2017 Fiscal Year Research-status Report
Functional analysis of Rad51 activation by Rad55-Rad57 and Swi5-Sfr1
| Project/Area Number |
17K15061
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| Research Institution | Tokyo Institute of Technology |
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Principal Investigator |
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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| Project Period (FY) |
2017-04-01 – 2020-03-31
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| Keywords | DNA repair / Homologous recombination / Rad51 / Swi5-Sfr1 |
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| Outline of Annual Research Achievements |
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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| Current Status of Research Progress |
Current Status of Research Progress
1: Research has progressed more than it was originally planned.
Reason
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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| Strategy for Future Research Activity |
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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Research Products
(3 results)
