Transient Stability of DNA Ends Allows Nonhomologous End Joining to Precede Homologous Recombination

Frank-Vaillant, Marie; Marcand, Stéphane

doi:10.1016/s1097-2765(02)00705-0

Cited by 174 publications

(137 citation statements)

References 72 publications

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“…Thus, canonical NHEJ is not intrinsically an error-prone process but a process that is as conservative as possible, wherein the occurrence of mutations depends on the structure of the DNA ends rather than on the accuracy of the NHEJ machinery itself. This model is consistent with results obtained in yeast (32). Even when ends are not fully complementary, instead of impairing end-joining completely, NHEJ permits approximate joining of the ends at the cost of limited mutagenesis at the junctions but protects against extensive nonconservative degradation.…”

Section: Discussionsupporting

confidence: 90%

Defects in XRCC4 and KU80 differentially affect the joining of distal nonhomologous ends

Guirouilh‐Barbat

Rass

Plo

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

152

183

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XRCC4-null mice have a more severe phenotype than KU80-null mice. Here, we address whether this difference in phenotype is connected to nonhomologous end-joining (NHEJ). We used intrachromosomal substrates to monitor NHEJ of two distal doublestrand breaks (DSBs) targeted by I-SceI, in living cells. In xrcc4-defective XR-1 cells, a residual but significant end-joining process exists, which primarily uses microhomologies distal from the DSB. However, NHEJ efficiency was strongly reduced in xrcc4-defective XR-1 cells versus complemented cells, contrasting with KU-deficient xrs6 cells, which showed levels of end-joining similar to those of complemented cells. Nevertheless, sequence analysis of the repair junctions indicated that the accuracy of end-joining was strongly affected in both xrcc4-deficient and KU-deficient cells. More specifically, these data showed that the KU80/XRCC4 pathway is conservative and not intrinsically error-prone but can accommodate non-fully complementary ends at the cost of limited mutagenesis.double-strand break repair ͉ genome rearrangements D NA double-strand breaks (DSBs) are harmful lesions generated by a variety of endogenous or exogenous stresses, potentially leading to genomic rearrangement. Nonhomologous endjoining (NHEJ) is a prominent pathway for DSB repair (1). Canonical NHEJ involves the successive intervention of the KU80-KU70 heterodimer, DNA-PKcs-Artemis, and, finally, ligase IV (Lig4) associated with its cofactors XRCC4 and Cernunnos/Xlf (2, 3). KU-independent NHEJ (KU-alt) has been described in vitro in both acellular extracts and cultured cells (1, 4-6).Alternative, XRCC4-independent DSB repair pathways (XRCC4-alt) have also been described, using episomic plasmid in cultured cells, using pulse field gel electrophoresis, or in in vitro biochemical experiments (5-10). One hypothesis could propose that XRCC4 and KU are implicated in the same canonical NHEJ pathway, whereas the alternate pathway is independent of both KU and XRCC4. However, in transgenic mice, the inactivation of XRCC4 or Lig4 results in a more severe phenotype than the inactivation of KU (11), suggesting that XRCC4 might have an additional essential function; however, studies show that XRCC4 and Lig4 do not have roles outside of NHEJ, whereas in contrast, KU acts in other processes such as transcription, apoptosis, and responses to the cell microenvironment (12)(13)(14).Alternatively, these varying phenotypes in mice may actually result from differences in DSB repair efficiencies, indicating that defects in XRCC4 might be more deleterious for DSB repair than defects in KU. What challenges this hypothesis, however, is that substantial class switch recombination (CSR) has recently been shown to occur in mouse B cells without XRCC4, whereas no CSR was recorded in cells devoid of KU (15-18).The relative contributions of XRCC4 and KU80 versus the XRCC4-alt and KU-alt pathways, respectively, to DSB repair remain unclear in wild-type cells. Contrasting results were obtained in living cells, using an episomic plasm...

