Abstract
DNA-protein cross-links (DPCs) are ubiquitous DNA lesions and are produced by ionizing radiation, UV light, aldehydes, and other genotoxic compounds. However, the repair mechanism of DPCs is largely unknown. In the accompanying genetic study, we have shown that DPCs are repaired by nucleotide excision repair (NER) and homologous recombination in E. coli. In the present study, we analyzed the biochemical aspects of DPC repair by NER. Since our genetic study suggested that the repair of DPCs by NER was dependent on the size of cross-linked proteins, we systematically constructed oligonucleotide substrates containing peptides and proteins of various sizes (0.2-44 kDa). DPCs were introduced as cross-links between oxanine in DNA and proteins or peptides. The DPC substrates were incubated with UvrABC nuclease (prokaryotic NER proteins), and products were analyzed by denaturing PAGE. As for other bulky DNA lesions, UvrABC incised the 8th and 5th phosphodiester bonds 5' and 3' to DPC, respectively, yielding a 12mer fragment. However, the incision efficiency decreased with increasing the size of cross-linked proteins. The incision efficiency was marginal for 14-16 kDa proteins, indicating that NER cannot process oversized DPCs. To confirm the in vivo role of NER, E. coli cells were treated with formaldehyde (FA), a DPC-inducing agent, and chromosomal DPCs were analyzed. DPCs were actively removed in wild type cells but poorly in NER-deficient cells.
