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1.
The endless tale of non-homologous end-joining   总被引:1,自引:0,他引:1  
Weterings E  Chen DJ 《Cell research》2008,18(1):114-124
DNA double-strand breaks (DSBs) are introduced in cells by ionizing radiation and reactive oxygen species. In addition, they are commonly generated during V(D)J recombination, an essential aspect of the developing immune system. Failure to effectively repair these DSBs can result in chromosome breakage, cell death, onset of cancer, and defects in the immune system of higher vertebrates. Fortunately, all mammalian cells possess two enzymatic pathways that mediate the repair of DSBs: homologous recombination and non-homologous end-joining (NHEJ). The NHEJ process utilizes enzymes that capture both ends of the broken DNA molecule, bring them together in a synaptic DNA-protein complex, and finally repair the DNA break. In this review, all the known enzymes that play a role in the NHEJ process are discussed and a working model for the co-operation of these enzymes during DSB repair is presented.  相似文献   

2.
The use of reporter systems to analyze DNA double-strand break(DSB) repairs,based on the enhanced green fluorescent protein (EGFP) and meganuclease such as I-Sce I,is usually carried out with cell lines.In this study,we developed three visual-plus quantitative assay systems for homologous recombination(HR),non-homologous end joining(NHEJ) and single-strand annealing(SSA) DSB repair pathways at the organismal level in zebrafish embryos.To initiate DNA DSB repair,we used two I-Sce I recognition sites in opposite orientation rather than the usual single site.The NHEJ,HR and SSA repair pathways were separately triggered by the injection of three corresponding I-Sce I-cut constructions,and the repair of DNA lesion caused by l-Sce I could be tracked by EGFP expression in the embryos.Apart from monitoring the intensity of green fluorescence,the repair frequencies could also be precisely measured by quantitative real-time polymerase chain reaction(qPCR).Analysis of DNA sequences at the DSB sites showed that NHEJ was predominant among these three repair pathways in zebrafish embryos.Furthermore,while HR and SSA reporter systems could be effectively decreased by the knockdown of rad51 and rad52,respectively,NHEJ could only be impaired by the knockdown of ligaseIV(lig4) when the NHEJ construct was cut by I-Sce I in vivo.More interestingly,blocking NHEJ with lig4-MO increased the frequency of HR,but decreased the frequency of SSA.Our studies demonstrate that the major mechanisms used to repair DNA DSBs are conserved from zebrafish to mammal,and zebrafish provides an excellent model for studying and manipulating DNA DSB repair at the organismal level.  相似文献   

3.
Homologous recombination in DNA repair and DNA damage tolerance   总被引:20,自引:0,他引:20  
Li X  Heyer WD 《Cell research》2008,18(1):99-113
Homologous recombination (HR) comprises a series of interrelated pathways that function in the repair of DNA double-stranded breaks (DSBs) and interstrand crosslinks (ICLs). In addition, recombination provides critical support for DNA replication in the recovery of stalled or broken replication forks, contributing to tolerance of DNA damage. A central core of proteins, most critically the RecA homolog Rad51, catalyzes the key reactions that typify HR: homology search and DNA strand invasion. The diverse functions of recombination are reflected in the need for context-specific factors that perform supplemental functions in conjunction with the core proteins. The inability to properly repair complex DNA damage and resolve DNA replication stress leads to genomic instability and contributes to cancer etiology. Mutations in the BRCA2 recombination gene cause predisposition to breast and ovarian cancer as well as Fanconi anemia, a cancer predisposition syndrome characterized by a defect in the repair of DNA interstrand crosslinks. The cellular functions of recombination are also germane to DNA-based treatment modalities of cancer, which target replicating cells by the direct or indirect induction of DNA lesions that are substrates for recombination pathways. This review focuses on mechanistic aspects of HR relating to DSB and ICL repair as well as replication fork support.  相似文献   

