Abstract
DNA-protein crosslinks (DPCs) generated by radiation and other genotoxic chemicals are formidable obstacles to DNA replication and transcription, and their persistence would lead to mutations and cell death. Thus, the repair or tolerance of DPCs is essential for cells. Recently, we have shown that nucleotide excision repair (NER) and homologous recombination (HR) are involved in the repair and tolerance of DPCs, yet much remains to be learned about how cells deal with these superbulky lesions. In this work, we utilized a genetic approach to obtain further insight into the mechanistic aspects of NER and HR of DPCs and to assess the roles of other repair mechanisms such as base excision repair (BER) and translesion synthesis (TLS). We measured the sensitivities of a panel of E. coli mutants to DPCs-inducing agents including formaldehyde (FA) and 5-azacytidine (azaC). The priA mutant displayed a marked sensitivity to FA and azaC, indicating a crucial role of the PriA helicase in the restart of stalled replication forks following HR. In contrast, other helicase mutants including priB, priC, and rep showed no to only marginal sensitivities. The mutants of recJ and recQ which are involved in the RecFOR-dependent HR pathway also showed marginal sensitivities to FA and azaC. This observation is consistent with our previous finding that the HR of DPCs proceeds exclusively through the RecBCD pathway. With respect to TLS polymerase mutants, polB, dinB, and umuDC exhibited no sensitivity to FA and azaC, eliminating their role in tolerance of DPCs. Regarding to the excision repair, the mutant deficient in the Cho endonulease (a UvrC homolog) showed moderate sensitivity to FA, suggesting a role of Cho in the excision of DPCs. The mutants of TCR (mfd) and BER (nth/nei and xth/nfo) were not sensitive to both DPC-inducing agents. The present study further confirms the critical role of NER and HR and eliminates the roles of BER and TLS in the repair and tolerance of DPCs.
