Structure-specific DNA endonuclease T7 endonuclease I cleaves DNA containing UV-induced DNA lesions

Abstract

Abstract The T7 gene 3 product, T7 endonuclease I, acts on various substrates with DNA structures, including Holliday junctions, heteroduplex DNAs and single-mismatch DNAs. Genetic analyses have suggested the occurrence of DNA recombination, replication and repair in Escherichia coli. In this study, T7 endonuclease I digested UV-irradiated covalently closed circular plasmid DNA into linear and nicked plasmid DNA, suggesting that the enzyme generates single- and double-strand breaks (SSB and DSB). To further investigate the biochemical functions of T7 endonuclease I, we have analysed endonuclease activity in UV-induced DNA substrates containing a single lesion, cyclobutane pyrimidine dimers (CPD) and 6–4 photoproducts (6–4PP). Interestingly, the leading cleavage site for CPD by T7 endonuclease I is at the second and fifth phosphodiester bonds that are 5′ to the lesion of CPD on the lesion strand. However, in the case of 6–4PP, the cleavage pattern on the lesion strand resembled that of CPD, and T7 endonuclease I could also cleave the second phosphodiester bond that is 5′ to the adenine–adenine residues opposite the lesion, indicating that the enzyme produces DSB in DNA containing 6–4PP. These findings suggest that T7endonuclease I accomplished successful UV damage repair by SSB in CPD and DSB in 6–4PP.