4.
DNA double-strand breaks (DSBs), which arise following exposure to a number of endogenous and exogenous agents, can be repaired by either the homologous recombination (HR) or non-homologous end-joining (NHEJ) pathways in eukaryotic cells. A vital step in HR repair is DNA end resection, which generates a long 30 single-stranded DNA (ssDNA) tail that can invade the homologous DNA strand. The generation of 30 ssDNA is not only essential for HR repair, but also promotes activation of the ataxia telangiectasia and Rad3-related protein (ATR). Multiple fac-tors, including the MRN/X complex, C-terminal-binding protein interacting protein (CtIP)/Sae2, exonuclease 1 (EXO1), Bloom syndrome protein (BLM)/Sgs1, DNA2 nuclease/helicase, and several chromatin remodelers, cooperate to complete the process of end resection. Here we review the basic machinery involved in DNA end resection in eukaryotic cells.  相似文献   

5.
The partitioning-defective 3 (Par3),a key component in the conserved Par3/Par6/aPKC complex,plays fundamentalroles in cell polarity.Herein we report the identification of Ku70 and Ku80 as novel Par3-interacting proteins throughan in vitro binding assay followed by liquid chromatography-tandem mass spectrometry.Ku70/Ku80 proteins are twokey regulatory subunits of the DNA-dependent protein kinase (DNA-PK),which plays an essential role in repairingdouble-strand DNA breaks (DSBs).We determined that the nuclear association of Par3 with Ku70/KuS0 was enhancedby y-irradiation (IR),a potent DSB inducer.Furthermore,DNA-PKcs,the catalytic subunit of DNA-PK,interacted withthe Par3/Ku70/Ku80 complex in response to IR.Par3 over-expression or knockdown was capable of up-or downregulat-ing DNA-PK activity,respectively.Moreover,the Par3 knockdown cells were found to be defective in random plasmidintegration,defective in DSB repair following IR,and radiosensitive,phenotypes similar to that of Ku70 knockdowncells.These findings identify Par3 as a novel component of the DNA-PK complex and implicate an unexpected link ofcell polarity to DSB repair.  相似文献   

6.
Li W  Ma H 《Cell research》2006,16(5):402-412
Meiotic prophase I is a long and complex phase. Homologous recombination is an important process that occurs between homologous chromosomes during meiotic prophase I. Formation of chiasmata, which hold homologous chromosomes together until the metaphase I to anaphase I transition, is critical for proper chromosome segregation. Recent studies have suggested that the SPO 11 proteins have conserved functions in a number of organisms in generating sites of double-stranded DNA breaks (DSBs) that are thought to be the starting points of homologous recombination. Processing of these sites of DSBs requires the function of RecA homologs, such as RAD5 1, DMC 1, and others, as suggested by mutant studies; thus the failure to repair these meiotic DSBs results in abnormal chromosomal alternations, leading to disrupted meiosis. Recent discoveries on the functions of these RecA homologs have improved the understanding of the mechanisms underlying meiotic homologous recombination.  相似文献   

7.
Embryonic stem cell maintenance, differentiation, and somatic cell reprogramming require the interplay of multiple pluripotency factors, epigenetic remodelers, and extracellular signaling pathways. RNA-binding proteins (RBPs) are involved in a wide range of regulatory pathways, from RNA metabolism to epigenetic modifications. In recent years we have witnessed more and more studies on the discovery of new RBPs and the assessment of their functions in a variety of biological systems, including stem cells. We review the current studies on RBPs and focus on those that have functional implications in pluripotency, differentiation, and/or reprogramming in both the human and mouse systems.  相似文献   

8.
Viruses are obligate intracellular parasites that subvert cellular metabolism and pathways to mediate their own replication—normally at the expense of the host cell. Polyomaviruses are a group of small DNA viruses, which have long been studied as a model for eukaryotic DNA replication. Polyomaviruses manipulate host replication proteins, as well as proteins involved in DNA maintenance and repair, to serve as essential cofactors for productive infection. Moreover, evidence suggests that polyomavirus infection poses a unique genotoxic threat to the host cell. In response to any source of DNA damage, cells must initiate an effective DNA damage response(DDR) to maintain genomic integrity, wherein two protein kinases, ataxia telangiectasia mutated(ATM) and ATM- and Rad3-related(ATR), are major regulators of DNA damage recognition and repair. Recent investigation suggests that these essential DDR proteins are required for productive polyomavirus infection. This review will focus on polyomaviruses and their interaction with ATMand ATR-mediated DNA damage responses and the effect of this interaction on host genomic stability.  相似文献   

