Recent studies show overlap between Fanconi anemia (FA) proteins and those involved in DNA repair mediated by homologous recombination (HR). However, the mechanism by which FA proteins affect HR is unclear. FA proteins (FancA/C/E/F/G/L) form a multiprotein complex, which is responsible for DNA damageinduced FancD2 monoubiquitination, a key event for cellular resistance to DNA damage. Here, we show that FANCD2-disrupted DT40 chicken B-cell line is defective in HR-mediated DNA double-strand break (DSB) repair, as well as gene conversion at the immunoglobulin light-chain locus, an event also mediated by HR. Gene conversions occurring in mutant cells were associated with decreased nontemplated mutations. In contrast to these defects, we also found increased spontaneous sister chromatid exchange (SCE) and intact Rad51 foci formation after DNA damage. Thus, we propose that FancD2 promotes a subpathway of HR that normally mediates gene conversion by a mechanism that avoids crossing over and hence SCEs.

The yeast SNM1/PSO2 gene specifically functions in DNA interstrand cross-link (ICL) repair, and its role has been suggested to be separate from other DNA repair pathways. In vertebrates, there are three homologs of SNM1 (SNM1A, SNM1B, and SNM1C/Artemis; SNM1 family proteins) whose functions are largely unknown. We disrupted each of the SNM1 family genes in the chicken B-cell line DT40. Both SNM1A-and SNM1B-deficient cells were sensitive to cisplatin but not to X-rays, whereas SNM1C/Artemis-deficient cells exhibited sensitivity to X-rays but not to cisplatin. SNM1A was nonepistatic with XRCC3 (homologous recombination), RAD18 (translesion synthesis), FANCC (Fanconi anemia), and SNM1B in ICL repair. SNM1A protein formed punctate nuclear foci depending on the conserved SNM1 (metallo-␤-lactamase) domain. PIAS1 was found to physically interact with SNM1A, and they colocalized at nuclear foci. Point mutations in the SNM1 domain, which disrupted the interaction with PIAS1, led to mislocalization of SNM1A in the nucleus and loss of complementation of snm1a cells. These results suggest that interaction between SNM1A and PIAS1 is required for ICL repair.DNA interstrand cross-links (ICLs) covalently bind the two complementary strands of the double helix of DNA. They severely impair fundamental processes of DNA metabolism such as transcription or replication, leading to cell death if they are left unrepaired. Current protocols for cancer chemotherapy often include ICL-inducing agents, i.e., mitomycin C (MMC) or cisplatin, for effective killing of malignant cells (reviewed in references 7 and 21).The molecular mechanism of ICL repair is still poorly understood. In the yeast Saccharomyces cerevisiae, three distinct pathways, including nucleotide excision repair (NER), homologous recombination (HR), and translesion synthesis (TLS), participate in ICL repair (7,20). In addition, yeast SNM1 (also known as PSO2) specifically functions in ICL repair without apparently participating directly in any of these pathways (2, 9). During the repair process, NER mediates incisions on both sides of the cross-linked DNA (sometimes termed "unhooking") (5), and double-strand breaks (DSBs), which are probably associated with the collapse of a replication fork, are formed. The DSBs are then repaired by HR mechanisms. The interdependence between unhooking and formation of DSBs remains unclear (7). Yeast snm1 mutants are proficient in unhooking but are unable to process DSB intermediates (7,19,40).In mammalian cells, the situation is even more complex.Among NER factors, XPF/ERCC1 endonuclease appears to be particularly important for the unhooking of ICLs (5,21,26,32). Hamster mutant cells lacking the RAD51 paralog XRCC2 or XRCC3 display extreme sensitivity to ICLs, indicating an important role of HR in mammalian ICL repair (17). Cells from Fanconi anemia (FA) patients are also highly sensitive to ICL reagents (33), but the role of FA proteins in ICL repair is still unclear (4). Furthermore, there are three homologs of SNM1 (referred to as SNM1A, S...

Recent studies implicate the transcription factor E2A in Ig diversification such as somatic hypermutation or gene conversion (GCV). GCV also requires active Ig transcription, expression of the activation-induced deaminase (AID) and a set of homologous recombination factors. We have disrupted the E2A gene in the chicken B-cell line DT40 and found greatly diminished rate of GCV without changes in the levels of transcripts from AID and Ig heavy chain or Ig light chain (IgL) genes. However, chromatin immunoprecipitation analysis revealed that the loss of E2A accompanies drastically reduced acetylation levels of the histone H4 in rearranged IgL locus. Furthermore, the defects in GCV were restored by trichostatin A treatment, which raised H4 acetylation to the normal levels. Thus, E2A may contribute to GCV by maintaining histone acetylation, which could be a prerequisite for targeting or full deaminase function of AID.