Abstract
Ionizing radiation produces oxidized bases and strand breaks as major DNA lesions. In addition, ionizing radiation also generates DNA-protein cross-links (DPCs) and interstrand cross-links. However, repair mechanisms of cross-linked lesions largely remain to be elucidated. We have shown previously that oxanine, a deamination product of guanine, reacts with polyamines and DNA binding proteins such as histone and DNA glycosylases, giving rise to DPCs. Furthermore, cross-link products between oxanine and spermine are excised from DNA by prokaryotic and eukaryotic nucleotide excision repair (NER) systems. DPCs bear large protein molecules and hence could hamper the access and/or assembly of NER proteins. In the present study, we have assessed the damage recognition and excision capacities of the NER system for DPCs using UvrABC nuclease. Oxanine was site-specifically incorporated into oligonucleotides and cross-linked to proteins of varied sizes. DPC-DNA was incubated with UvrABC, and incision products were analyzed by denaturing PAGE. DPC-DNA was also incubated with UvrAB, and the formation of DNA-protein complex was analyzed by native PAGE. The dual incision activity of UvrABC for DPC-DNA varied significantly depending on the size of cross-linked proteins. The efficiency of DNA-UvrB complex formation was also dependent on the size of cross-linked proteins. These results indicate that the damage recognition step by UvrA2B is key to the excision of DPCs by UvrABC nuclease.