9.
Hunter N 《Cell research》2008,18(3):328-330
Homologous recombination occurs when a damaged chromosome uses an intact homologous chromosome as a template for its repair. The main steps of recombination are most readily illustrated for the repair of a DNA doublestrand-break (DSB). First, DSB-ends are processed to form single-stranded tails, which assemble into nucleoprotein complexes comprising an oligomeric filament of a RecA-family protein (Rad51 in eukaryotes) and associated factors. Rad51 filaments catalyze homologous pairing and strand-exchange between a DSB-end and a double-stranded template to form a joint molecule intermediate. This structure allows de novo priming of DNA synthesis to restore sequences that were lost or damaged at the site of the original lesion. Recombination also underpins chromosome replication by facilitating the repair of broken replication forks. In this case, joint molecule formation allows rep- lication to reinitiate. At the final step of recombination, strand-exchange "Holliday" junctions that connect the involved chromosomes are resolved so that segregation can ensue. Joint molecule resolution can occur with one of two outcomes: a crossover, in which chromosome arms are exchanged; or a noncrossover without exchange.  相似文献   

10.
Regulation of cell survival and death during Flavivirus infections   总被引:3,自引:0,他引:3  
Flaviviruses, ss(+) RNA viruses, include many of mankind's most important pathogens. Their pathogenicity derives from their ability to infect many types of cells including neurons, to replicate, and eventually to kill the cells. Flaviviruses can activate tumor necrosis factor α and both intrinsic(Bax-mediated) and extrinsic pathways to apoptosis. Thus they can use many approaches for activating these pathways. Infection can lead to necrosis if viral load is extremely high or to other types of cell death if routes to apoptosis are blocked. Dengue and Japanese Encephalitis Virus can also activate autophagy. In this case the autophagy temporarily spares the infected cell, allowing a longer period of reproduction for the virus, and the autophagy further protects the cell against other stresses such as those caused by reactive oxygen species. Several of the viral proteins have been shown to induce apoptosis or autophagy on their own, independent of the presence of other viral proteins. Given the versatility of these viruses to adapt to and manipulate the metabolism, and thus to control the survival of, the infected cells, we need to understand much better how the specific viral proteins affect the pathways to apoptosis and autophagy. Only in this manner will we be able to minimize the pathology that they cause.  相似文献   

11.
DNA double-strand breaks (DSBs) are repaired by nonhomologous end-joining (NHEJ) and homologous recombination (HR). The NHEJ/HR decision is under complex regulation and involves DNA-dependent protein kinase (DNA-PKcs). HR is elevated in DNA-PKcs null cells, but suppressed by DNA-PKcs kinase inhibitors, suggesting that kinase-inactive DNA-PKcs (DNA-PKcs-KR) would suppress HR. Here we use a direct repeat assay to monitor HR repair of DSBs induced by I-SceI nuclease. Surprisingly, DSB-induced HR in DNA-PKcs-KR cells was 2- to 3-fold above the elevated HR level of DNA-PKcs null cells, and ~4- to 7-fold above cells expressing wild-type DNA-PKcs. The hyperrecombination in DNA-PKcs-KR cells compared to DNA-PKcs null cells was also apparent as increased resistance to DNA crosslinks induced by mitomycin C. ATM phosphorylates many HR proteins, and ATM is expressed at a low level in cells lacking DNA-PKcs, but restored to wild-type level in cells expressing DNA-PKcs-KR. Several clusters of phosphorylation sites in DNA-PKcs, including the T2609 cluster, which is phosphorylated by DNA-PKcs and ATM, regulate access of repair factors to broken ends. Our results indicate that ATM-dependent phosphorylation of DNA-PKcs-KR contributes to the hyperrecombination phenotype. Interestingly, DNA-PKcs null cells showed more persistent ionizing radiation-induced RAD51 foci (but lower HR levels) compared to DNA-PKcs-KR cells, consistent with HR completion requiring RAD51 turnover. ATM may promote RAD51 turnover, suggesting a second (not mutually exclusive) mechanism by which restored ATM contributes to hyperrecombination in DNA-PKcs-KR cells. We propose a model in which DNA-PKcs and ATM coordinately regulate DSB repair by NHEJ and HR.  相似文献   

