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Kentaro Ito, Yasuto Murayama, Masayuki Takahashi, Hiroshi Iwasaki
During homologous recombination, Rad51 forms a nucleoprotein filament with single-stranded DNA (ssDNA) that undergoes strand exchange with homologous double-stranded DNA (dsDNA). Here, we use real-time analysis to show that strand exchange by fission yeast Rad51 proceeds via two distinct three-strand intermediates, C1 and C2. Both intermediates contain Rad51, but whereas the donor duplex remains intact in C1, the ssDNA strand is intertwined with the complementary strand of the donor duplex in C2. Swi5-Sfr1, an evolutionarily conserved recombination activator, facilitates the C1-C2 transition and subsequent ssDNA release from C2 to complete strand exchange in an ATP-hydrolysis-dependent manner...
January 2018: Nature Structural & Molecular Biology
Bilge Argunhan, Yasuto Murayama, Hiroshi Iwasaki
Homologous recombination (HR) is the process whereby two DNA molecules that share high sequence similarity are able to recombine to generate hybrid DNA molecules. Throughout evolution, the ability of HR to identify highly similar DNA sequences has been adopted for numerous biological phenomena including DNA repair, meiosis, telomere maintenance, ribosomal DNA amplification and immunological diversity. Although Rad51 and Dmc1 are the key proteins that promote HR in mitotic and meiotic cells, respectively, accessory proteins that allow Rad51 and Dmc1 to effectively fulfil their functions have been identified in all examined model systems...
July 2017: FEBS Letters
Guan-Chin Su, Hsin-Yi Yeh, Sheng-Wei Lin, Chan-I Chung, Yu-Shan Huang, Yi-Chung Liu, Ping-Chiang Lyu, Peter Chi
During DNA double-strand break and replication fork repair by homologous recombination, the RAD51 recombinase catalyzes the DNA strand exchange reaction via a helical polymer assembled on single-stranded DNA, termed the presynaptic filament. Our published work has demonstrated a dual function of the SWI5-SFR1 complex in RAD51-mediated DNA strand exchange, namely, by stabilizing the presynaptic filament and maintaining the catalytically active ATP-bound state of the filament via enhancement of ADP release. In this study, we have strived to determine the basis for physical and functional interactions between Mus musculus SWI5-SFR1 and RAD51...
July 27, 2016: Nucleic Acids Research
Alexander Lorenz, Alizée Mehats, Fekret Osman, Matthew C Whitby
During meiosis programmed DNA double-strand breaks (DSBs) are repaired by homologous recombination using the sister chromatid or the homologous chromosome (homolog) as a template. This repair results in crossover (CO) and non-crossover (NCO) recombinants. Only CO formation between homologs provides the physical linkages guiding correct chromosome segregation, which are essential to produce healthy gametes. The factors that determine the CO/NCO decision are still poorly understood. Using Schizosaccharomyces pombe as a model we show that the Rad51/Dmc1-paralog complexes Rad55-Rad57 and Rdl1-Rlp1-Sws1 together with Swi5-Sfr1 play a major role in antagonizing both the FANCM-family DNA helicase/translocase Fml1 and the RecQ-type DNA helicase Rqh1 to limit hybrid DNA formation and promote Mus81-Eme1-dependent COs...
December 16, 2014: Nucleic Acids Research
Yasuhiro Tsutsui, Yumiko Kurokawa, Kentaro Ito, Md Shahjahan P Siddique, Yumiko Kawano, Fumiaki Yamao, Hiroshi Iwasaki
Fbh1, an F-box helicase related to bacterial UvrD, has been proposed to modulate homologous recombination in fission yeast. We provide several lines of evidence for such modulation. Fbh1, but not the related helicases Srs2 and Rqh1, suppressed the formation of crossover recombinants from single HO-induced DNA double-strand breaks. Purified Fbh1 in complex with Skp1 (Fbh1-Skp1 complex) inhibited Rad51-driven DNA strand exchange by disrupting Rad51 nucleoprotein filaments in an ATP-dependent manner; this disruption was alleviated by the Swi5-Sfr1 complex, an auxiliary activator of Rad51...
