KINDAI UNIVERSITY


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SHINOHARA Miki

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FacultyDepartment of Advanced Bioscience / Graduate School of Agriculture / KINDAI Research Institute for Agricultural Technology and Innovation
PositionProfessor
DegreePh.D in Medicine
Commentator Guidehttps://www.kindai.ac.jp/meikan/2071-shinohara-miki.html
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Last Updated :2020/09/03

Education and Career

Education

  •   1988 04  - 1992 03 , Osaka University, School of Science
  •   1992 04  - 1994 03 , Osaka University, Graduate School of Science
  •   1994 04  - 1998 03 , Osaka University, Graduate School of Medicine

Academic & Professional Experience

  •   2017 ,  - 現在, Faculty of Agriculture, Kindai University
  •   2007 ,  - 2017 , Institute for Protein Research, Osaka University
  •   2004 ,  - 2007 , Institute for Protein Research, Osaka University
  •   2001 ,  - 2004 , RIRBM, Hiroshima University
  •   1998 ,  - 2000 , HFSP Long-term Fellow, Radiation Oncology, University of Chicago
  •   1998 ,  - 1998 , JSPS Research fellow PD, National Institute of Genetics
  •   1996 ,  - 1998 , JSPS research fellow DC, Graduate School of Medicine, Osaka University

