2017 Fiscal Year Annual Research Report
Molecular mechanism of mating-type switching in Schizosaccharomyces pombe
Project/Area Number |
17J04051
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Research Institution | Tokyo Institute of Technology |
Principal Investigator |
AFSHAR NEGAR 東京工業大学, 生命理工学研究科, 特別研究員(DC2)
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Project Period (FY) |
2017-04-26 – 2019-03-31
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Keywords | mating-type switching / homologous recombination / fission yeast / mediators / Rad51 |
Outline of Annual Research Achievements |
Mating-type switching (MTS) in Schizosaccharomyces pombe is initiated by a self-programmed DNA double strand break (DSB) associated with DNA replication stall at the mat locus. The DSB is repaired by a mechanism involving homologous recombination (HR), resulting in gene conversion between mat1 and mat2/3. In this project, I initially planned to study the molecular functions of Mcl1 and Rad51-related proteins, which plays a role in the initial and central steps of HR-mediated MTS, respectively. In 2017, I started biochemical analysis of Rad51-related proteins, including the Rad55-Rad57 heterodimer complex and Shu complex consisting of Sws1, Rlp1 and Rdl1. Both Rad55-Rad57 and Rlp1-Rdl1, but not Sws1, are Rad51 paralogs that share structural similarities with Rad51. A prominent feature of Sws1 is a SWIM domain. I have established purification protocols of both complexes; Rad55-Rad57 and Rlp1-Rdl1 are purified from S. pombe and recombinant Sws1 is purified from Escherichia coli. Preliminary results show that Rad55-Rad57 has strong affinity for long single-stranded and double-stranded DNA substrates while Shu complex binds quite weakly to both DNA substrates. In addition, both complexes have a very weak ATPase activity, which is independent of single-stranded or double-stranded DNA. I am now studying impacts on DNA binding and ATPase activities of Rad51by these complexes.
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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
Having managed to optimize efficient protocols to purify Rad55-Rad57 and the Shu complex, I have initiated their biochemical analysis. Rad51 is a single-stranded DNA-dependent ATPase, which plays a key role in the strand exchange reaction, a central reaction in HR. Rad55-Rad57 heterodimer and Shu complex both contain Walker A motifs, which is a signature of ATPase activity. Thus, I first demonstrated that both complexes can indeed hydrolyze ATP. Furthermore, I have determined that Rad55-Rad57 binds conventional DNA structures with relatively high affinity, whereas the Shu complex does so only weakly. These results comprise the initial biochemical characterization of Rad55-Rad57 and Shu complex from S. pombe. Therefore, I assess my progress to be much better than the initial plan.
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Strategy for Future Research Activity |
I will try several in vitro experiments to assess the functions of Rad55-Rad57 and Shu complex towards Rad51. Assays such as D-loop, which measures early HR, and strand exchange, which measures mid HR, will be utilized. Testing the DNA binding affinity of Shu complex to a variety of DNA substrates is necessary in understanding the involvement of this complex in Rad51 activation. In addition, combing the in vivo phenotype with in vitro experiments will elucidate the physiological relevance of each of the complexes in the context of Rad51-mediated strand exchange. In summary, by better understanding the molecular roles of these complexes in Rad51-driven strand exchange, I can gain new insights into MTS, as well as other biological processes that require HR.
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Research Products
(1 results)