Ionizing radiation produces a wide spectrum of DNA damages including base and sugar damages, single and double strand breaks, abasic sites, DNA-protein and DNA-DNA crosslinks as well as multiply damage sites(MIDS). Double strand breaks are repaired either by homologous recombination or non-homologous end-joining mechanism. Despite the fact that double strand break is a lethal lesion, lethality induced by ionizing radiation cannot be fully explained by the amount of double strand breaks formed. It was suggested by Ward that a significant amount of cell killing by low LET radiation at biological relevant doses is due to the production of MDS, a cluster of damages within a localized region. There is increasing evidence that MDS are biologically important and might contribute significantly to lethality and mutagenesis induced by ionizing radiation. The long-term goal for this project is to understand the biological consequences of MDS. Two approaches are taken in order to achieve this goal.
Aims 1 to 2 are directed to elucidate the in vivo biological consequences of MDS. A yeast shuttle plasmid, pRS413, containing various MDS will be constructed and used to transform E. coli of various repair backgrounds. The lethality conferred by various MDS will be scored by measuring the survival of damaged pRS413 and the mutagenicity of MDS by direct sequencing of the mutant progeny plasmid obtained after transformation. In addition, in vitro processing of these MDS will be studied in Aims 3 and 4.
Aim 3 is directed to examine the in vitro processing of DNA containing tandem lesions by E. coli BER enzymes including endonucleases III and VIII, formamidopyrimidine N-glycosylase and 5? AP endonucleases from E.coli. The nature of the reaction products and the kinetics of removal for each of the lesions within the MDS will be elucidated. In addition, a complete in vitro analysis of the possible enzymatic steps involved in the repair of tandem lesions will also be performed.
Aim 4 is directed to examine whether E. coli DNA binding protein, such as HU protein can mediate the sequential repair of closely opposed lesion. It is believed that a comprehensive study involving in vivo biological and in vitro enzymatic studies will provide significant insight into understanding the biological consequences of MDS, and thus the genotoxic and mutagenic effect of ionizing radiation.
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