Research Activities

Research Areas

  • Life sciences, Genetics
  • Life sciences, Molecular biology

Published Papers

  • Distinct Functions in Regulation of Meiotic Crossovers for DNA Damage Response Clamp Loader Rad24(Rad17) and Mec1(ATR) Kinase, Shinohara, Miki, Bishop, Douglas K., Shinohara, Akira, GENETICS, GENETICS, 213(4), 1255 - 1269, Dec. 2019 , Refereed
    Summary:The number and distribution of meiotic crossovers (COs) are highly regulated, reflecting the requirement for COs during the first round of meiotic chromosome segregation. CO control includes CO assurance and CO interference, which promote at least one CO per chromosome bivalent and evenly-spaced COs, respectively. Previous studies revealed a role for the DNA damage response (DDR) clamp and the clamp loader in CO formation by promoting interfering COs and interhomolog recombination, and also by suppressing ectopic recombination. In this study, we use classical tetrad analysis of Saccharomyces cerevisiae to show that a mutant defective in , which encodes the DDR clamp loader ( in other organisms), displayed reduced CO frequencies on two shorter chromosomes (III and V), but not on a long chromosome (chromosome VII). The residual COs in the mutant do not show interference. In contrast to , mutants defective in the ATR kinase homolog , including a null and a kinase-dead mutant, show slight or few defects in CO frequency. On the other hand, COs show defects in interference, similar to the mutant. Our results support a model in which the DDR clamp and clamp-loader proteins promote interfering COs by recruiting pro-CO Zip, Mer, and Msh proteins to recombination sites, while the kinase regulates CO distribution by a distinct mechanism. Moreover, CO formation and its control are implemented in a chromosome-specific manner, which may reflect a role for chromosome size in regulation.
  • Meiotic prophase-like pathway for cleavage-independent removal of cohesin for chromosome morphogenesis, Kiran Challa, M. Shinohara, Shinohara Akira, Current Genetics, Current Genetics, 65(4), 817 - 827, Aug. 2019 , Refereed
  • Meiosis-specific cohesin component, Rec8, promotes the localization of Mps3 SUN domain protein on the nuclear envelope, Bommi, Jagadeeswara Rao, Rao, Hanumanthu Bala Durga Prasada, Challa, Kiran, Higashide, Mika, Shinmyozu, Kaori, Nakayama, Jun-ichi, Shinohara, Miki, Shinohara, Akira, GENES TO CELLS, GENES TO CELLS, 24(1), 94 - 106, Jan. 2019 , Refereed
    Summary:Proteins in the nuclear envelope (NE) play a role in the dynamics and functions of the nucleus and of chromosomes during mitosis and meiosis. Mps3, a yeast NE protein with a conserved SUN domain, predominantly localizes on a yeast centrosome equivalent, spindle pole body (SPB), in mitotic cells. During meiosis, Mps3, together with SPB, forms a distinct multiple ensemble on NE. How meiosis-specific NE localization of Mps3 is regulated remains largely unknown. In this study, we found that a meiosis-specific component of the protein complex essential for sister chromatid cohesion, Rec8, binds to Mps3 during meiosis and controls Mps3 localization and proper dynamics on NE. Ectopic expression of Rec8 in mitotic yeast cells induced the formation of Mps3 patches/foci on NE. This required the cohesin regulator, WAPL ortholog, Rad61/Wpl1, suggesting that a meiosis-specific cohesin complex with Rec8 controls NE localization of Mps3. We also observed that two domains of the nucleoplasmic region of Mps3 are essential for NE localization of Mps3 in mitotic as well as meiotic cells. We speculate that the interaction of Mps3 with the meiosis-specific cohesin in the nucleoplasm is a key determinant for NE localization/function of Mps3.
  • Casein kinase II phosphorylates the C-terminal region of Lif1 to promote the Lif1-Xrs2 interaction needed for non-homologous end joining, Matsuzaki, Kenichiro, Shinohara, Miki, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 501(4), 1080 - 1084, Jul. 2018 , Refereed
    Summary:A DNA double strand break (DSB) is one of the most cytotoxic DNA lesions, but it can be repaired by non homologous end joining (NHEJ) or by homologous recombination. The choice between these two repair pathways depends on the cell cycle stage. Although NHEJ constitutes a simple re-ligation reaction, the regulatory mechanism(s) controlling its activity has not been fully characterized. Lif1 is a regulatory subunit of the NHEJ-specific DNA ligase IV and interacts with Xrs2 of the MRX complex which is a key factor in DSB repair. Specifically, the C-terminal region of Lif1, which contains a CK2-specific phosphorylation motif, interacts with the FHA domain of Xrs2 during canonical-NHEJ (C-NHEJ). Herein, we show that Lif1 and Cka2, a catalytic subunit of yeast CK2, interact and that the C-terminal phosphorylation consensus motif in Lift is phosphorylated by recombinant CK2. These observations suggest that phosphorylation of Lift by CK2 at a DSB site promotes the Lif1-Xrs2 interaction and facilitates C-NHEJ. (C) 2018 The Authors. Published by Elsevier Inc.
