Structural and Biochemical Studies of Lesion Recognition in Bacterial DNA Repair

DNA is constantly under threat of chemical modification by endogenous and exogenous sources of DNA damage. Mutations resulting from these modifications have deleterious consequences for the cell, resulting in genome instability and the threat of unregulated cell growth in the form of cancer.;Nucleotide excision repair (NER) is unique among the various systems of DNA repair in its ability to repair a wide variety of structurally diverse lesions, including sterically bulky adducts that are not accessible to other systems of DNA repair. In bacteria, repair is carried out in an ATP-dependent process by UvrA, UvrB, and UvrC. We developed a disulfide crosslinking system for trapping covalent complexes of UvrB on DNA, which allowed us to capture two new structures of wild-type and a Y95W mutant of UvrB bound to DNA. In addition, we developed a crosslinking assay to map the interaction between UvrB and lesion-containing DNA. The results from this work suggest UvrB interacts specifically with the lesion-containing strand during lesion recognition, and provides a new model for recognition of different types of NER lesions by UvrB.;Base excision repair (BER) comprises a group DNA glycosylases that are each specialized towards removing a particular base modification to the DNA. Repair of oxidative damage in bacteria is mediated by MutM/Fpg, which excises 8-oxo-7,8-dihydroguanine (oxoG) from oxoG:C base pairs. Recently, several crystal structures of MutM trapped onto fully duplex DNA (using disulfide crosslinking) containing an oxoG established the first mechanism for intrahelical lesion recognition by a DNA glycosylase, via detection of lesion-dependent remodeling of the DNA backbone at the lesion site. Using X-ray crystallography, we investigated the effect of sequence context on intrahelical lesion recognition by systematically varying the sequence on the 5' flank of the lesion base. These structures show the direct influence of base stacking interactions on intrahelical lesion recognition and base extrusion, with implications for the general efficiency of DNA repair in different sequence contexts.