August 2014: PLoS Genetics
Louise H Fornander, Axelle Renodon-Cornière, Naoyuki Kuwabara, Kentaro Ito, Yasuhiro Tsutsui, Toshiyuki Shimizu, Hiroshi Iwasaki, Bengt Nordén, Masayuki Takahashi
The Swi5-Sfr1 heterodimer protein stimulates the Rad51-promoted DNA strand exchange reaction, a crucial step in homologous recombination. To clarify how this accessory protein acts on the strand exchange reaction, we have analyzed how the structure of the primary reaction intermediate, the Rad51/single-stranded DNA (ssDNA) complex filament formed in the presence of ATP, is affected by Swi5-Sfr1. Using flow linear dichroism spectroscopy, we observe that the nucleobases of the ssDNA are more perpendicularly aligned to the filament axis in the presence of Swi5-Sfr1, whereas the bases are more randomly oriented in the absence of Swi5-Sfr1...
February 2014: Nucleic Acids Research
Yasuto Murayama, Yumiko Kurokawa, Yasuhiro Tsutsui, Hiroshi Iwasaki
Both ubiquitously expressed Rad51 and meiosis-specific Dmc1 are required for crossover production during meiotic recombination. The budding yeast Rad52 and its fission yeast ortholog, Rad22, are "mediators;" i.e., they help load Rad51 onto ssDNA coated with replication protein A (RPA). Here we show that the Swi5-Sfr1 complex from fission yeast is both a mediator that loads Dmc1 onto ssDNA and a direct "activator" of DNA strand exchange by Dmc1. In stark contrast, Rad22 inhibits Dmc1 action by competing for its binding to RPA-coated ssDNA...
November 1, 2013: Genes & Development
Guan-Chin Su, Chan-I Chung, Chia-Yu Liao, Sheng-Wei Lin, Cheng-Ting Tsai, Tao Huang, Hung-Wen Li, Peter Chi
Homologous recombination catalyzed by the RAD51 recombinase eliminates deleterious DNA lesions from the genome. In the presence of ATP, RAD51 forms a nucleoprotein filament on single-stranded DNA, termed the presynaptic filament, to initiate homologous recombination-mediated DNA double-strand break repair. The SWI5-SFR1 complex stabilizes the presynaptic filament and enhances its ability to mediate the homologous DNA pairing reaction. Here we characterize the RAD51 presynaptic filament stabilization function of the SWI5-SFR1 complex using optical tweezers...
January 2014: Nucleic Acids Research
Dorota Dziadkowiec, Karol Kramarz, Karolina Kanik, Piotr Wisniewski, Antony M Carr
Previously we identified Rrp1 and Rrp2 as two proteins required for the Sfr1/Swi5-dependent branch of homologous recombination (HR) in Schizosaccharomyces pombe. Here we use a yeast two-hybrid approach to demonstrate that Rrp1 and Rrp2 can interact with each other and with Swi5, an HR mediator protein. Rrp1 and Rrp2 form co-localizing methyl methanesulphonate-induced foci in nuclei, further suggesting they function as a complex. To place the Rrp1/2 proteins more accurately within HR sub-pathways, we carried out extensive epistasis analysis between mutants defining Rrp1/2, Rad51 (recombinase), Swi5 and Rad57 (HR-mediators) plus the anti-recombinogenic helicases Srs2 and Rqh1...
September 2013: Nucleic Acids Research
Kazumi Saikusa, Naoyuki Kuwabara, Yuichi Kokabu, Yu Inoue, Mamoru Sato, Hiroshi Iwasaki, Toshiyuki Shimizu, Mitsunori Ikeguchi, Satoko Akashi
It is now recognized that intrinsically disordered proteins (IDPs) play important roles as hubs in intracellular networks, and their structural characterisation is of significance. However, due to their highly dynamic features, it is challenging to investigate the structures of IDPs solely by conventional methods. In the present study, we demonstrate a novel method to characterise protein complexes using electrospray ionization ion mobility mass spectrometry (ESI-IM-MS) in combination with small-angle X-ray scattering (SAXS)...
