Free radical-mediated DNA double-strand breaks (DSBs) induced by ionizing radiation, radiomimetic natural products, and oxidative stress have chemically modified termini such as 32-phosphoglycolates that must be removed before the break can be rejoined. In addition, accurate rejoining of these breaks requires replacement of fragmented nucleotides by gap filling on aligned DSB ends. Radiation-induced DSBs are also often accompanied by nearby oxidative base damage that can interfere with rejoining. DSBs induced by topoisomerase inhibitors have protein-linked termini that likewise must be removed for repair. The overall goal of the proposed project is to determine how these damaged DSB ends are resolved for repair by nonhomologous end joining, and how they affect the overall joining process. Candidate enzymes for processing damaged ends include tyrosyl-DNA phosphodiesterase (TDP1) and the DNA end-specific endonucleases Mre11, Artemis, Metnase and CtIP. Mouse and human cells with genetic defects in Artemis, Metnase, TDP1, Mre11 and combinations thereof will be augmented with siRNA knockdown to generate cells with various combinations of end-processing deficiencies. These cells will be subjected to cytotoxicity, cytogenetic and DSB repair assays, as well as a newly developed real-time PCR assay for measuring the persistence of blocked termini in cells. These studies will determine whether these repair factors provide alternative pathways for resolving blocked DNA termini, and the degree of overlap between them. Defined DSB substrates, bearing ends with 32-phosphoglycolates or oxidatively modified bases in various contexts, will be used to determine the specificities of Artemis and Metnase in trimming damaged ends. The efficiency with which the resulting trimmed ends progress to the gap filling and ligation steps in cell extracts will be determined. Tolerance for damaged bases in gap-filling on aligned DSB ends by DNA polymerase;will be determined, as well as the possible cooperation and competition between polymerase;and Artemis in resolving different types of damaged DNA termini.
Because radiotherapy and some types of chemotherapy kill tumor cells by inducing DNA double- strand breaks, enzymes that process damage at the ends of such breaks represent potential therapeutic targets that could be exploited to improve efficacy and therapeutic index. Moreover, some repair enzymes delete segments of normal DNA in the process of removing damage from the ends, and therefore the accuracy of repair can depend on the specific repair enzymes used, which is the focus of the proposed research. Because inaccurate double-strand break repair can lead to genomic alterations that promote malignancy, it is essential in terms of cancer prevention to understand how the choice of repair enzymes and repair systems is made by the cell for specific types of double-strand breaks.
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