| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2004: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2003: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Research Abstract |
The aim of the project was to analyze behavior and function of RecQ family helicases and their interacting proteins during repair processes of DNA damages by means of Xenopus egg extract cell free experimental system. A product of Bloom syndrome causative gene, Blm, was bound to chromatin during the process of DNA replication in the extract, and the chromatin binding of Blm was mutually dependent on chromatin loading of DNA topoisomerase III α (Top3α), which is known as one of interacting proteins of Blm. In addition, significant suppression of DNA replication was observed after an immuno-depletion of Blm or Top3α. Further analysis indicated that the suppression was not due to an activation of checkpoint pathway. Causative gene products of Werner and Rothmund-Thomson syndromes, Wrn and RecQL4, respectively, were associated onto chromatin in response to an induction of DNA double-strand breaks (DSB). The association of the helicases was completely dependent on the presence of RPA, a single-stranded DNA binding protein complex in eukaryotic cells. For quantitative analyses for activities to repair DSB, I constructed experimental systems to measure activities of homologous recombination repair (HRR) and non-homologous end-joining (NHEJ) in the egg extract using linearized plasmid DNA as substrates. Results of the assay revealed that 1)repair through NHEJ was almost 1000-fold more effective than HRR, that 2)NHFJ activity was partially sensitive to aphidicolin, an inhibitor of DNA polymerise α, δ and ε, or wortmannin, an inhibitor of DNA-dependent protein kinase, and that 3)HRR activity was hardly detected after the addition of aphidicolin. As a result of the assay after immuno-depletion of RecQL4 from the egg extract, marked suppression of NHFJ activity and significant promotion of HRR activity were observed in the depleted extarct. Thus, it was suggested that RecQL4 is concerning to DSB repair by stimulating NHEJ and suppressing HRR.
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