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DNA-protein crosslinks (DPCs)-where proteins are covalently trapped on the DNA strand-block the progression of replication and transcription machineries and hence hamper the faithful transfer of genetic information. However, the repair mechanism of DPCs remains largely elusive. Here we have analyzed the roles of nucleotide excision repair (NER) and homologous recombination (HR) in the repair of DPCs both in vitro and in vivo using a bacterial system. Several lines of biochemical and genetic evidence show that both NER and HR commit to the repair or tolerance of DPCs, but differentially. NER repairs DPCs with crosslinked proteins of sizes less than 12-14 kDa, whereas oversized DPCs are processed exclusively by RecBCD-dependent HR. These results highlight how NER and HR are coordinated when cells need to deal with unusually bulky DNA lesions such as DPCs.

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.