Abstract
Ionizing radiation, ultraviolet light, and certain chemical mutagens induce DNA-protein crosslinks (DPCs). DPCs are by far larger than conventional bulky DNA lesions such as pyrimidine dimers and aromatic base adducts. Since DPCs are extremely bulky, they will strongly block the progression of replication and transcription machineries and hence exert adverse effects on cells. We have previously shown that in E. coli DPCs containing relatively small crosslinked ptoteins (CLPs) (12 kDa) were processed by RecBCD-dependent homologous recombination (HR). Thus, E. coli cells use repair (NER) and damage tolerance (HR) mechanisms to alleviate the genotoxic effects of DPCs. In the present study, we have asked whether mammalian cells use a similar strategy to ensure survival in the face of DPCs. The activity assays with cell extracts showed that the upper size limit of CLPs amenable to mammalian NER was about 8 kDa, which was considerably smaller than that of E. coli. NER-proficient and deficient cells were treated with DPC-inducing agents, and the repair of genomic DPCs was analyzed. However, NER-dependent removal of DPC was not observed. Thus, NER is not involved in the repair of DPCs in mammalian cells. We also found that genomic DPCs were not polyubiqutinated for proteasomal degradation prior to NER. Cnversely, irs1SF (XRCC3) and 51D1 (RAD51D) cells deficient in HR were hypersensitive to DPC-inducing agents, indicating the involvement of the HR-dependent damage tolerance mechanism for DPCs. In keeping with this observation, cells accumulated nuclear RAD51 and γ-H2AX foci following the treatment with DPC-inducing agents. These results show the differential involvement of NER in the repair of DPCs in bacterial and mammalian cells, and demonstrate the conserved role of HR in tolerance to DPCs among species.
