Which Of The Following Mechanisms Can Serve To Repair Double-stranded Breaks In Dna

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The integrity of genomic DNA is crucial for its function. And nevertheless, DNA in living cells is inherently unstable. It is subject to mechanical stress and to many types of chemical modification that may lead to breaks in 1 or both strands of the double helix. Within the prison cell, reactive oxygen species generated past normal respiratory metabolism can crusade double-strand breaks, as can stalled DNA replication. External agents that crusade double-strand breaks include ionizing radiations and certain chemotherapeutic drugs. Deoxyribonucleic acid double-strand breaks are also made and repaired during meiosis when recombination takes place between paired homologous chromosomes, during the rearrangement of immunoglobulin factor segments in lymphocyte development and during integration of certain mobile genetic elements and viruses into the host cell Dna.

It is difficult to know how ofttimes double-strand breaks occur in the genome of a cell not exposed to external Deoxyribonucleic acid-damaging agents, but we know from work with yeast cells that one persistent Dna double-strand break can be sufficient to trigger the death of a cell. If double-strand breaks go unrepaired in mammalian cells, they can too cause gene deletion, chromosome loss and other chromosomal aberrations that might ultimately produce cancers.

DNA double-strand breaks are repaired by ways of two main mechanisms: nonhomologous end joining and homologous recombination (see Figure 1). Both mechanisms operate in all eukaryotic cells that accept been examined simply the relative contribution of each mechanism varies. For example, most mammalian cells seem to favour nonhomologous finish joining (also chosen 'illegitimate recombination'), whereas homologous recombination is more common in the budding yeast Saccharomyces cerevisiae. I possible reason for this departure might be the prevalence in mammalian cells of repetitive sequences, which could lead to gene distension or deletion if homologous recombination were mutual. In addition to these principal mechanisms, Dna double-strand breaks can exist repaired past means of single-strand annealing between adjacent repeated Deoxyribonucleic acid sequences, which involves deletion of the intervening DNA (encounter Figure one). This article focuses mainly on nonhomologous end joining, the all-time-characterized mammalian DNA double-strand break repair mechanism.

Figure ane Culling pathways of DNA double-strand break repair. Homologous recombination is the preferred route in yeast. It involves invasion of the broken Dna strands into a homologous Deoxyribonucleic acid duplex molecule. This process requires Rad52 (a DNA end-binding protein), Rad51 (which forms filaments along the unwound DNA strands), Dna polymerases and other less well-characterized cistron products. The Dna ends are ligated by DNA ligase I and the interwound Deoxyribonucleic acid strands are separated, probably by another poly peptide complex, with no loss of genetic information. Only i of many possible recombination products is shown here. Single-strand annealing takes identify between two homologous DNA sequences in tandem (yellow and orange boxes) by a less well-studied mechanism. It too requires Rad52, and extensive degradation of the two unannealed strands results in considerable loss of genetic material. Nonhomologous end joining rejoins the two broken ends directly. It requires the DNA end-binding protein Ku which, in mammalian cells, forms a complex with Deoxyribonucleic acid-PKcs (red). Other steps in this procedure involve the Rad50–Mre11–Nbs1 complex in mammals (which may also exist involved in homologous recombination, at least in yeast) and the XRCC4–Deoxyribonucleic acid ligase 4 complex. Few, or no, bases are missing from the products of nonhomologous end joining.

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Responses to Dna damage

The presence of Deoxyribonucleic acid double-strand breaks — and other types of Deoxyribonucleic acid impairment — in a dividing prison cell is registered by cell-cycle surveillance mechanisms (besides chosen checkpoint mechanisms) and leads to arrest of the cell division wheel at ane of several points. This arrest stops progress through Thousand phase, so preventing possible chromosome loss or manual of harm to the next cellular generation, and progress into or through S phase, when errors in DNA replication could be sustained. In one case the Deoxyribonucleic acid impairment is repaired, the cell bike usually resumes.

Dna double-strand breaks tin can as well lead to cell death by apoptosis. Apoptosis is thought to issue when the level of DNA harm is as well groovy to warrant repair, still, the level of damage that tin can be tolerated by unlike jail cell types varies markedly.

Homologous recombination

Homologous recombination involves unwinding of the damaged DNA helix and invasion of the damaged strands into a homologous Dna duplex molecule that may exist the sister chromatid, homologous chromosome or, for example, a vector or viral genome with homology to the genomic Dna. Repair of a DNA double-strand break by homologous recombination takes place by means of replication, using the homologous strand equally template (run across Effigy 1). Homologous recombination therefore requires extensive regions of DNA homology in another DNA duplex, and no loss of genetic information normally results.