  • Budding Yeast SLX4 Contributes to the Appropriate Distribution of Crossovers and Meiotic Double-Strand Break Formation on Bivalents During Meiosis, Higashide, Mika, Shinohara, Miki, G3-GENES GENOMES GENETICS, G3-GENES GENOMES GENETICS, 6(7), 2033 - 2042, Jul. 2016 , Refereed
    Summary:The number and distribution of meiosis crossover (CO) events on each bivalent are strictly controlled by multiple mechanisms to assure proper chromosome segregation during the first meiotic division. In Saccharomyces cerevisiae, Slx4 is a multi-functional scaffold protein for structure-selective endonucleases, such as Slx1 and Rad1 (which are involved in DNA damage repair), and is also a negative regulator of the Rad9-dependent signaling pathway with Rtt107. Slx4 has been believed to play only a minor role in meiotic recombination. Here, we report that Slx4 is involved in proper intrachromosomal distribution of meiotic CO formation, especially in regions near centromeres. We observed an increase in uncontrolled CO formation only in a region near the centromere in the slx4 Delta mutant. Interestingly, this phenomenon was not observed in the slx1 Delta, rad1 Delta, or rtt107 Delta mutants. In addition, we observed a reduced number of DNA double-strand breaks (DSBs) and altered meiotic DSB distribution on chromosomes in the slx4 Delta mutant. This suggests that the multi-functional Slx4 is required for proper CO formation and meiotic DSB formation.
  • The MRX Complex Ensures NHEJ Fidelity through Multiple Pathways Including Xrs2-FHA-Dependent Tel1 Activation, Iwasaki, Daichi, Hayashihara, Kayoko, Shima, Hiroki, Higashide, Mika, Terasawa, Masahiro, Gasser, Susan M., Shinohara, Miki, PLOS GENETICS, PLOS GENETICS, 12(3), e1005942, Mar. 2016 , Refereed
    Summary:Because DNA double-strand breaks (DSBs) are one of the most cytotoxic DNA lesions and often cause genomic instability, precise repair of DSBs is vital for the maintenance of genomic stability. Xrs2/Nbs1 is a multi-functional regulatory subunit of the Mre11-Rad50-Xrs2/Nbs1 (MRX/N) complex, and its function is critical for the primary step of DSB repair, whether by homologous recombination (HR) or non-homologous end joining. In human NBS1, mutations result truncation of the N-terminus region, which contains a forkhead-associated (FHA) domain, cause Nijmegen breakage syndrome. Here we show that the Xrs2 FHA domain of budding yeast is required both to suppress the imprecise repair of DSBs and to promote the robust activation of Tel1 in the DNA damage response pathway. The role of the Xrs2 FHA domain in Tel1 activation was independent of the Tel1-binding activity of the Xrs2 C terminus, which mediates Tel1 recruitment to DSB ends. Both the Xrs2 FHA domain and Tel1 were required for the timely removal of the Ku complex from DSB ends, which correlates with a reduced frequency of imprecise end-joining. Thus, the Xrs2 FHA domain and Tel1 kinase work in a coordinated manner to maintain DSB repair fidelity.
  • Chromosome Synapsis Alleviates Mek1-Dependent Suppression of Meiotic DNA Repair, Subramanian, Vijayalakshmi V., MacQueen, Amy J., Vader, Gerben, Shinohara, Miki, Sanchez, Aurore, Borde, Valerie, Shinohara, Akira, Hochwagen, Andreas, PLOS BIOLOGY, PLOS BIOLOGY, 14(2), e1002369, Feb. 2016 , Refereed
    Summary:Faithful meiotic chromosome segregation and fertility require meiotic recombination between homologous chromosomes rather than the equally available sister chromatid, a bias that in Saccharomyces cerevisiae depends on the meiotic kinase, Mek1. Mek1 is thought to mediate repair template bias by specifically suppressing sister-directed repair. Instead, we found that when Mek1 persists on closely paired ( synapsed) homologues, DNA repair is severely delayed, suggesting that Mek1 suppresses any proximal repair template. Accordingly, Mek1 is excluded from synapsed homologues in wild-type cells. Exclusion requires the AAA(+)-ATPase Pch2 and is directly coupled to synaptonemal complex assembly. Stage-specific depletion experiments further demonstrate that DNA repair in the context of synapsed homologues requires Rad54, a repair factor inhibited by Mek1. These data indicate that the sister template is distinguished from the homologue primarily by its closer proximity to inhibitory Mek1 activity. We propose that once pairing or synapsis juxtaposes homologues, exclusion of Mek1 is necessary to avoid suppression of all templates and accelerate repair progression.
  • Canonical Non-Homologous End Joining in Mitosis Induces Genome Instability and Is Suppressed by M-phase-Specific Phosphorylation of XRCC4, Terasawa, Masahiro, Shinohara, Akira, Shinohara, Miki, PLOS GENETICS, PLOS GENETICS, 10(8), e1004563, Aug. 2014 , Refereed