March 7, 2013: Analyst
Alexander Lorenz, Fekret Osman, Weili Sun, Saikat Nandi, Roland Steinacher, Matthew C Whitby
The formation of healthy gametes depends on programmed DNA double-strand breaks (DSBs), which are each repaired as a crossover (CO) or non-crossover (NCO) from a homologous template. Although most of these DSBs are repaired without giving COs, little is known about the genetic requirements of NCO-specific recombination. We show that Fml1, the Fanconi anemia complementation group M (FANCM)-ortholog of Schizosaccharomyces pombe, directs the formation of NCOs during meiosis in competition with the Mus81-dependent pro-CO pathway...
June 22, 2012: Science
Shang-Pu Tsai, Guan-Chin Su, Sheng-Wei Lin, Chan-I Chung, Xiaoyu Xue, Myun Hwa Dunlop, Yufuko Akamatsu, Maria Jasin, Patrick Sung, Peter Chi
Homologous recombination (HR) represents a major error-free pathway to eliminate pre-carcinogenic chromosomal lesions. The DNA strand invasion reaction in HR is mediated by a helical filament of the Rad51 recombinase assembled on single-stranded DNA that is derived from the nucleolytic processing of the primary lesion. Recent studies have found that the human and mouse Swi5 and Sfr1 proteins form a complex that influences Rad51-mediated HR in cells. Here, we provide biophysical evidence that the mouse Swi5-Sfr1 complex has a 1:1 stoichiometry...
August 2012: Nucleic Acids Research
Naoyuki Kuwabara, Yasuto Murayama, Hiroshi Hashimoto, Yuuichi Kokabu, Mitsunori Ikeguchi, Mamoru Sato, Kouta Mayanagi, Yasuhiro Tsutsui, Hiroshi Iwasaki, Toshiyuki Shimizu
Rad51 forms a helical filament on single-stranded DNA and promotes strand exchange between two homologous DNA molecules during homologous recombination. The Swi5-Sfr1 complex interacts directly with Rad51 and stimulates strand exchange. Here we describe structural and functional aspects of the complex. Swi5 and the C-terminal core domain of Sfr1 form an essential activator complex with a parallel coiled-coil heterodimer joined firmly together via two previously uncharacterized leucine-zipper motifs and a bundle...
March 7, 2012: Structure
Yuichi Kokabu, Yasuto Murayama, Naoyuki Kuwabara, Tomotaka Oroguchi, Hiroshi Hashimoto, Yasuhiro Tsutsui, Naohito Nozaki, Satoko Akashi, Satoru Unzai, Toshiyuki Shimizu, Hiroshi Iwasaki, Mamoru Sato, Mitsunori Ikeguchi
In eukaryotes, DNA strand exchange is the central reaction of homologous recombination, which is promoted by Rad51 recombinases forming a right-handed nucleoprotein filament on single-stranded DNA, also known as a presynaptic filament. Accessory proteins known as recombination mediators are required for the formation of the active presynaptic filament. One such mediator in the fission yeast Schizosaccharomyces pombe is the Swi5-Sfr1 complex, which has been identified as an activator of Rad51 that assists in presynaptic filament formation and stimulates its strand exchange reaction...
December 16, 2011: Journal of Biological Chemistry
Yasuto Murayama, Yasuhiro Tsutsui, Hiroshi Iwasaki
Homologous recombination proceeds via the formation of several intermediates including Holliday junctions (HJs), which are important for creating crossover products. DNA strand exchange is a core reaction that produces these intermediates that is directly catalyzed by RecA family recombinases, of which there are two types in eukaryotes: universal Rad51 and meiosis-specific Dmc1. We demonstrated previously that Rad51 promotes four-strand exchange, mimicking the formation and branch migration of HJs. Here we show that Dmc1 from fission yeast has a similar activity, which requires ATP hydrolysis and is independent of an absolute requirement for the Swi5-Sfr1 complex...
