2018 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 / Rad55-Rad57 / Rad51 paralogs |
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| Outline of Annual Research Achievements |
Homologous recombination (HR) is essential for the repair of DNA double-strand breaks, which can lead to genomic instability, a hallmark of cancer. Although Rad51 is the key protein in HR, several groups of accessory proteins exist to regulate its activity. In fission yeast, two such complexes are Swi5-Sfr1 (SS) and the Rad51 paralogs Rad55-Rad57 (RR). The physical and functional interaction of SS with Rad51 has been characterized in depth through a multidisciplinary approach, and the manuscript describing this work is currently in preparation. A protocol for the efficient purification of RR has now been established. Thus, comprehensive biochemical analysis is currently underway, including the intrinsic biochemical nature of RR as well as its stimulatory effect on Rad51.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
The SS component of the project has progressed very smoothly and is being prepared for publication now. However, characterization of RR has proven more challenging. After much difficulty, I was able to overcome the first major obstacle by purifying the complex. The biochemical behavior of the complex has been surprising and unexpected. My preliminary findings are interesting and potentially very insightful. Several parallel experiments are underway to explain these observations, including the use of different assay systems to monitor Rad51 stimulation by RR (e.g., different DNA substrates) and different buffer conditions (e.g., different monovalent and divalent cations).
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| Strategy for Future Research Activity |
Once the SS manuscript is published, I will focus primarily on the biochemical characterization of RR. It may be necessary to purify other recombination factors to reveal the function of RR, so these will be purified accordingly. Furthermore, because RR has been purified by overexpression from fission yeast (i.e., its native host), it may be interesting to monitor the postranslational modifications of RR by mass spectrometry. Along a similar line, it is known that the Rad51 interaction sites in SS, which we identified by NMR and site-specific crosslinking, are flanked by CDK phosphorylation sites, raising the possibility that the interaction between Rad51 and SS is regulated in a cell cycle-dependent manner by phosphorylation. This will be my secondary focus for the upcoming year.
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Research Products
(2 results)