    Summary:DNA double-strand breaks (DSBs) can be repaired by one of two major pathways-non-homologous end-joining (NHEJ) and homologous recombination (HR)-depending on whether cells are in G1 or S/G2 phase, respectively. However, the mechanisms of DSB repair during M phase remain largely unclear. In this study, we demonstrate that transient treatment of M-phase cells with the chemotherapeutic topoisomerase inhibitor etoposide induced DSBs that were often associated with anaphase bridge formation and genome instability such as dicentric chromosomes. Although most of the DSBs were carried over into the next G1 phase, some were repaired during M phase. Both NHEJ and HR, in particular NHEJ, promoted anaphase-bridge formation, suggesting that these repair pathways can induce genome instability during M phase. On the other hand, C-terminal-binding protein interacting protein (CtIP) suppressed anaphase bridge formation, implying that CtIP function prevents genome instability during mitosis. We also observed M-phase-specific phosphorylation of XRCC4, a regulatory subunit of the ligase IV complex specialized for NHEJ. This phosphorylation required cyclin-dependent kinase (CDK) activity as well as polo-like kinase 1 (Plk1). A phosphorylation-defective XRCC4 mutant showed more efficient M-phase DSB repair accompanied with an increase in anaphase bridge formation. These results suggest that phosphorylation of XRCC4 suppresses DSB repair by modulating ligase IV function to prevent genome instability during M phase. Taken together, our results indicate that XRCC4 is required not only for the promotion of NHEJ during interphase but also for its M-phase-specific suppression of DSB repair.
  • Dot1-Dependent Histone H3K79 Methylation Promotes the Formation of Meiotic Double-Strand Breaks in the Absence of Histone H3K4 Methylation in Budding Yeast, Ismail, Mohammad Bani, Shinohara, Miki, Shinohara, Akira, PLOS ONE, PLOS ONE, 9(5), e96648, May 2014 , Refereed
    Summary:Epigenetic marks such as histone modifications play roles in various chromosome dynamics in mitosis and meiosis. Methylation of histones H3 at positions K4 and K79 is involved in the initiation of recombination and the recombination checkpoint, respectively, during meiosis in the budding yeast. Set1 promotes H3K4 methylation while Dot1 promotes H3K79 methylation. In this study, we carried out detailed analyses of meiosis in mutants of the SET1 and DOT1 genes as well as methylation-defective mutants of histone H3. We confirmed the role of Set1-dependent H3K4 methylation in the formation of double-strand breaks (DSBs) in meiosis for the initiation of meiotic recombination, and we showed the involvement of Dot1 (H3K79 methylation) in DSB formation in the absence of Set1-dependent H3K4 methylation. In addition, we showed that the histone H3K4 methylation-defective mutants are defective in SC elongation, although they seem to have moderate reduction of DSBs. This suggests that high levels of DSBs mediated by histone H3K4 methylation promote SC elongation.
  • Mps3 SUN domain is important for chromosome motion and juxtaposition of homologous chromosomes during meiosis, Rao, Hanumanthu B. D. Prasada, Shinohara, Miki, Shinohara, Akira, GENES TO CELLS, GENES TO CELLS, 16(11), 1081 - 1096, Nov. 2011 , Refereed
    Summary:In budding yeast, Mps3 is essential for duplicating the spindle pole body (SPB) and is critical for promoting chromosome motion during meiosis. It is a member of the SUN (Sad1-Unc-84) domain family of proteins that localizes to the inner nuclear envelope (NE) in many eukaryotic organisms and preferentially localizes to the SPB in vegetative growth; in meiotic prophase I, it redistributes to many sites within the NE. We constructed an mps3 mutant, mps3-sun, which completely lacks the SUN domain. Surprisingly, the mps3-sun mutation does not disrupt SPB duplication or Mps3 localization to the NE in meiosis. However, it confers several defects during meiotic prophase I including reduced chromosome motion, premature synapsis between homologous chromosomes, and reduced levels of closely juxtaposed homologous loci in pachytene. These findings suggest that in meiosis, the Mps3 SUN domain is important for modulating chromosome motion events that act in meiotic chromosome juxtaposition and by extension, promoting proper morphogenesis of the synaptonemal complex.
  • Cyclin-dependent kinase promotes formation of the synaptonemal complex in yeast meiosis, Zhu, Zhihui, Mori, Saori, Oshiumi, Hiroyuki, Matsuzaki, Kenichiro, Shinohara, Miki, Shinohara, Akira, GENES TO CELLS, GENES TO CELLS, 15(10), 1036 - 1050, Oct. 2010 , Refereed
    Summary:Cyclin-dependent protein kinases (CDKs) are required for various cell cycle events both in mitosis and in meiosis. During the meiotic prophase of Saccharomyces cerevisiae, only one CDK, Cdc28, which forms a complex with B-type cyclins, Clb5 or Clb6, promotes not only the onset of premeiotic DNA replication but also the formation of meiotic double-strand breaks (DSBs). In this study, we showed that Cdc28 exhibits punctate staining on chromosomes during meiotic prophase I. Chromosomal localization of Cdc28, dependent on Clb5 and/or Clb6, is frequently observed in zygotene and pachytene, when formation of the synaptonemal complex (SC) occurs. Interestingly, the CDK localization is independent of DSB formation, but rather dependent on meiosis-specific chromosome components such as Red1, Hop1 and a cohesin subunit Rec8. Compromised CDK activity in meiotic prophase leads to defective SC formation without affecting DSB formation. These results suggest that CDK-dependent phosphorylation regulates meiotic chromosome morphogenesis.
