Homology-directed repair of DNA damage has recently emerged as a major mechanism for the maintenance of genomic integrity in mammalian cells. The highly conserved strand transferase, Rad51, is expected to be critical for this process. XRCC3 possesses a limited sequence similarity to Rad51 and interacts with it. Using a novel fluorescence-based assay, we demonstrate here that error-free homology-directed repair of DNA double-strand breaks is decreased 25-fold in an XRCC3-deficient hamster cell line and can be restored to wild-type levels through XRCC3 expression. These results establish that XRCC3-mediated homologous recombination can reverse DNA damage that would otherwise be mutagenic or lethal.

The BRCA2 tumor suppressor has been implicated in the maintenance of chromosomal stability through a function in DNA repair. In this report, we examine human and mouse cell lines containing different BRCA2 mutations for their ability to repair chromosomal breaks by homologous recombination. Using the I-SceI endonuclease to introduce a double-strand break at a specific chromosomal locus, we find that BRCA2 mutant cell lines are recombination deficient, such that homology-directed repair is reduced 6- to >100-fold, depending on the cell line. Thus, BRCA2 is essential for efficient homology-directed repair, presumably in conjunction with the Rad51 recombinase. We propose that impaired homology-directed repair caused by BRCA2 deficiency leads to chromosomal instability and, possibly, tumorigenesis, through lack of repair or misrepair of DNA damage.

Repair of chromosomal breaks is essential for cellular viability, but misrepair generates mutations and gross chromosomal rearrangements. We investigated the interrelationship between two homologous-repair pathways, i.e., mutagenic single-strand annealing (SSA) and precise homology-directed repair (HDR). For this, we analyzed the efficiency of repair in mammalian cells in which double-strand break (DSB) repair components were disrupted. We observed an inverse relationship between HDR and SSA when RAD51 or BRCA2 was impaired, i.e., HDR was reduced but SSA was increased. In particular, expression of an ATP-binding mutant of RAD51 led to a >90-fold shift to mutagenic SSA repair. Additionally, we found that expression of an ATP hydrolysis mutant of RAD51 resulted in more extensive gene conversion, which increases genetic loss during HDR. Disruption of two other DSB repair components affected both SSA and HDR, but in opposite directions: SSA and HDR were reduced by mutation of Brca1, which, like Brca2, predisposes to breast cancer, whereas SSA and HDR were increased by Ku70 mutation, which affects nonhomologous end joining. Disruption of the BRCA1-associated protein BARD1 had effects similar to those of mutation of BRCA1. Thus, BRCA1/BARD1 has a role in homologous repair before the branch point of HDR and SSA. Interestingly, we found that Ku70 mutation partially suppresses the homologous-repair defects of BARD1 disruption. We also examined the role of RAD52 in homologous repair. In contrast to yeast, Rad52 ؊/؊ mouse cells had no detectable HDR defect, although SSA was decreased. These results imply that the proper genetic interplay of repair factors is essential to limit the mutagenic potential of DSB repair.

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...

RAD51 is one of six mitotic human homologs of the E. coli RecA protein (RAD51-Paralogs) that play a central role in homologous recombination and repair of DNA double-strand breaks (DSBs). Here we demonstrate that RAD51 is important for resistance to cisplatin and mitomycin C in cells expressing the BCR/ABL oncogenic tyrosine kinase. BCR/ABL significantly enhances the expression of RAD51 and several RAD51-Paralogs. RAD51 overexpression is mediated by a STAT5-dependent transcription as well as by inhibition of caspase-3-dependent cleavage. Phosphorylation of the RAD51 Tyr-315 residue by BCR/ABL appears essential for enhanced DSB repair and drug resistance. Induction of the mammalian RecA homologs establishes a unique mechanism for DNA damage resistance in mammalian cells transformed by an oncogenic tyrosine kinase.

Fanconi anemia (FA) is a recessive disorder characterized by congenital abnormalities, progressive bone-marrow failure, and cancer susceptibility. Cells from FA patients are hypersensitive to agents that produce DNA crosslinks and, after treatment with these agents, have pronounced chromosome breakage and other cytogenetic abnormalities. Eight FANC genes have been cloned, and the encoded proteins interact in a common cellular pathway. DNA-damaging agents activate the monoubiquitination of FANCD2, resulting in its targeting to nuclear foci that also contain BRCA1 and BRCA2͞FANCD1, proteins involved in homologydirected DNA repair. Given the interaction of the FANC proteins with BRCA1 and BRCA2, we tested whether cells from FA patients (groups A, G, and D2) and mouse Fanca ؊/؊ cells with a targeted mutation are impaired for this repair pathway. We find that both the upstream (FANCA and FANCG) and downstream (FANCD2) FA pathway components promote homology-directed repair of chromosomal double-strand breaks (DSBs). The FANCD2 monoubiquitination site is critical for normal levels of repair, whereas the ATM phosphorylation site is not. The defect in these cells, however, is mild, differentiating them from BRCA1 and BRCA2 mutant cells. Surprisingly, we provide evidence that these proteins, like BRCA1 but unlike BRCA2, promote a second DSB repair pathway involving homology, i.e., single-strand annealing. These results suggest an early role for the FANC proteins in homologous DSB repair pathway choice.double-strand break repair ͉ FANC ͉ homologous recombination ͉ mammalian cells

Decreased BRCA1 expression in the absence of genetic mutation is observed frequently in sporadic cancers of the breast and other sites, although little is known regarding the mechanisms by which the expression of this gene can be repressed. Here, we show that activating and repressive E2Fs simultaneously bind the BRCA1 promoter at two adjacent E2F sites in vivo, and that hypoxia induces a dynamic redistribution of promoter occupancy by these factors resulting in the transcriptional repression of BRCA1 expression. Functionally, we show that hypoxia is associated with impaired homologous recombination, whereas the nonhomologous end-joining (NHEJ) repair pathway is unaffected under these conditions. Repression of BRCA1 expression by hypoxia represents an intriguing mechanism of functional BRCA1 inactivation in the absence of genetic mutation. We propose that hypoxiainduced decreases in BRCA1 expression and consequent suppression of homologous recombination may lead to genetic instability by shifting the balance between the highfidelity homologous recombination pathway and the errorprone NHEJ pathway of DNA repair. Furthermore, these findings provide a novel link between E2Fs and the transcriptional response to hypoxia and provide insight into the mechanisms by which the tumor microenvironment can contribute to genetic instability in cancer. (Cancer Res 2005; 65(24): 11597-604)

SUMMARY FANCM is a Fanconi anemia nuclear core complex protein required for the functional integrity of the FANC-BRCA pathway of DNA damage response and repair. Here we report the isolation and characterization of two histone-fold-containing FANCM-associated proteins, MHF1 and MHF2. We show that suppression of MHF1 expression results in 1) destabilization of FANCM and MHF2, 2) impairment of DNA damage-induced monoubiquitination and foci formation of FANCD2, 3) defective chromatin localization of FA nuclear core complex proteins, 4) elevated MMC-induced chromosome aberrations, and 5) sensitivity to MMC and camptothecin. We also provide biochemical evidence that MHF1 and MHF2 assemble into a heterodimer that binds DNA and enhances the DNA branch migration activity of FANCM. These findings reveal critical roles of the MHF1-MHF2 dimer in DNA damage repair and genome maintenance through FANCM.