Many of the genes and proteins involved in homologous recombination have been identified, mainly past ways of yeast genetics, using mutants that are hypersensitive to ionizing radiation. These proteins include the products of the RAD51, RAD52, RAD54, RAD55, RAD57, RAD50, MRE11 and XRS2 genes, and homologues of most of them accept been cloned from mammalian species.

Nonhomologous terminate joining

In contrast to homologous recombination, nonhomologous end joining requires no homology with a second DNA duplex and no, or only a few base of operations pairs of, homology between the two broken DNA ends.

In mammalian cells and in yeast, the first protein to recognize a DNA double-strand intermission seems to be Ku, a heterodimer of related ∼70 kDa and ∼fourscore kDa polypeptides that together bind to the ends of broken DNA (see Figure 1). It is not yet clear whether one Ku heterodimer is sufficient to bind to the 2 ends at a break or whether one or more heterodimers demark to each end.

The mammalian Ku protein forms a complex with the Dna-dependent poly peptide kinase catalytic subunit (called DNA-PKcs) and, in the presence of Dna ends in vitro, activates its serine/threonine kinase activity. Thus, in mammalian cells, Ku probably recruits Dna-PKcs to the break, where the kinase phosphorylates proteins bound to the DNA effectually the break, and possibly also phosphorylates soluble proteins in the nucleus. DNA-PKcs is a large protein (∼465 kDa) but, aside from its kinase domain, the functions of the bulk of the poly peptide are not known. Information technology might tether the ii ends together while they are rejoined, regulate other repair molecules, control the chromatin structure around the break, and/or be involved in signal transduction. Intriguingly, there is no obvious Ku-binding equivalent of Dna-PKcs in yeast cells. Information technology is not still articulate whether this reflects a fundamental difference between nonhomologous end joining in yeast and mammals.

Nigh DNA double-strand breaks are not blunt-concluded merely take unmarried-stranded overhangs. These DNA ends might need to be trimmed by exonucleases and/or endonucleases before they tin be rejoined. Indeed, a protein circuitous containing a Dna endonuclease and/or exonuclease seems to be involved in DNA double-strand interruption repair. In yeast, this complex contains Mre11p, Rad50p and Xrs2p (and possibly other polypeptides). Mre11p and Rad50p are homologous to a bacterial endo/exonuclease called SbcCD, and human Mre11 has nuclease activity in vitro. Genetic studies of yeast indicate that these three cistron products function in both homologous recombination and nonhomologous end joining but exactly how they work in the ii processes is unknown. Mammalian cells take homologues of Rad50p and Mre11p only a different protein, Nbs1, seems to replace Xrs2p. Nbs1 seems to point the presence of Dna damage to the cell cycle checkpoint machinery and to regulate the activity of the Mre11–Rad50 circuitous.

Finally, any remaining gaps in the DNA must exist filled by a Deoxyribonucleic acid polymerase (precisely which polymerase(s) perform this job is not known) and the ends must be ligated. Nonhomologous end joining uses DNA ligase Iv in mammalian cells and its homologue Lig4p in yeast. In mammalian cells, DNA ligase Four binds tightly to a protein chosen XRCC4, which is a substrate for DNA-PKcs in vitro and might regulate the activity of the ligase. The yeast equivalent of XRCC4 is Lif1p.

Two relatives of Deoxyribonucleic acid-PKcs in mammalian cells, ATM and ATR, are involved in Dna damage signalling. Biochemical studies have suggested that ATM binds to DNA and phosphorylates the tumour suppressor poly peptide p53 at sites that become phosphorylated in vivo when cells are irradiated. Also, phosphorylation of p53 in response to agents that produce double-strand breaks is dumb in ATM-deficient cells. This provides strong back up for the idea that ATM signals DNA damage to the checkpoint machinery, whereas such disarming show is defective for DNA-PKcs. ATR also phosphorylates p53 on physiologically relevant sites in vitro. It is not yet clear whether ATM, ATR and DNA-PKcs recognize different types of DNA damage or operate at different stages of the cell cycle or in different nuclear compartments.

