Title:
Molecular mechanism of recombinase-mediated strand exchange reaction, a central reaction of homologous recombination

Hiroshi Iwasaki, PhD
School and Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology

Abstract:
In general, recombinases bind to single-stranded DNA (ssDNA), leading to a formation of a helical presynaptic filament that promotes homology search and homologous pairing of a complementary DNA strand. This reaction is a fundamental reaction of homologous recombination and the elementary step of it is called as “DNA strand exchange”. We have established an in vitro reconstitution of the central step of DNA stand exchange, which consists of purified proteins, including Rad51 recombinase (which is also called as Rhp51), the two mediators (Rad22 and the Swi5-Sfr1 protein complex) and replication protein A (RPA) from fission yeast and model substrate DNAs. Our genetic and biochemical studies have demonstrated that the Swi5-Sfr1 protein complex is an activator and stabilizer of Rad51 recombinase. Rad22 (fission yeast Rad52 homologue) alone cannot activate the Rad51 presynaptic filament, but rather it seems to work as a recruiter to target Rad51 onto RPA-bound ssDNA regions (1, 2, 3). In addition, we have demonstrated in vitro formation of Holliday junction, which is a common DNA intermediate of Homologous recombination (4). I will discuss about the molecular mechanisms of these strand-exchange-based processes in this session.

References:
1.Reconstitution of DNA strand exchange mediated by Rhp51 recombinase and two mediators. Kurokawa Y, Murayama Y, Haruta-Takahashi N, Urabe I and Iwasaki H. PLoS Biol (2008) 6: e88.

2. The Swi5-Sfr1 complex stimulates Rad51- and Dmc1-mediated DNA strand exchange in vitro . Haruta N, Kurokawa Y, Murayama Y, Akamatsu Y, Unzai S, Tsutsui Y and Iwasaki H. Nature Struct. Mol. Biol. (2006) 13: 823-730.

3. Two different Swi5-containing protein complexes are involved in mating-type switching and recombination repair in fission yeast. Akamatsu Y, Dziadkowiec D, Ikeguchi M, Shinagawa H and Iwasaki H. Proc. Natl. Acad. Sci. USA. (2003) 100: 15770-15775.

4. Formation and branch migration of Holliday junctions mediated by eukaryotic recombinases .Murayama Y, Kurokawa Y, Mayanagi K and Iwasaki H. Nature (2008) 451: 1018-1021.