  • Genetic Analysis of Baker's Yeast Msh4-Msh5 Reveals a Threshold Crossover Level for Meiotic Viability, Nishant, K. T., Chen, Cheng, Shinohara, Miki, Shinohara, Akira, Alani, Eric, PLOS GENETICS, PLOS GENETICS, 6(8), Aug. 2010 , Refereed
    Summary:During meiosis, the Msh4-Msh5 complex is thought to stabilize single-end invasion intermediates that form during early stages of recombination and subsequently bind to Holliday junctions to facilitate crossover formation. To analyze Msh4-Msh5 function, we mutagenized 57 residues in Saccharomyces cerevisiae Msh4 and Msh5 that are either conserved across all Msh4/5 family members or are specific to Msh4 and Msh5. The Msh5 subunit appeared more sensitive to mutagenesis. We identified msh4 and msh5 threshold (msh4/5-t) mutants that showed wild-type spore viability and crossover interference but displayed, compared to wild-type, up to a two-fold decrease in crossing over on large and medium sized chromosomes (XV, VII, VIII). Crossing over on a small chromosome, however, approached wild-type levels. The msh4/5-t mutants also displayed synaptonemal complex assembly defects. A triple mutant containing a msh4/5-t allele and mutations that decreased meiotic double-strand break levels (spo11-HA) and crossover interference (pch2D) showed synergistic defects in spore viability. Together these results indicate that the baker's yeast meiotic cell does not require the,90 crossovers maintained by crossover homeostasis to form viable spores. They also show that Pch2-mediated crossover interference is important to maintain meiotic viability when crossovers become limiting.
  • Meiotic recombination-related DNA synthesis and its implications for cross-over and non-cross-over recombinant formation, Terasawa, Masahiro, Ogawa, Hideyuki, Tsukamoto, Yasumasa, Shinohara, Miki, Shirahige, Katsuhiko, Kleckner, Nancy, Ogawa, Tomoko, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 104(14), 5965 - 5970, Apr. 2007 , Refereed
    Summary:Meiotic recombination-related DNA synthesis (MRDS) was analyzed in Saccharomyces cerevisiae by specifically timed incorporation of thymidine analogs into chromosomes. Lengths and positions of incorporation tracts were determined relative to a known recombination hot spot along DNA, as was the timing and localization of incorporation relative to forming and formed synaptonemal complex in spread chromosomes. Distinct patterns could be specifically associated with the majority cross-over and non-cross-over recombination processes. The results obtained provide direct evidence for key aspects of current consensus recombination models, provide information regarding temporal and spatial relationships between non-cross-over formation and the synaptonemal complex, and raise the possibility that removal of RecA homolog Rad51 plays a key role in regulating onset of MRDS. Finally, classical observations on MRDS in Drosophila, mouse, and lily are readily mapped onto the findings presented here, providing further evidence for a broadly conserved meiotic recombination process.
  • Genetic Interactions of Histone Modification Machinery Set1 and PAF1C with the Recombination Complex Rec114-Mer2-Mei4 in the Formation of Meiotic DNA Double-Strand Breaks., Ying Zhang, Takuya Suzuki, Ke Li, Santosh K Gothwal, Miki Shinohara, Akira Shinohara, International journal of molecular sciences, International journal of molecular sciences, 21(8), Apr. 12 2020 , Refereed
    Summary:Homologous recombination is essential for chromosome segregation during meiosis I. Meiotic recombination is initiated by the introduction of double-strand breaks (DSBs) at specific genomic locations called hotspots, which are catalyzed by Spo11 and its partners. DSB hotspots during meiosis are marked with Set1-mediated histone H3K4 methylation. The Spo11 partner complex, Rec114-Mer2-Mei4, essential for the DSB formation, localizes to the chromosome axes. For efficient DSB formation, a hotspot with histone H3K4 methylation on the chromatin loops is tethered to the chromosome axis through the H3K4 methylation reader protein, Spp1, on the axes, which interacts with Mer2. In this study, we found genetic interaction of mutants in a histone modification protein complex called PAF1C with the REC114 and MER2 in the DSB formation in budding yeast Saccharomyces cerevisiae. Namely, the paf1c mutations rtf1 and cdc73 showed synthetic defects in meiotic DSB formation only when combined with a wild-type-like tagged allele of either the REC114 or MER2. The synthetic defect of the tagged REC114 allele in the DSB formation was seen also with the set1, but not with spp1 deletion. These results suggest a novel role of histone modification machinery in DSB formation during meiosis, which is independent of Spp1-mediated loop-axis tethering.