12.
DNA non-homologous end joining (NHEJ) and homologous recombination (HR) function to repair DNA double-strand breaks (DSBs) in G2 phase with HR preferentially repairing heterochromatin-associated DSBs (HC-DSBs). Here, we examine the regulation of repair pathway usage at two-ended DSBs in G2. We identify the speed of DSB repair as a major component influencing repair pathway usage showing that DNA damage and chromatin complexity are factors influencing DSB repair rate and pathway choice. Loss of NHEJ proteins also slows DSB repair allowing increased resection. However, expression of an autophosphorylation-defective DNA-PKcs mutant, which binds DSBs but precludes the completion of NHEJ, dramatically reduces DSB end resection at all DSBs. In contrast, loss of HR does not impair repair by NHEJ although CtIP-dependent end resection precludes NHEJ usage. We propose that NHEJ initially attempts to repair DSBs and, if rapid rejoining does not ensue, then resection occurs promoting repair by HR. Finally, we identify novel roles for ATM in regulating DSB end resection; an indirect role in promoting KAP-1-dependent chromatin relaxation and a direct role in phosphorylating and activating CtIP.  相似文献   

13.
Non-homologous end-joining (NHEJ) and homologous recombination (HR) represent the two main pathways for repairing DNA double-strand breaks (DSBs). During the G2 phase of the mammalian cell cycle, both processes can operate and chromatin structure is one important factor which determines DSB repair pathway choice. ATM facilitates the repair of heterochromatic DSBs by phosphorylating and inactivating the heterochromatin building factor KAP-1, leading to local chromatin relaxation. Here, we show that ATM accumulation and activity is strongly diminished at DSBs undergoing end-resection during HR. Such DSBs remain unrepaired in cells devoid of the HR factors BRCA2, XRCC3 or RAD51. Strikingly, depletion of KAP-1 or expression of phospho-mimic KAP-1 allows repair of resected DSBs in the absence of BRCA2, XRCC3 or RAD51 by an erroneous PARP-dependent alt-NHEJ process. We suggest that DSBs in heterochromatin elicit initial local heterochromatin relaxation which is reversed during HR due to the release of ATM from resection break ends. The restored heterochromatic structure facilitates HR and prevents usage of error-prone alternative processes.  相似文献   

14.
Homologous recombination (HR) and non‐homologous end joining (NHEJ) represent distinct pathways for repairing DNA double‐strand breaks (DSBs). Previous work implicated Artemis and ATM in an NHEJ‐dependent process, which repairs a defined subset of radiation‐induced DSBs in G1‐phase. Here, we show that in G2, as in G1, NHEJ represents the major DSB‐repair pathway whereas HR is only essential for repair of ~15% of X‐ or γ‐ray‐induced DSBs. In addition to requiring the known HR proteins, Brca2, Rad51 and Rad54, repair of radiation‐induced DSBs by HR in G2 also involves Artemis and ATM suggesting that they promote NHEJ during G1 but HR during G2. The dependency for ATM for repair is relieved by depleting KAP‐1, providing evidence that HR in G2 repairs heterochromatin‐associated DSBs. Although not core HR proteins, ATM and Artemis are required for efficient formation of single‐stranded DNA and Rad51 foci at radiation‐induced DSBs in G2 with Artemis function requiring its endonuclease activity. We suggest that Artemis endonuclease removes lesions or secondary structures, which inhibit end resection and preclude the completion of HR or NHEJ.  相似文献   

