题 目: FRET-based real-time analysis provides new insights into Rad51-driven DNA strand exchange reaction
报告人: Professor, Hiroshi Iwasaki
Tokyo Institute of Technology
地 点: ZOOM线上报告
主持人: 齐志 研究员
Homologous recombination (HR) is a critical mechanism responsible for accurately DNA double-strand breaks (DSBs) and for restoring stalled DNA replication forks. Rad51, the RecA-family recombinase, is the key protein that drives HR in eukaryotic cells. Several auxiliary factors interact with Rad51 to potentiate its activity including Rad52, Rad54, the Rad51 paralogs Rad55-Rad57, and Swi5-Sfr1.
HR is initiated by the formation of 3’ single-stranded DNA (ssDNA) at the DSB site. This ssDNA is first bound by RPA, then by the Rad51, which forms a right-handed nucleoprotein filament. This filament named presynaptic filament captures double-stranded DNA (dsDNA), searches for homology, and promotes the transfer of the complementary strand when homology is found, producing a new heteroduplex.
We previously developed a real-time assay to monitor DNA strand exchange reaction by fission yeast Rad51. The result shows that strand exchange by Rad51 proceeds via two distinct three-strand intermediates, C1 and C2. The first complex C1 contains the intact donor duplex. On the other hand, the second complex C2 contains newly formed heteroduplex DNA (ref 1). RecA-family recombinases including Rad51 possess the conserved DNA binding sites, Site I and Site II. Site I contains the conserved motifs, loop 1 (L1) and loop 2 (L2). Our mutant analysis with a mutation of the conserved Arg in L1 suggests that L1 is involved in the C1 complex formation. On the other hand, mutant analysis with a mutation of the conserved Val in L2 suggests that L2 mediates the C1-C2 transition, producing the heteroduplex. In addition, mutant analysis suggests that Site II serves as the DNA entry position for initial Rad51 filament formation, as well as for donor dsDNA incorporation (ref 2).
In addition to this mechanism, I will also discuss the molecular role of the Swi5-Sfr1 complex, a Rad51 auxiliary factor, in the strand exchange reaction.
1. Two three-strand intermediates are processed during Rad51-driven DNA strand exchange. Ito K, Murayama Y, Takahashi M, Iwasaki H. Nature Struct Mole Biol (2018) 25: 29–36.
2. Real-time tracking reveals catalytic roles for the two DNA binding sites of Rad51. Ito K, Murayama Y, Kurokawa Y, Kanamaru S, Kokabu Y, Maki T, Argunhan B, Tsubouchi H, Ikeguchi M, Takahashi M, Iwasaki H. Nat Commun. (2020) 11. 2950.
Professor Hiroshi Iwasaki obtained his Ph.D. and started his faculty position in Osaka University in 1991. In 2001, he joined and spent eight years in Yokohama City University, where he had been a Professor from 2007. In 2009, Prof. Iwasaki began to work in the School and Graduate School of Bioscience and Biotechnology at Tokyo Institute of Technology. In 2016, he turned to Institute of Innovative Research of Tokyo Institute of Technology. Prof. Iwasaki is also Adjunct Professor of Yokohama City University and the president of the Genetics Society of Japan. His laboratory is studying on molecular mechanism of homologous recombination.