March 1, 2011: Genes & Development
Jingsong Yuan, Junjie Chen
The Swi5-Mei5 complex and its homologues are involved in specialized recombination pathways in budding and fission yeasts. Although the fission yeast homologue Swi5-Sfr1 is critical for homologous recombination repair, the budding yeast counterpart Sae3-Mei5 is meiosis-specific, interacts with Dmc1, and promotes assembly of Dmc1 on meiotic chromosomes. Here, we identify and characterize the human SWI5-MEI5 (C9orf119-C10orf78) complex. We showed that SWI5 and MEI5 form a stable complex in vitro and in vivo. The C-terminal Swi5 domain of SWI5 and the middle coiled-coil region of MEI5 dictate this conserved interaction...
March 18, 2011: Journal of Biological Chemistry
Yufuko Akamatsu, Maria Jasin
In fission yeast, the Swi5-Sfr1 complex plays an important role in homologous recombination (HR), a pathway crucial for the maintenance of genomic integrity. Here we identify and characterize mammalian Swi5 and Sfr1 homologues. Mouse Swi5 and Sfr1 are nuclear proteins that form a complex in vivo and in vitro. Swi5 interacts in vitro with Rad51, the DNA strand-exchange protein which functions during HR. By generating Swi5(-/-) and Sfr1(-/-) embryonic stem cell lines, we found that both proteins are mutually interdependent for their stability...
October 14, 2010: PLoS Genetics
Naoyuki Kuwabara, Hiroshi Hashimoto, Noriyo Yamada, Satoru Unzai, Mitsunori Ikeguchi, Mamoru Sato, Yasuto Murayama, Hiroshi Iwasaki, Toshiyuki Shimizu
The assembly of the presynaptic filament of recombinases represents the most important step in homologous recombination. The formation of the filament requires assistance from mediator proteins. Swi5 and Sfr1 have been identified as mediators in fission yeast and these proteins form a complex that stimulates strand exchange. Here, the expression, purification and crystallization of Swi5 and its complex with an N-terminally truncated form of Sfr1 (DeltaN180Sfr1) are presented. Analytical ultracentrifugation of the purified samples showed that Swi5 and the protein complex exist as tetramers and heterodimers in solution, respectively...
September 1, 2010: Acta Crystallographica. Section F, Structural Biology and Crystallization Communications
Lubos Cipak, Mario Spirek, Maria Novatchkova, Zhiming Chen, Cornelia Rumpf, Wolfgang Lugmayr, Karl Mechtler, Gustav Ammerer, Edina Csaszar, Juraj Gregan
Tandem affinity purification (TAP) is a method that allows rapid purification of native protein complexes. We developed an improved technique to fuse the fission yeast genes with a TAP tag. Our technique is based on tagging constructs that contain regions homologous to the target gene cloned into vectors carrying a TAP tag. We used this technique to design strategies for TAP-tagging of predicted Schizosaccharomyces pombe genes ( To validate the approach, we purified the proteins, which associated with two evolutionarily conserved proteins Swi5 and Sfr1 as well as three protein kinases Ksg1, Orb6 and Sid1...
October 2009: Proteomics
Dorota Dziadkowiec, Edyta Petters, Agnieszka Dyjankiewicz, Paweł Karpiński, Valerie Garcia, Adam Watson, Antony M Carr
We identified two predicted proteins in Schizosaccharomyces pombe, Rrp1 (SPAC17A2.12) and Rrp2 (SPBC23E6.02) that share 34% and 36% similarity to Saccharomyces cerevisiae Ris1p, respectively. Ris1p is a DNA-dependent ATP-ase involved in gene silencing and DNA repair. Rrp1 and Rrp2 also share similarity with S. cerevisiae Rad5 and S. pombe Rad8, containing SNF2-N, RING finger and Helicase-C domains. To investigate the function of the Rrp proteins, we studied the DNA damage sensitivities and genetic interactions of null mutants with known DNA repair mutants...
May 1, 2009: DNA Repair
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