15.
Repair of DNA double strand breaks (DSBs) plays a critical role in the maintenance of the genome. DSB arise frequently as a consequence of replication fork stalling and also due to the attack of exogenous agents. Repair of broken DNA is essential for survival. Two major pathways, homologous recombination (HR) and non-homologous end-joining (NHEJ) have evolved to deal with these lesions, and are conserved from yeast to vertebrates. Despite the conservation of these pathways, their relative contribution to DSB repair varies greatly between these two species. HR plays a dominant role in any DSB repair in yeast, whereas NHEJ significantly contributes to DSB repair in vertebrates. This active NHEJ requires a regulatory mechanism to choose HR or NHEJ in vertebrate cells. In this review, we illustrate how HR and NHEJ are differentially regulated depending on the phase of cell cycle and on the nature of the DSB.  相似文献   

16.
Goodarzi AA  Jeggo P  Lobrich M 《DNA Repair》2010,9(12):1273-1282
DNA non-homologous end-joining (NHEJ) and homologous recombination (HR) represent the major DNA double strand break (DSB) pathways in mammalian cells, whilst ataxia telangiectasia mutated (ATM) lies at the core of the DSB signalling response. ATM signalling plays a major role in modifying chromatin structure in the vicinity of the DSB and increasing evidence suggests that this function influences the DSB rejoining process. DSBs have long been known to be repaired with two (or more) component kinetics. The majority (~85%) of DSBs are repaired with fast kinetics in a predominantly ATM-independent manner. In contrast, ~15% of radiation-induced DSBs are repaired with markedly slower kinetics via a process that requires ATM and those mediator proteins, such as MDC1 or 53BP1, that accumulate at ionising radiation induced foci (IRIF). DSBs repaired with slow kinetics predominantly localise to the periphery of genomic heterochromatin (HC). Indeed, there is mounting evidence that chromatin complexity and not damage complexity confers slow DSB repair kinetics. ATM's role in HC-DSB repair involves the direct phosphorylation of KAP-1, a key HC formation factor. KAP-1 phosphorylation (pKAP-1) arises in both a pan-nuclear and a focal manner after radiation and ATM-dependent pKAP-1 is essential for DSB repair within HC regions. Mediator proteins such as 53BP1, which are also essential for HC-DSB repair, are expendable for pan-nuclear pKAP-1 whilst being essential for pKAP-1 formation at IRIF. Data suggests that the essential function of the mediator proteins is to promote the retention of activated ATM at DSBs, concentrating the phosphorylation of KAP-1 at HC DSBs. DSBs arising in G2 phase are also repaired with fast and slow kinetics but, in contrast to G0/G1 where they all DSBs are repaired by NHEJ, the slow component of DSB repair in G2 phase represents an HR process involving the Artemis endonuclease. Results suggest that whilst NHEJ repairs the majority of DSBs in G2 phase, Artemis-dependent HR uniquely repairs HC DSBs. Collectively, these recent studies highlight not only how chromatin complexity influences the factors required for DSB repair but also the pathway choice.  相似文献   

17.
DNA double-strand breaks (DSBs) are the major lethal lesion induced by ionizing radiation or by replication block. However, cells can take advantage of DSB-induced recombination in order to generate genetic diversity in physiological processes such as meiosis and V(D)J recombination. Two main alternative pathways compete for DSB repair: homologous recombination (HR) and non-homologous end-joining (NHEJ). This review will briefly present the mechanisms and the enzymatic complex for HR and NHEJ. The signalling of the DSB through the ATM pathway will be presented. Then, we will focus on the case of the RAD51 protein, which plays a pivotal role in HR and is conserved from bacteria to humans. Post-translational regulation of RAD51 is presented. Two contrasting situations are discussed: one with up-regulation (expression of the oncogene BCR/ABL) and one with a down-regulation (expression of the oncogene BCL-2) of RAD51, associated with apoptosis inhibition and tumour predisposition.  相似文献   

