Chromosomal double-strand breaks (DSBs) in mammalian cells are repaired by either homology-directed repair (HDR), using a homologous sequence as a repair template, or nonhomologous end-joining (NHEJ), which often involves sequence alterations at the DSB site. To characterize the interrelationship of these two pathways, we analyzed HDR of a DSB in cells deficient for NHEJ components. We find that the HDR frequency is enhanced in Ku70 Double-strand breaks (DSBs) are potentially catastrophic lesions that if not repaired will lead to loss of genetic information and mutagenesis or cell death. In mammalian cells, two major pathways exist to repair DSBshomologous recombination and nonhomologous endjoining (NHEJ;Liang et al. 1998). NHEJ, the rejoining of DNA ends with the use of little or no sequence homology, involves the processing of ends such that nucleotides are often deleted or inserted at the break site prior to ligation (Jeggo 1998). Such modifications are likely central to the ability of mammalian cells to rejoin DNA ends with a variety of structures. Homology-directed repair (HDR) of a DSB, in contrast, requires significant lengths of sequence homology so that a DNA end from one molecule can invade a homologous sequence and prime repair synthesis (Pâques and Haber 1999). Processing of DNA ends also occurs with HDR; however, repair is typically precise, because a homologous sequence templates the repair event.Several processes exist in which repair of a DSB is restricted to either NHEJ or HDR. For example, DSBs introduced by the RAG proteins to generate antigen receptor diversity during V(D)J rearrangement are repaired by the NHEJ pathway (Jeggo 1998), whereas those introduced during meiosis by the Spo11 protein are repaired by the HDR pathway (Keeney 2001). The restriction in type of DSB repair raises the question as to how pathway choice is regulated. Several studies point to cell cycle phase as one factor that modulates repair pathway choice. In chicken cells, HDR has been found to play a dominant role in repairing radiation-induced DSBs in late S/G 2 phase, whereas NHEJ is preferentially used during G 1 /early S phase (Takata et al. 1998). Consistent with this in mammalian cells, the preferred homologous template for HDR-the sister chromatid (Johnson and Jasin 2001)-is present only during the S/G 2 phase of the cell cycle. Despite a cell cycle preference, HDR and NHEJ can nevertheless be coupled for the repair of a single DSB in mammalian cells (Richardson and Jasin 2000), indicating that the two repair pathways are not completely restricted to different cell cycle phases and that other factors influence pathway choice.Based on in vitro studies of DNA end-binding proteins such as RAD52, it has been suggested that end-binding proteins may direct entry into alternative DSB repair pathways (Van Dyck et al. 1999). However, no evidence yet exists in vivo to support this model, and mutation of the Rad52 gene in mouse does not confer a cellular DSB repair phenotype (Rijkers et al. 1998). NHEJ proteins, that is, th...

Mammalian cells have been presumed to repair potentially lethal chromosomal double-strand breaks (DSBs) in large part by processes that do not require homology to the break site. This contrasts with Saccharomyces cerevisiae where the major DSB repair pathway is homologous recombination. Recently, it has been determined that DSBs in genomic DNA in mammalian cells can stimulate homologous recombination as much as 3 or 4 orders of magnitude, suggesting that homology-directed repair may play an important role in the repair of chromosomal breaks. To determine whether mammalian cells use recombinational repair at a significant level, we have analyzed the spectrum of repair events at a defined chromosomal break by using direct physical analysis of repair products. When an endonucleasegenerated DSB is introduced into one of two direct repeats, homologous repair is found to account for 30-50% of observed repair events. Both noncrossover and deletional homologous repair products are detected, at approximately a 1:3 ratio. These results demonstrate the importance of homologous recombination in the repair of DSBs in mammalian cells. In the remaining observed repair events, DSBs are repaired by nonhomologous processes. The nonhomologous repair events generally result in small deletions or insertions at the break site, although a small fraction of events result in larger chromosomal rearrangements. Interestingly, in two insertions, GT repeats were integrated at one of the broken chromosome ends, suggesting that DSB repair can contribute to the spread of microsatellite sequences in mammalian genomes.

The BRCA2 tumor suppressor plays an important role in the repair of DNA damage by homologous recombination, also termed homology-directed repair (HDR). Human BRCA2 is 3,418 aa and is composed of several domains. The central part of the protein contains multiple copies of a motif that binds the Rad51 recombinase (the BRC repeat), and the C terminus contains domains that have structural similarity to domains in the ssDNA-binding protein replication protein A (RPA). To gain insight into the role of BRCA2 in the repair of DNA damage, we fused a single (BRC3, BRC4) or multiple BRC motifs to the large RPA subunit. Expression of any of these protein fusions in Brca2 mutant cells substantially improved HDR while suppressing mutagenic repair. A fusion containing a Rad52 ssDNA-binding domain also was active in HDR. Mutations that reduced ssDNA or Rad51 binding impaired the ability of the fusion proteins to function in HDR. The high level of spontaneous chromosomal aberrations in Brca2 mutant cells was largely suppressed by the BRC-RPA fusion proteins, supporting the notion that the primary role of BRCA2 in maintaining genomic integrity is in HDR, specifically to deliver Rad51 to ssDNA. The fusion proteins also restored Rad51 focus formation and cellular survival in response to DNA damaging agents. Because as little as 2% of BRCA2 fused to RPA is sufficient to suppress cellular defects found in Brca2-mutant mammalian cells, these results provide insight into the recently discovered diversity of BRCA2 domain structures in different organisms.double-strand break ͉ mammalian cells ͉ Rad51 ͉ homologous recombination ͉ BRC repeat

Non-homologous end joining (NHEJ) and homologous recombination (HR) are two alternative/competitor pathways for the repair of DNA double-strand breaks (DSBs). To gain further insights into the regulation of DSB repair, we detail here the different HR pathways affected by (i) the inactivation of DNA-PK activity, by treatment with Wortmannin, and (ii) a mutation in the xrcc4 gene, involved in a late NHEJ step, using the XR-1 cell line. Here we have analyzed not only the impact of NHEJ inactivation on recombination induced by a single DSB targeted to the recombination substrate (using I-SceI endonuclease) but also on gamma-ray- and UV-C-induced and spontaneous recombination and finally on Rad51 foci formation, i.e. on the assembly of the homologous recombination complex, at the molecular level. The results presented here show that in contrast to embryonic stem cells, the xrcc4 mutation strongly stimulates I-SceI-induced HR in adult hamster cells. More precisely, we show here that both single strand annealing and gene conversion are stimulated. In contrast, Wortmannin does not affect I-SceI-induced HR. In addition, gamma-ray-induced recombination is stimulated by both xrcc4 mutation and Wortmannin treatment in an epistatic-like manner. In contrast, neither spontaneous nor UV-C-induced recombination was affected by xrcc4 mutation, showing that the channeling from NHEJ to HR is specific to DSBs. Finally, we show here that xrcc4 mutation or Wortmannin treatment results in a stimulation of Rad51 foci assembly, thus that a late NHEJ step is able to affect Rad51 recombination complex assembly. The present data suggest a model according to which NHEJ and HR do not simply compete for DSB repair but can act sequentially: a defect in a late NHEJ step is not a dead end and can make DSB available for subsequent Rad51 recombination complex assembly.