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Section: Discussionsupporting

confidence: 90%

Defects in XRCC4 and KU80 differentially affect the joining of distal nonhomologous ends

Guirouilh‐Barbat

Rass

Plo

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

152

183

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“…2f). These results support and extend previous suggestions 18,19 that the higher stability of DSB ends in G1-arrested cells stimulates NHEJ,…”

Section: Hhs Public Accesssupporting

confidence: 92%

“…2f). These results support and extend previous suggestions 18,19 that the higher stability of DSB ends in G1-arrested cells stimulates NHEJ, which we propose might result from a lack of resection of these ends. In addition, NHEJ is probably aided by the retention of the end-joining protein Mre11 at DSB ends (Fig.…”

supporting

confidence: 93%

DNA end resection, homologous recombination and DNA damage checkpoint activation require CDK1

Ira

Pellicioli

Balijja

et al. 2004

Nature

656

684

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A single double-strand break (DSB) induced by HO endonuclease triggers both repair by homologous recombination and activation of the Mec1-dependent DNA damage checkpoint in budding yeast [1][2][3][4][5][6] . Here we report that DNA damage checkpoint activation by a DSB requires the cyclin-dependent kinase CDK1 (Cdc28) in budding yeast. CDK1 is also required for DSB-induced homologous recombination at any cell cycle stage. Inhibition of homologous recombination by using an analogue-sensitive CDK1 protein 7,8 results in a compensatory increase in nonhomologous end joining. CDK1 is required for efficient 5′ to 3′ resection of DSB ends and for the recruitment of both the single-stranded DNA-binding complex, RPA, and the Rad51 recombination protein. In contrast, Mre11 protein, part of the MRX complex, accumulates at unresected DSB ends. CDK1 is not required when the DNA damage checkpoint is initiated by lesions that are processed by nucleotide excision repair. Maintenance of the DSB-induced checkpoint requires continuing CDK1 activity that ensures continuing end resection. CDK1 is also important for a later step in homologous recombination, after strand invasion and before the initiation of new DNA synthesis.In budding yeast, a chromosomal DSB created by HO endonuclease has been used both to study the kinetics and efficiency of DSB repair and to analyse the induction of the DNA damage checkpoint dependent on Mec1 (an ATR homologue). In cells carrying HML or HMR mating-type switching donor sequences, a DSB at the MAT locus is efficiently repaired by gene conversion. In strains lacking donor sequences, induction of an unrepairable DSB causes arrest of cell cycle progression before anaphase 1,2 . In bothCorrespondence and requests for materials should be addressed to M.F. (marco.foiani@ifom-ieo-campus.it) or J.E.H. † Present address: Rockefeller University, 1230 York Avenue, New York, New York 10021-6399, USA. ★ These authors contributed equally to this work Supplementary Information accompanies the paper on www.nature.com/nature. Competing interests statementThe authors declare that they have no competing financial interests. instances, a key step is the 5′ to 3′ resection of DSB ends to produce single-stranded DNA (ssDNA), which is bound by the RPA complex. RPA binding is essential both for association of Mec1 checkpoint kinase 9 and for loading of Rad51 recombination protein 6 . HHS Public AccessActivation of the Mec1-dependent DNA damage checkpoint after a DSB is regulated by the cell cycle 3 , with no activation in G1-arrested cells. A DSB induced in cells that have been arrested in G1, and then released into S phase, results in hyperphosphorylation of the Mec1 target Rad53 after the completion of S phase, in G2 ( Supplementary Fig. S1a). To test whether the checkpoint depends on the activity of cyclin-dependent kinases, we inactivated CDK1 in nocodazole-blocked G2 cells. We overexpressed the CDK1/Clb inhibitor, Sic1 (ref. 10), in G2 cells at the same time that an unrepairable DSB was induced at MAT. CDK1 i...

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“…In vivo analysis of the recruitment of DNA repair proteins at the sites of laser-induced DNA lesions shows a transient assembly of NHEJ repair factors, which precedes a prolonged occupancy by HR factors, such as RAD51 [33]. These and other authors conclude that NHEJ and HR are not competing pathways, NHEJ being an immediate early repair pathway that precedes a more prolonged attempt to repair persistent DNA lesions by HR [33,34]. Thus, in both external and replication-induced DSBs, the cytologically visible stabilization of damaged-induced ssDNA by RAD51 polymerization is considered a marker of HR activity, even if the strand invasion process and the completion of DNA repair do not occur until a homologous template is available.…”

Section: Introductionmentioning

confidence: 76%

Homologous Recombination Conserves DNA Sequence Integrity Throughout the Cell Cycle in Embryonic Stem Cells

Serrano

Liang

Chang

et al. 2011

Stem Cells and Development

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Transient Stability of DNA Ends Allows Nonhomologous End Joining to Precede Homologous Recombination

Cited by 174 publications

References 72 publications

Defects in XRCC4 and KU80 differentially affect the joining of distal nonhomologous ends

Defects in XRCC4 and KU80 differentially affect the joining of distal nonhomologous ends

DNA end resection, homologous recombination and DNA damage checkpoint activation require CDK1

Homologous Recombination Conserves DNA Sequence Integrity Throughout the Cell Cycle in Embryonic Stem Cells

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