18.
Hyperthermia has a radiosensitizing effect, which is one of the most important biological bases for its use in cancer therapy with radiation. Although the mechanism of this effect has not been clarified in molecular terms, possible involvement of either one or both of two major DNA double-strand break (DSB) repair pathways, i.e. nonhomologous end joining (NHEJ) and homologous recombination (HR), has been speculated. To test this possibility, we examined cells of the chicken B-lymphocyte cell line DT40 and its derivatives lacking NHEJ and/or HR: KU70(-/-), DNA-PKcs(-/-/-), RAD54(-/-) and KU70(-/-)/RAD54(-/-). Radiosensitization by hyperthermia could be seen in all of the mutants, including KU70(-/-)/RAD54(-/-), which lacked both NHEJ and HR. Therefore, radiosensitization by hyperthermia cannot be explained simply by its inhibitory effects, if any, on NHEJ and/or HR alone. However, in NHEJ-defective KU70(-/-) and DNA-PKcs(-/-/-), consisting of two subpopulations with distinct radiosensitivity, the radiosensitive subpopulation, which is considered to be cells in G(1) and early S, was not sensitized. Substantial sensitization was seen only in the radioresistant subpopulation, which is considered to be cells in late S and G(2), capable of repairing DSBs through HR. This observation did not exclude possible involvement of NHEJ in G(1) and early S phase and also suggested inhibitory effects of hyperthermia on HR. Thus partial contribution of NHEJ and HR in radiosensitization by hyperthermia, especially that depending on the cell cycle stage, remains to be considered.  相似文献   

19.
Joyce EF  Paul A  Chen KE  Tanneti N  McKim KS 《Genetics》2012,191(3):739-746
Repair of meiotic double-strand breaks (DSBs) uses the homolog and recombination to yield crossovers while alternative pathways such as nonhomologous end joining (NHEJ) are suppressed. Our results indicate that NHEJ is blocked at two steps of DSB repair during meiotic prophase: first by the activity of the MCM-like protein MEI-218, which is required for crossover formation, and, second, by Rad51-related proteins SPN-B (XRCC3) and SPN-D (RAD51C), which physically interact and promote homologous recombination (HR). We further show that the MCM-like proteins also promote the activity of the DSB repair checkpoint pathway, indicating an early requirement for these proteins in DSB processing. We propose that when a meiotic DSB is formed in the absence of both MEI-218 and SPN-B or SPN-D, a DSB substrate is generated that can enter the NHEJ repair pathway. Indeed, due to its high error rate, multiple barriers may have evolved to prevent NHEJ activity during meiosis.  相似文献   

20.
DNA-dependent protein kinase (DNA-PK), composed of Ku70, Ku80, and the catalytic subunit (DNA-PKcs), is involved in double-strand break (DSB) repair by non-homologous end joining (NHEJ). DNA-PKcs defects confer ionizing radiation sensitivity and increase homologous recombination (HR). Increased HR is consistent with passive shunting of DSBs from NHEJ to HR. We therefore predicted that inhibiting the DNA-PKcs kinase would increase HR. A novel DNA-PKcs inhibitor (1-(2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone; designated IC86621) increased ionizing radiation sensitivity but surprisingly decreased spontaneous and DSB-induced HR. Wortmannin also inhibits DNA-PKcs and reduces DSB-induced HR. IC86621 did not affect HR product outcome, indicating that it affects HR initiation. Thus, HR is increased in the absence of DNA-PKcs, but decreased when DNA-PKcs is catalytically inactive, suggesting interactive competition between HR and NHEJ. The effects of IC86621 and wortmannin were proportional to the level of DNA-PKcs, consistent with inhibited DNA-PKcs acting in a dominant negative manner. We propose that inhibition of DNA-PKcs blocks its autophosphorylation, prevents dissociation of DNA-PKcs from DNA ends, and thereby blocks both HR and NHEJ. By blocking the two major DSB repair pathways, DNA-PKcs inhibitors should radiosensitize at all cell-cycle stages and are therefore excellent candidates for augmenting cancer radiotherapy.  相似文献   

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