| 研究課題/領域番号 |
17K15061
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| 研究機関 | 東京工業大学 |
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研究代表者 |
Argunhan Bilge 東京工業大学, 科学技術創成研究院, 特任助教 (30792759)
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| 研究期間 (年度) |
2017-04-01 – 2020-03-31
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| キーワード | DNA repair / Homologous recombination / Rad51 / Swi5-Sfr1 |
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| 研究実績の概要 |
Homologous recombination (HR) is important for repairing DNA double-strand breaks, which can lead to genomic instability and cancer. Although Rad51 is the key protein in HR, several groups of accessory proteins are required to modulate the activity of Rad51. Two such protein complexes in fission yeast are Swi5-Sfr1 (SS) and the Rad51 paralogs Rad55-Rad57 (RR). I have optimized the purification of RR and can achieve good yield and purity. I have identified residues in SS that are required for the functional interaction with Rad51. When these residues are mutated, the stimulation of Rad51-mediated HR is defective in vitro. Consistent with this, a strain encoding the mutant SS protein is defective for DNA repair. I will continue this work to uncover the molecular mechanisms underlying these defects.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
1: 当初の計画以上に進展している
理由
The purification of RR was a big challenge. However, I was able to successfully purify a good yield of RR, far more than I expected. In regards to SS, the in vivo crosslinking experiments initially proposed were conducted with relative ease. Furthermore, I was able to observe the in vivo defects of SS mutants with some relatively simple genetic manipulation. The in vitro experiments that have been performed so far are established in the lab of Prof. Hiroshi Iwasaki, so there has been little/no obstacle to the progress of such experiments. In conclusion, the progress has been encouraging and I hope this will continue into the second year of the project.
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| 今後の研究の推進方策 |
The biochemical characterization of RR can begin in earnest now that the purification protocol is optimized. Using conventional biochemistry and genetics, I have demonstrated that SS mutants are defective in the functional interaction with Rad51. Next, I aim to employ a real-time assay that was developed by our lab to elucidate the nature of SS mutant defects at the molecular level. Furthermore, the motifs of SS that interact with Rad51 are flanked by phosphorylation sites. I will investigate whether such sites regulate the interaction with Rad51. Finally, by combining in vivo crosslinking with mass spectrometry, I have identified putative Rad51 residues that interact with SS; future work will involve validating this result and performing complementary analysis of Rad51 mutants.
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