DNA interstrand cross-links are complex lesions that covalently link both strands of the duplex DNA. Lesion removal is proposed to be initiated via the UvrABC nucleotide excision repair complex; however, less is known about the subsequent steps of this complex repair pathway. In this study, we characterized the contribution of nucleotide excision repair mutants to survival in the presence of psoralen-induced damage. Unexpectedly, we observed that the nucleotide excision repair mutants exhibit differential sensitivity to psoralen-induced damage, with uvrC mutants being less sensitive than either uvrA or uvrB. We show that Cho, an alternative endonuclease, acts with UvrAB and is responsible for the reduced hypersensitivity of uvrC mutants. We find that Cho's contribution to survival correlates with the presence of DNA interstrand cross-links, rather than monoadducts, and operates at a step after, or independently from, the initial incision during the global repair of psoralen DNA adducts from the genome. IMPORTANCEDNA interstrand cross-links are complex lesions that covalently bind to both strands of the duplex DNA and whose mechanism of repair remains poorly understood. In this study, we show that Cho, an alternative endonuclease, acts with UvrAB and participates in the repair of DNA interstrand cross-links formed in the presence of photoactivated psoralens. Cho's contribution to survival correlates with the presence of DNA interstrand cross-links and operates at a step after, or independently from, the initial incision during the repair process. P soralens are tricyclic asymmetrical compounds containing furan and pyrone rings and bind DNA nonspecifically, with a preference for pyrimidines to form noncovalent bonds (1-3). Upon absorption of UV-A light, a covalent bond forms through photoaddition between the C-5AC-6 double bond of the pyrimidine and the C-4=AC-5= furan double bond or C-3=AC-4= pyrone double bond of psoralen. Absorption of a second photon results in photoaddition on the remaining furan or pyrone with a second pyrimidine, creating a DNA interstrand cross-link. Thus, psoralen-induced damage consists of both monoadducts and DNA interstrand cross-links. Because DNA interstrand crosslinks covalently bind both strands of the duplex DNA, they inhibit cellular processes that require strand denaturation, including transcription and replication. This inhibition is generally considered to be the reason for the potency of psoralens, and this class of compounds is used in treating different skin diseases, such as vitiligo, psoriasis, and as a chemotherapeutic for some forms of cancer (4-6).Several models have been proposed for DNA interstrand crosslink repair. A feature common to most models is that the repair process is initiated by nucleotide excision repair, followed by the sequential action of other DNA repair processes, such as recombination or translesion synthesis, which function to provide an undamaged template that replaces the incised sequence. In these models, a second round of nucleotide exc...
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. iii ACKNOWLEDGMENTS I would like to express my sincere gratitude to my adviser, Dr.Justin Courcelle for granting me the opportunity to work under him and providing me guidance throughout my graduate tenure. Secondly, I would like to thank my committee members Dr.Michael Bartlett and Dr.Rahul Raghavan for the excellent feedback and assistance with my written thesis. In addition, I would also like to thank Dr.Charmain Courcelle for her immense support and guidance. Lastly, I would like to extend my thanks to all my friends and family members for their support and encouragement.
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