Enzymatic properties of purified Escherichia coli uvrABC proteins.

Abstract

The cloned uvrA and uvrB genes of E. coli K-12 were amplified by linkage to the PL promoter of plasmid pKC30. The uvrC gene was amplified in the high-copy-number plasmid pRLM 24. The 3 gene products (purified in each case to > 95% purity) and ATP are required to effectively incise UV-damaged DNA. The uvrABC proteins bind tightly to damaged sites in DNA, requiring the initial attachment of the uvrA protein in the presence of ATP before productive binding of the uvrB and uvrC proteins. Using a cloned tandem double tandem double insert of the lac p-o region as a damaged DNA substrate for the uvrABC complex and analyzing the incision both 5'' and 3'' to each pyrimidine dimer, it was found the 1 break occurs 7 nucleotides 5'' to a pyrimidine dimer and a 2nd break is made 3-4 nucleotides 3'' from the same pair of pyrimidines in the dimer. No such breaks are found in the strand complementary to the dimer. The size of the incised fragment in the DNA suggests that incision may be coordinated with excision reactions in repair processes.