Defective double-strand interruption repair

Cells and animals with mutations in genes that encode components of nonhomologous end joining are radiosensitive, that is, they dice every bit a effect of much lower doses of radiation than do normal cells. The mutant mouse line called the severe combined immunodeficient (SCID) mouse is as well radiosensitive. These mice are immunodeficient considering a mutation in the gene encoding Deoxyribonucleic acid-PKcs prevents their lymphocytes from making the immunoglobulin V(D)J gene rearrangements that are necessary to produce the normal repertoire of antibodies and T-cell receptors during development. This surprising finding revealed that the DNA double-strand breaks made during this programmed gene rearrangement are repaired by nonhomologous end joining. Consequent with this, targeted disruption of the genes that encode DNA-PKcs, Ku70 and Ku80 in mice effect in SCID. No similar cause of human immunodeficiency has been reported, however, suggesting that defects in nonhomologous end joining either are rare or cause embryonic lethality in humans.

Merely defects in other components of the nonhomologous end joining machinery might crusade human being disease. For example, a defect in DNA ligase Four was recently discovered in the cells of a patient with childhood leukaemia who died from overreaction to standard radiotherapy. Likewise, Nijmegen breakage syndrome is caused by a defect in the cistron encoding Nbs1. The symptoms of this syndrome include genetic instability and a predisposition to cancers, especially to lymphoid cancers. Another human genetic instability and lymphoid cancer predisposition syndrome called ataxia telangiectasia is acquired past mutations in the gene that encodes the signalling protein ATM. Many of the symptoms of this disorder may be explained past a failure to signal cell-bicycle arrest in the presence of double-strand breaks, leading to genetic instability that ultimately produces cancer or jail cell expiry.

Deoxyribonucleic acid break repair in development

Targeted disruption of the genes encoding DNA ligase Iv, XRCC4 and Rad50 in mice results in embryonic lethality, indicating that Dna double-strand break repair is crucial for development. This might simply reflect a loftier rate of double-strand suspension formation in rapidly proliferating cells and, therefore, a demand for very efficient DNA repair mechanisms in embryogenesis. Alternatively, it might indicate that DNA rearrangement takes place early in embryogenesis. No developmental Dna rearrangement other than 5(D)J recombination is known in mammals merely a contempo discovery hints that the cadherin superfamily of genes might undergo similar rearrangement. As the cadherins are implicated in neurogenesis, disruption of this procedure might consequence in embryonic lethality. Consequent with this idea, the main defect in DNA ligase Four-deficient embryos and in XRCC4-scarce embryos seems to be in the fundamental nervous system.

Structural considerations

The establishment of multiprotein complexes and the state of packing of the Deoxyribonucleic acid are probably crucial to DNA repair. An initial indication of this was the discovery that the yeast silencing proteins Sir2p, Sir3p and Sir4p — which inhibit the transcription of certain yeast loci, presumably by packaging the DNA in a form inaccessible to the transcriptional appliance — are required for nonhomologous stop joining. I popular model to explain the role of Sir proteins in nonhomologous end joining is that this procedure requires 'inactivation' of the DNA around a double-strand break, probably by recruitment of heterochromatin-forming proteins. By dissimilarity, homologous recombination probably requires a more open chromatin conformation to allow strand invasion. At that place is little testify to back up these conjectures, nonetheless.

On exposure of a human cell to ionizing radiation, Mre11 and Rad50 seem to relocate from a diffuse distribution in the nucleus to foci, in a process that requires the ATM gene product. These foci may stand for loci of individual double-strand breaks, pools of mobilized Deoxyribonucleic acid repair factors, or domains of DNA repair factories.

DNA repair factors at telomeres

In yeast, the genes that encode Ku70 and Ku80 are essential to maintain the length of the telomeres — the specialized chromatin structures at the ends of chromosomes. Yeast Ku might act at the telomeres past protecting the DNA finish from degradation and by clustering telomeres together at the nuclear periphery. At first sight, the presence of Ku at telomeres seems incongruous; Ku promotes DNA terminate joining, yet the DNA ends of telomeres must be prevented from joining to other DNA ends. The office of Ku at telomeres is probably to promote some specific telomeric chromatin structure. Alternatively, the telomeres might be a storage site for Ku, Sir proteins and perhaps other factors involved in double-strand suspension repair. Indeed, in yeast the germination of double-strand breaks disperses Ku and the Sir proteins from the telomeres. This relocation response tin be induced by equally little every bit one double-strand break, and requires a Dna impairment-signalling pathway including Mec1p and Rad9p.

From this brief survey, it is clear that recent years have witnessed tremendous progress in our understanding of the molecular basis by which DNA double-strand breaks are repaired in eukaryotic cells. This progress has created an exciting and speedily expanding field that is offset to influence many areas of pure and practical biology.

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Biography

C Featherstone and SP Jackson, The Wellcome/CRC Institute, Tennis Courtroom Road, Cambridge CB2 1QR, UK.

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DOI: https://doi.org/x.1016/S0960-9822(00)80005-half dozen

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