Genes Affecting the Repair and Survival of Escherichia coli Following Psoralen-Induced Damage: a DNA Interstrand Crosslinking Agent
Portland State University. Department of Biology
Date of Publication
Master of Science (M.S.) in Biology
Escherichia coli -- Research, Psoralens, DNA repair, Mutagens, Mutation (Biology)
1 online resource (vi, 79 pages)
Photoactivated psoralens and other agents that form DNA interstrand crosslinks are highly cytotoxic and are useful in treating a range of diseases, including vitiligo, psoriasis, and some forms of cancer. Unlike many lesions that damage only one strand of the duplex DNA, DNA interstrand crosslinks form covalent bonds with both strands. Thus, repairing these lesions is complicated both by the lack of an undamaged strand to serve as a template for resynthesis following excision, as well as the potential to form double strand breaks if both strands are incised. A number of models have proposed that repair is likely to couple nucleotide excision repair with other repair pathways such as recombination, and/or translesion synthesis. However, several aspects of these models remain speculative, and how these medically relevant lesions are repaired by cells still remains elusive. In this study, I use Escherichia coli as a model organism to characterize which gene products contribute to survival in the presence of psoralen-induced DNA interstrand crosslinks.
In Chapter II, I demonstrate that although nucleotide excision repair initiates repair, not all subunits contribute equally to survival. Notably, uvrC is less sensitive to psoralen-induced damage than either uvrA or uvrB. I found that Cho, an alternative endonuclease, accounts for the increased resistance of uvrC mutants and contributes to survival in the presence of UvrABC. Cho was not required following angelicin treatment, a psoralen derivative that only forms monoadducts, suggesting that Cho function is specific for interstrand crosslink repair. However, Cho, by itself, is not required for the initial incision and only modestly enhances the rate that psoralen crosslinks are incised in vivo.
Following incision, many of the intermediates in the repair process remain speculative. In Chapter III, I examine how recombination and translesion synthesis mutants contribute to survival of psoralen-induced damage. I show that both recBC and recF contribute to survival, but that neither mutant is as hypersensitive as recA, potentially suggesting that pathways involving either single strand gaps or double strand break intermediates can occur during repair. Finally, I show that Polymerase V is responsible for the translesion synthesis that contributes to survival in the case of psoralen-induced damage in E.coli.
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Perera, Anthonige Vidya, "Genes Affecting the Repair and Survival of Escherichia coli Following Psoralen-Induced Damage: a DNA Interstrand Crosslinking Agent" (2015). Dissertations and Theses. Paper 2195.