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Yang, Ning

Microbiology and Molecular Genetics

2005

PhD

A significant proportion of cellular DNA damages induced by ionizing radiation are produced in clusters, also called multiply damaged sites. Cluster damage sites consist of closely spaced single lesions (oxidized purine and pyrimidine bases, abasic sites or strand breaks) within one or two helical turns of DNA. It has been demonstrated by in vitro repair studies using model clustered damages and purified enzymes or cell extracts and in bacteria that many bistranded clustered damages can be converted to lethal double strand breaks by oxidative DNA glycosylases during attempted base excision repair. To determine whether DNA glycosylases could produce double strand breaks at radiation-induced clustered damages in human cells, stably transformed human lymphoblastoid TK6 cells that inducibly overexpress the oxidative DNA glycosylases/AP lyases, hNTHl and hOGG1, were assessed for their radiation responses, including survival, mutation induction and the enzymatic production of double strand breaks post-irradiation. We found that additional double strand breaks were generated during post-irradiation incubation in uninduced TK6 control cells. Moreover, overproduction of either DNA glycosylases resulted in significantly increased double strand break formation, which correlated with an elevated sensitivity to the cytotoxic and mutagenic effects of ionizing radiation. We also down-regulated the expression of hNTHl or hOGGl in TK6 cells using intracellularly expressed siRNAs and showed that down-regulation of hNTHl or hOGG1 resulted in decreased DSB formation post-irradiation. In case of hOGG1 whose main substrate is the mutagenic but not cytotoxic 8-oxoguanine, the decreased DSB formation correlated with a decrease in radiosensitivity. Cells deficient in hNTHl, the DNA glycosylase that repairs a major lethal single free radical damage, thymine glycol, were more sensitive to ionizing radiation. In addition, in hydrogen peroxide-treated cells where the majority of free radical DNA damages are single lesions, overexpression of hNTHl and hOGG1 resulted in reduced cell killing while suppression of glycosylase expression resulted in elevated cell death. These data demonstrate that attempted repair of radiation-induced clustered damage sites by the oxidative DNA glycosylases can lead to the formation of potentially lethal and mutagenic double strand breaks in human cells.