| dc.description.abstract | DNA is a fundamental biomolecule of life which stores the genetic information of organisms and drives many of the evolutionary processes in nature. Despite this, DNA is subject to physical and chemical damage from environmental sources that impair the ability to access the genomic information. Unsurprisingly, organisms have evolved unique biochemical adaptations to produce antibiotic chemical agents that can modify or alter DNA (genotoxins). Consequently, it is essential for these organisms to possess specialized DNA repair pathways to protect the genome from these unique sources of damage, although these pathways remained unexplored. In azinomycin B (AZB) biosynthesis- an exceptionally potent DNA interstrand crosslinking (ICL) agent, the identification of the first bacterial DNA glycosylase (AlkZ) that can unhook crosslinked DNA to initiate the base excision repair (BER) was observed, although the molecular basis for this unprecedented activity was lacking. Using X-ray crystallography, we determined the structure of Streptomyces sahachiroi AlkZ which revealed a novel fold for DNA glycosylases (winged helix-turn-helix) and provided insight on the mechanism of base excision of crosslinked nucleobases by this family of enzymes. An evolutionarily related, yet functionally diverged ortholog of AlkZ in E. coli was identified through phylogenetics and domain analysis, and I demonstrated that YcaQ is a DNA glycosylase that unhooks a variety of ICLs from different bifunctional alkylating agents. We established through cellular experiments that YcaQ is part of an alternative ICL repair pathway in E. coli which relies on the BER machinery and is separate from canonical nucleotide excision repair (NER) of DNA crosslinks. We next performed a phylogenetic, taxonomic, and genome mining analysis of AZL (AlkZ-like) and YQL (YcaQ-like) proteins in bacteria, and showed an intimate association of AZL genes with biosynthetic gene clusters, and elucidated the DNA glycosylase-mediated antibiotic resistance mechanisms of three diverse bacterial genotoxins (hedamycin, LLD-family, and the trioxacarcins) using various experimental techniques. The work presented here in this dissertation contributes to the expanding roles of DNA glycosylases and the BER pathway with implications in antimicrobial self-resistance and ICL repair of distinct genotoxins encountered in nature. | |
