Abstract
Ionizing radiation, UV light, and a certain class of mutagens irreversibly trap proteins on the DNA strand and induce DNA-protein crosslink (DPC) lesions. DPCs are extremely bulky as compared to oxidative base damage and UV-induced pyrimdine dimers so that they would impede the progression of replication as well as transcription machineries, thereby exerting adverse effects on cells. The presence of DPCs as DNA lesions has been long known, but it has been largely unknown how they are repaired in cells. Recently, we have analyzed the repair mechanism of DPCs in vitro and in vivo using E. coli as a model system and shown that nucleotide excision repair (NER) and homologous recombination (HR) work cooperatively to cope with the genotoxic effect of DPCs. However, it remains elusive whether DPCs are also processed in a similar manner in eukaryotic cells. In the present study, we analyzed the repair mechanism of DPCs in vitro and in vivo using mammalian cells. When model DPC substrates were incubated with cell-free extracts from HeLa cells, a dual incision indicative of NER occurred on the 5´ and 3´ sides of DPCs. The incision efficiency decreased with increasing the size of crosslinked proteins (CLPs). The upper size limit of CLPs amenable to NER was 8-10 kDa, which was smaller than that observed for E. coli NER. We also isolated chromosomal DNA from cells treated with formaldehyde, a typical DPC-inducing agent, and analyzed the removal of DPCs by SDS-PAGE. The rates of removal of DPCs were similar for wild type and NER-deificient XPA cells. These in vitro and in vivo data suggest that unlike in bacteria cells, NER is not involved in the repair of DPCs in mammalian cells. We are currently assessing the role of HR and will present the result in the meeting.
