The E3 ubiquitin ligase RNF20 regulates chromatin structure by monoubiquitinating histone H2B in transcription. Here, we show that RNF20 is localized to double-stranded DNA breaks (DSBs) independently of H2AX and is required for the DSB-induced H2B ubiquitination. In addition, RNF20 is required for the methylation of H3K4 at DSBs and the recruitment of the chromatin-remodeling factor SNF2h. Depletion of RNF20, depletion of SNF2h, or expression of the H2B mutant lacking the ubiquitination site (K120R) compromises resection of DNA ends and recruitment of RAD51 and BRCA1. Consequently, cells lacking RNF20 or SNF2h and cells expressing H2B K120R exhibit pronounced defects in homologous recombination repair (HRR) and enhanced sensitivity to radiation. Finally, the function of RNF20 in HRR can be partially bypassed by forced chromatin relaxation. Thus, the RNF20-mediated H2B ubiquitination at DSBs plays a critical role in HRR through chromatin remodeling.

DNA double-strand breaks represent the most potentially serious damage to a genome; hence, many repair proteins are recruited to nuclear damage sites by as yet poorly characterized sensor mechanisms. Here, we show that NBS1, the gene product defective in Nijmegen breakage syndrome (NBS), physically interacts with histone, rather than damaged DNA, by direct binding to gamma-H2AX. We also demonstrate that NBS1 binding can occur in the absence of interaction with hMRE11 or BRCA1. Furthermore, this NBS1 physical interaction was reduced when anti-gamma-H2AX antibody was introduced into normal cells and was also delayed in AT cells, which lack the kinase activity for phosphorylation of H2AX. NBS1 has no DNA binding region but carries a combination of the fork-head associated (FHA) and the BRCA1 C-terminal domains (BRCT). We show that the FHA/BRCT domain of NBS1 is essential for this physical interaction, since NBS1 lacking this domain failed to bind to gamma-H2AX in cells, and a recombinant FHA/BRCT domain alone can bind to recombinant gamma-H2AX. Consequently, the FHA/BRCT domain is likely to have a crucial role for both binding to histone and for relocalization of hMRE11/hRAD50 nuclease complex to the vicinity of DNA damage.

Double-strand breaks occur during DNA replication and are also induced by ionizing radiation. There are at least two pathways which can repair such breaks: non-homologous end joining and homologous recombination (HR). Although these pathways are essentially independent of one another, it is possible that the proteins Mre11, Rad50 and Xrs2 are involved in both pathways in Saccharomyces cerevisiae. In vertebrate cells, little is known about the exact function of the Mre11-Rad50-Nbs1 complex in the repair of double-strand breaks because Mre11- and Rad50-null mutations are lethal. Here we show that Nbs1 is essential for HR-mediated repair in higher vertebrate cells. The disruption of Nbs1 reduces gene conversion and sister chromatid exchanges, similar to other HR-deficient mutants. In fact, a site-specific double-strand break repair assay showed a notable reduction of HR events following generation of such breaks in Nbs1-disrupted cells. The rare recombinants observed in the Nbs1-disrupted cells were frequently found to have aberrant structures, which possibly arise from unusual crossover events, suggesting that the Nbs1 complex might be required to process recombination intermediates.

DNA-protein cross-links (DPCsaccount for a class of the most ubiquitous DNA lesions and are known to be produced by chemical agents, such as formaldehyde (FA) and transition metals, and by physical agents, such as ionizing radiation and UV light (1). DPCs are also produced by anticancer drugs, such as 5-aza-2Ј-deoxycytidine (azadC) and cisplatin (1, 2). Although some classes of DPCs contain a flanking strand break (e.g. covalently trapped topoisomerases) (3), typical (and probably the most common) DPCs contain proteins irreversibly trapped on the uninterrupted DNA strand. It is readily inferred from the unusually bulky nature of crosslinked proteins (CLPs) that steric hindrance imposed by CLPs on proteins involved in DNA transactions would hamper replication, transcription, and repair. Consistent with this notion, DPCs incorporated into oligonucleotides and plasmid DNA block DNA replication in vitro (4, 5) and in vivo (6, 7), respectively. Moreover, CLPs attenuate the binding of the damage recognition protein (UvrB) involved in bacterial nucleotide excision repair (NER) in a size-dependent manner (7).Conversely, it has been largely elusive how cells circumvent the genotoxic effects of DPCs. We recently showed that NER and homologous recombination (HR) play pivotal roles in mitigating the genotoxic effects of DPCs in bacteria (7). Interestingly, the two pathways contribute differentially to the tolerance of DPCs. In NER catalyzed by UvrABC, the excision efficiency for DPCs varies dramatically with the size of CLPs both in vitro and in vivo and is attenuated by steric hindrance of CLPs. The upper size limit of CLPs amenable to NER in vitro was around 16 kDa, but the biologically relevant size limit was lower in vivo, at around 11 kDa. DPCs with oversized CLPs are processed exclusively by RecB-dependent HR. Given that HR * This work was supported in part by Grants-in-aid for Scientific Research from the Japan Society for the Promotion of Science (to T. N., H. T., and H. I.) and by a Grant-in-aid for the Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology (to H. I.

Nijmegen breakage syndrome (NBS), also known as ataxia-telangiectasia (AT) variant, is an autosomal recessive disorder characterized by microcephaly, growth retardation, severe combined immunodeficiency and a high incidence of lymphoid cancers. Cells from NBS patients display chromosome instability, hypersensitivity to ionizing radiation and abnormal cell-cycle regulation after irradiation, all of which are characteristics shared with AT. Recently, the NBS locus was mapped at 8q21 by two independent approaches, complementation studies and linkage analysis. Here, we report the positional cloning of the NBS gene, NBS1, from an 800-kb candidate region. The gene comprises 50 kb and encodes a protein of 754 amino acids. The amino-terminal region of the protein shows weak homology to the yeast XRS2, MEK1, CDS1 and SPK1 proteins. The gene is expressed at high levels in the testes, suggesting that it might be involved in meiotic recombination. We detected the same 5-bp deletion in 13 individuals, and conclude that it is likely to be a founder mutation.