH and PMS proteins that coordinate events after recognition. The other proteins involved in MMR include PCNA, exonucleases such as EXO1 and DNA polymerases δ and DNA polymerases ε, as well as replication factors such as RPA. The replication complex proofreads during replication. After replication, MMR carries out a backup proofreading.

Defects in the MMR genes result in a higher risk of colon and other cancers. Germ-line mutations in any of the five human MMR genes give rise to hereditary nonpolyposis colon cancer (HNPCC), which accounts for 1–5% of all cases of colon cancer. Individuals affected with HNPCC have a high lifetime risk of developing colorectal, endometrial and other cancers known collectively as the HNPCC tumor spectrum. MMR proteins may be involved in damage sensing and apoptosis. Persistent attempts by the MMR pathway to remove mismatched bases at sites of damage may lead to persistent DNA strand breaks. These act as a signal to the cell to induce apoptosis. A TCR mechanism is associated with MMR although the mechanistic details are unknown. A link between defects in MMR and aging has been noted. See also Mismatch Repair Genes

Recombinational Repair

  1. Top of page
  2. Introduction
  3. Nucleotide Excision Repair
  4. Base Excision Repair
  5. Mismatch Repair
  6. Recombinational Repair
  7. See also
  8. References
  9. Further Reading
  10. Web Links

The recombinational repair pathway responds to the potentially lethal formation of DSBs that occur in the genome through the direct action of ionizing radiation, exposure to certain chemicals, or the product of blocked replication forks. There are actually three DSB repair pathways that operate in the cell, and defects in each of these pathways have serious consequences for human health (Karran, 2000). The mechanism of two of the pathways uses homologous recombination, whereas the other does not. See also DNA Recombination

Nonhomologous end rejoining

Nonhomologous end rejoining (NHEJ) is effected without the need for extensive homology between the DNA ends that are to be joined, and it has been considered the principal pathway for DSB repair in mammalian cells (Durocher and Jackson, 2001). This pathway also processes the site-specific DSBs created during variant (diversity) joining (V(D)J) recombination – an essential component for the developmental maturation of the immune system. Defects in NHEJ result in the disorder severe combined immune deficiency (SCID) and in sensitivity to ionizing radiation. The mechanism of end rejoining involves the Ku70/80 heterodimer that binds the ends of the double-stranded DNA to be rejoined. This activates the catalytic subunit of DNA-PK, a protein kinase that stabilizes the interaction with the DNA ends. Rejoining is then carried out by the DNA ligase IV/XRCC4 heterodimer.

DNA-PK is a component of the DNA damage sensor and it is part of the family of phosphatidylinositol 3-kinase-like (PIKK) kinases that includes the ataxia telangiectasia mutated (ATM) and ataxia telangiectasia Rad3-related (ATR) signaling proteins that function in the homologous recombination pathway (see below). It is thought that PIKKs associate with accessory lesion-binding proteins and act as DNA damage sensors for a range of different DNA repair pathways. See also DNA Repair: Disorders

Homologous recombination and single-strand annealing

Two pathways use homologous recombination (HR) as a mechanism for DSB repair. HR uses the RAD52 group of proteins to form and resolve Holliday junctions. Other proteins involved include RAD51, RPA, XRCC2 and XRCC3. Mammalian cells carry out HR during mitotic recombination and repair DSBs by HR in late S and G2 phases of the cell cycle, where an undamaged sister chromatid is available. The human breast cancer susceptibility genes breast cancer 1, early onset (BRCA1) and breast cancer 2, early onset (BRCA2) are involved in recombinational repair together with RAD51 homolog (RAD51). In cells with damaged DNA, these three proteins relocate to regions that contain PCNA at sites of DNA damage. ATM, which is mutated in the radiation-sensitive disorder ataxia telangiectasia, is proposed to be a damage sensor of DSBs in the HR pathway. ATM phosphorylates BRCA1, which triggers the response to ionizing radiation in mammalian cells. BRCA1 has also been implicated in the repair of oxidative damage through the TCR pathway, although its role there is not understood. See also Transcription-coupled DNA Repair

The single-strand annealing (SSA) pathway is a subpathway of HR, which relies on regions of homology to align the strands of DNA to be rejoined. The proteins involved in the pathway are RAD50, MRE11 and NBS1. MRE11, a 3′–5′ exonuclease, possibly removes damaged or mismatched DNA ends to expose short lengths of single-stranded DNA. In the absence of a sister chromatid, sites of limited homology in the resected region may anneal to begin repair through SSA. Mutation of the Nijmegen breakage syndrome 1 (NBS1) gene results in the radiation-sensitive disease Nijmegen breakage syndrome. Affected individuals have immune deficiency, chromosome instability and lymphoreticular tumors. Mutations in the meiotic recombination 11 homolog A (MRE11A) gene that encodes MRE11 have also been found. The clinical symptoms are similar to the symptoms of ataxia telangiectasia, thus emphasizing the importance of the SSA pathway. Remarkably, in both MRE11A- and NBS1-defective cells there is no defect in DSB rejoining despite the severe phenotype. Crosstalk between the repair systems probably accounts for this and there is evidence that components from both the NER and MMR pathways can function during SSA.

A newly emerging area of research links the DNA repair and damage sensing pathways to the aging process. Most normal human somatic cells lose 50–200 base pairs from the telomeric repeat sequences found at the ends of their chromosomes. This loss has been linked causally to replicative senescensce. The inability to correctly sense or repair short telomeres can not only affect the aging process but also contribute to genetic instability that leads to malignant transformation. Molecules that have been implicated in sensing short telomeres include ATM, Ku70/80 and their downstream targets such as p53, DNA-PK and p21. The molecules involved in repairing short telomeres are likely to differ depending on the cell type. The telomerase-independent pathway probably uses these proteins together with BRCA1, the RAD50–MRE11–p95 complex and two DNA helicases, which when mutated cause Bloom disease and Werner disease, two human syndromes that are associated with premature aging. See also Telomeres and Telomerase in Ageing and Cancer

DNA repair has a fundamental role in DNA metabolism. Its importance in maintaining genome stability not only protects against the onset of malignant transformation but also contributes to the normal development of the organism.

References

  1. Top of page
  2. Introduction
  3. Nucleotide Excision Repair
  4. Base Excision Repair
  5. Mismatch Repair
  6. Recombinational Repair
  7. See also
  8. References
  9. Further Reading
  10. Web Links

Further Reading

  1. Top of page
  2. Introduction
  3. Nucleotide Excision Repair
  4. Base Excision Repair
  5. Mismatch Repair
  6. Recombinational Repair
  7. See also
  8. References
  9. Further Reading
  10. Web Links

Web Links

  1. Top of page
  2. Introduction
  3. Nucleotide Excision Repair
  4. Base Excision Repair
  5. Mismatch Repair
  6. Recombinational Repair
  7. See also
  8. References
  9. Further Reading
  10. Web Links