Hierarchy of DNA Damage Recognition in Escherichia coli Nucleotide Excision Repair

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Abstract
DNA damage recognition plays a central role in nucleotide excision repair (NER). Here we present evidence that in Escherichia coli NER, DNA damage is recognized through at least two separate but successive steps, with the first focused on distortions from the normal structure of the DNA double helix (initial recognition) and the second specifically recognizing the type of DNA base modifications (second recognition), after an initial local separation of the DNA strands. DNA substrates containing stereoisomeric (+)- or (−)-trans- or (+)- or (−)-cis-BPDE-N2-dG lesions in DNA duplexes of known conformations were incised by UvrABC nuclease with efficiencies varying by up to 3-fold. However, these stereoisomeric adducts, when positioned in an opened, single-stranded DNA region, were all incised with similar efficiencies and with enhanced rates (by factors of 1.4−6). These bubble substrates were also equally and efficiently incised by UvrBC nuclease without UvrA. Furthermore, removal of the Watson−Crick partner cytosine residue (leaving an abasic site) in the complementary strand opposite a (+)-cis-BPDE-N2-dG lesion led to a significant reduction in both the binding of UvrA and the incision efficiency of UvrABC by a factor of 5. These data suggest that E. coli NER features a dynamic two-stage recognition mechanism.