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Chair(s): Mitra, Sankar
(SY003) Molecular mechanism of BER-mediated double strand DNA break formation.
Satoh, Masahiko*,1, 1 Division of Health and Environmental Research, Ste-Foy, Quebec, Canada
ABSTRACT- Double-strand DNA break (DSB) are the most lethal type of DNA damage. To protect cells from the lethal effects of DSBs, cells repair DSB through non-homologous end joining or recombination. In fact, the number of ionizing radiation-induced DSBs in mammalian cells are rapidly reduced during the post-exposure period. These DSBs are however only detectable in cells exposed to high doses of ionizing radiation; but it is unlikely that DSB repair has evolved to rejoin DSBs produced under such artificial experimental conditions. If one considers the fact that DSB repair is well conserved among eukaryotes, DSB is likely produced in cells, even though these cells receive only a low dose of radiation from the environment. Because hydroxyl radicals generated by ionizing radiation form DNA damage clusters, in which multiple DNA damages are located within a close distance, it has been suggested that DSB is produced by an attempt to repair these damages. To investigate the mechanism of DSB formation, we established a cell-free DSB formation assay with a plasmid containing two damages on opposite DNA strands. By incubation of the plasmid with cell-free extract from 46BR cells, which form long DNA repair patches due to an abnormality in DNA ligase I, we found increased formation of DSBs. Thus we proposed that DSBs can be produced through collision of two repair machineries by simultaneous repair of two damages. Particularly, we proposed that collision of two DNA polymerases may result in DSB formation. However, we recently found that DSBs are produced even in the absence of DNA strand synthesis. Thus, we analyzed the structure of DSB ends and found removal of DNA strand in the 5' to 3'direction, suggesting that cleavage of DNA strands by Fen-1 plays a critical role in DSB formation. In fact, addition of Fen-1 to the cell-free DSB formation assay, even with extracts from normal cells, resulted in a promotion of DSB formation. Furthermore, we found an increased number of DSBs in cells overexpressing Fen-1 after exposure of cells to -rays. Thus these results demonstrate that DSBs can be produced by a novel pathway mediated by Fen-1.
Key words: Double-strand DNA breaks, DNA repair, FEN-1
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