| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2006: ¥2,400,000 (Direct Cost: ¥2,400,000)
Fiscal Year 2005: ¥1,100,000 (Direct Cost: ¥1,100,000)
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| Research Abstract |
To understand the functional relationship between BRCA2 and homologous recombination (HR) related molecules, we disrupted the Rad51 cofactors XRCC3, RAD52, and BRCA1 in brca2 null cells. We also inactivated the FANC-C gene, one of Fanconi anemia genes responsible for human chromosome breakage syndrome in brca2 null cells. The number of spontaneously arising chromosome breaks was higher in brca2/fanc-c clones than in the single mutants. In contrast, brca2/xrcc3, brca2/rad52, brca2/brca1 cells displayed similar number of chromosome breaks as brca2 cells did. These observations suggest that the Fanconi pathway but not the other HR related molecules can contribute to genome maintenance during the cell cycle, presumably independently of BRCA2. Remarkably, the brca2/xrcc3, brca2/rad52, brca2/brca1, and brca2/fanc-c mutants showed the same level of cisplatin sensitivity as did brca2 cells. These genetic data reveal that XRCC3, RAD52, BRCA1, and FANC-C proteins may function in cooperation with BRCA2 in the interstrand crosslink repair. Other way round, brca1 cells showed higher sensitivity compared to brca2 cells in cellular response to camptothecin. Camptothecin inhibits topoisomerase I and gives rise to DSBs, which can be repaired by HR. Thus BRCA1 and BRCA2 contribute to HR also independently of each other depending on the type of DNA damage.
To improve monitoring of DNA repair process, we generated DSBs by laser microiradiation. The advantage of the microlaser approach is its ability to generate multiple DSBs at defined regions in the cell nucleus. Importantly RAD51 was definitely colocalized with yH2AX even in the absence of BRCA2. From these results we conclude that RAD51 is recruited to DNA damage sites independently of BRCA2. We also developed chromatin immunoprecipitation to monitor the in vivo phosphorylation of H2AX after DNA double strand breaks induced by I-SceI endonuclease digestion in DT40 cells.
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