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.

Long interspersed nucleotide element-1 (L1) is a retroelement comprising about 17% of the human genome, of which 80-100 copies are competent as mobile elements (retrotransposition: L1-RTP). Although the genetic structures modified during L1-RTP have been clarified, little is known about the cellular signaling cascades involved. Herein we found that 6-formylindolo[3,2-b]carbazole (FICZ), a tryptophan photoproduct postulated as a candidate physiological ligand of the aryl hydrocarbon receptor (AhR), induces L1-RTP. Notably, RNA-interference experiments combined with back-transfection of siRNA-resistant cDNAs revealed that the induction of L1-RTP by FICZ is dependent on AhR nuclear translocator-1 (ARNT1), a binding partner of AhR, and the activation of cAMP-responsive elementbinding protein. However, our extensive analyses suggested that AhR is not required for L1-RTP. FICZ stimulated the interaction of the L1-encoded open reading frame-1 (ORF1) and ARNT1, and recruited ORF1 to chromatin in a manner dependent on the activation of mitogen-activated protein kinase. Along with our additional observations that the cellular cascades for FICZ-induced L1-RTP were different from those of L1-RTP triggered by DNA damage, we propose that the presence of the cellular machinery of ARNT1 mediates L1-RTP. A possible role of ARNT1-mediated L1-RTP in the adaptation of living organisms to environmental changes is discussed.

The proteins responsible for radiation sensitive disorders, NBS1, kinase ataxia-telangiectasia-(A-T)-mutated (ATM) and MRE11, interact through the C-terminus of NBS1 in response to the generation of DNA double-strand breaks (DSBs) and are all implicated in checkpoint regulation and DSB repair, such as homologous recombination (HR). We measured the ability of several NBS1 mutant clones and A-T cells to regulate HR repair using the DR-GFP or SCneo systems. ATM deficiency did not reduce the HR repair frequency of an induced DSB, and it was confirmed by findings that HR frequencies are only slightly affected by deletion of ATM-binding site at the extreme Cterminus of NBS1. In contrast, The HR-regulating ability is dramatically reduced by deletion of the MRE11-binding domain at the C-terminus of NBS1 and markedly inhibited by mutations in the FHA/BRCT domains at the N-terminus. This impaired capability in HR is consistent with a failure to observe MRE11 foci formation. Furthermore, normal HR using sister chromatid was completely inhibited by the absence of FHA/BRCT domains. These results suggested that the N-and C-terminal domains of NBS1 are the major regulatory domains for HR pathways, very likely through the recruitment and retention of the MRE11 nuclease to DSB sites in an ATMindependent fashion.

In order to preserve and protect genetic information, eukaryotic cells have developed a signaling or communications network to help the cell respond to DNA damage, and ATM and NBS1 are key players in this network. ATM is a protein kinase which is activated immediately after a DNA double strand break (DSB) is formed, and the resulting signal cascade generated in response to cellular DSBs is regulated by post-translational protein modifications such as phosphorylation and acetylation. In addition, to ensure the efficient functioning of DNA repair and cell cycle checkpoints, the highly ordered structure of eukaryotic chromatin must be appropriately altered to permit access of repair-related factors to DNA. These alterations are termed chromatin remodeling, and are executed by a specific remodeling complex in conjunction with histone modifications. Current advances in the molecular analysis of DNA damage responses have shown that the auto-phosphorylation of ATM and the interaction between ATM and NBS1 are key steps for ATM activation, and that the association of ATM and NBS1 is involved in chromatin remodeling. Identification of novel factors which function in ubiquitination (RNF8, Ubc13, Rap80, etc.) has also enabled us to understand more details of the early stages in DNA repair pathways which respond to DSBs. In this review, the focus is on the role of ATM and the RAD50/MRE11/NBS1 complex in DSB response pathways, and their role in DSB repair and in the regulation of chromatin remodeling.

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