Research performed here is for a better understanding of UV- mutagenesis in E. coli and the interplay with DNA repair and other genetic activities in the cells. This general area of basic research has provided the foundation for understanding repair and mutagenesis and the hallmarks of ultraviolet radiation damage in human cells. The area is even now supporting research protocols for clinical cancer treatment. The studies emphasize activities of DNA photolyase that occur without the light that normally energizes monomerization of cyclobutane pyrimidine dimers (CPD), the most stable photo-product produced by ultraviolet radiation in DNA. The close and selective fit between DNA photolyase and CPD in DNA can in some cases prevent the formation of mutations normally induced at such lesions even though the photo-product is not repaired. A centrally hypothesis to be examined here is that idea that photolyase-CPD complexes interfere with some aspect of translesion DNA synthesis which normally establishes mutated DNA sequences. Thus the photolyase-CPD complex would act as a probe of this critical step in mutagenesis. Three types of dark effect by photolyase-CPD complexes will be studied: (a) disruption of UV- mutagenesis targeted by CPD (producing glutamine tRNA suppressor mutations), (b) enhanced UV-inactivation of cell viability and (c) signaling from UV-induced damage (CPD) in the genome that is recognized by RexAB proteins and produces cell death by membrane depolarization. The three different effects may reflect a common mechanism for disruption of translesion DNA synthesis and/or replication restart (recombination tolerance) and therefore prove mutually revealing. An assay for the migration of RecA protein from cytoplasm to cell membrane will be established with mini-cells and used to cell whether photolyase-CPD complexes and other components alter this migration. A newly defined gene and protein activity that influences the consequence(s) of photolyase-CPD complexes in DNA in E. coli will be characterized genetically and physiologically.
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