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

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 E.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 analyzed 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 T7 endonuclease I accomplished successful UV damage repair by SSB in CPD and DSB in 6-4PP.