  • Enhanced homologous recombination by the modulation of targeting vector ends., Shinji Hirotsune, Hiroshi Kiyonari, Mingyue Jin, Kanako Kumamoto, Kayo Yoshida, Miki Shinohara, Hitomi Watanabe, Anthony Wynshaw-Boris, Fumio Matsuzaki, Scientific reports, Scientific reports, 10(1), 2518 - 2518, Feb. 13 2020 , Refereed
    Summary:The field of genome editing was founded on the establishment of methods, such as the clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein (CRISPR/Cas) system, used to target DNA double-strand breaks (DSBs). However, the efficiency of genome editing also largely depends on the endogenous cellular repair machinery. Here, we report that the specific modulation of targeting vectors to provide 3' overhangs at both ends increased the efficiency of homology-directed repair (HDR) in embryonic stem cells. We applied the modulated targeting vectors to produce homologous recombinant mice directly by pronuclear injection, but the frequency of HDR was low. Furthermore, we combined our method with the CRISPR/Cas9 system, resulting in a significant increase in HDR frequency. Thus, our HDR-based method, enhanced homologous recombination for genome targeting (eHOT), is a new and powerful method for genome engineering.
  • Srs2 helicase prevents the formation of toxic DNA damage during late prophase I of yeast meiosis., Hiroyuki Sasanuma, Hana Subhan M Sakurai, Yuko Furihata, Kiran Challa, Lira Palmer, Susan M Gasser, Miki Shinohara, Akira Shinohara, Chromosoma, Chromosoma, 128(3), 453 - 471, Sep. 2019 , Refereed
    Summary:Proper repair of double-strand breaks (DSBs) is key to ensure proper chromosome segregation. In this study, we found that the deletion of the SRS2 gene, which encodes a DNA helicase necessary for the control of homologous recombination, induces aberrant chromosome segregation during budding yeast meiosis. This abnormal chromosome segregation in srs2 cells accompanies the formation of a novel DNA damage induced during late meiotic prophase I. The damage may contain long stretches of single-stranded DNAs (ssDNAs), which lead to aggregate formation of a ssDNA binding protein, RPA, and a RecA homolog, Rad51, as well as other recombination proteins inside of the nuclei, but not that of a meiosis-specific Dmc1. The Rad51 aggregate formation in the srs2 mutant depends on the initiation of meiotic recombination and occurs in the absence of chromosome segregation. Importantly, as an early recombination intermediate, we detected a thin bridge of Rad51 between two Rad51 foci in the srs2 mutant, which is rarely seen in wild type. These might be cytological manifestation of the connection of two DSB ends and/or multi-invasion. The DNA damage with Rad51 aggregates in the srs2 mutant is passed through anaphases I and II, suggesting the absence of DNA damage-induced cell cycle arrest after the pachytene stage. We propose that Srs2 helicase resolves early protein-DNA recombination intermediates to suppress the formation of aberrant lethal DNA damage during late prophase I.
  • Molecular Camouflage of Plasmodium falciparum Merozoites by Binding of Host Vitronectin to P47 Fragment of SERA5., Takahiro Tougan, Jyotheeswara R Edula, Eizo Takashima, Masayuki Morita, Miki Shinohara, Akira Shinohara, Takafumi Tsuboi, Toshihiro Horii, Scientific reports, Scientific reports, 8(1), 5052 - 5052, Mar. 22 2018 , Refereed
    Summary:The malaria parasite Plasmodium falciparum proliferates in the blood stream where the host immune system is most active. To escape from host immunity, P. falciparum has developed a number of evasion mechanisms. Serine repeat antigen 5 (SERA5) is a blood stage antigen highly expressed at late trophozoite and schizont stages. The P47 N-terminal domain of SERA5, the basis of SE36 antigen of the blood stage vaccine candidate under clinical trials, covers the merozoite surface. Exploring the role of the P47 domain, screening of serum proteins showed that vitronectin (VTN) directly binds to 20 residues in the C-terminal region of SE36. VTN co-localized with P47 domain in the schizont and merozoite stages. Phagocytosis assay using THP-1 cells demonstrated that VTN bound to SE36 prevented engulfment of SE36-beads. In addition, several serum proteins localized on the merozoite surface, suggesting that host proteins camouflage merozoites against host immunity via binding to VTN.
  • RPA Mediates Recruitment of MRX to Forks and Double-Strand Breaks to Hold Sister Chromatids Together, Andrew Seeber, Anna Maria Hegnauer, Nicole Hustedt, Ishan Deshpande, Jerome Poli, Jan Eglinger, Philippe Pasero, Heinz Gut, Miki Shinohara, Karl-Peter Hopfner, Kenji Shimada, Susan M. Gasser, MOLECULAR CELL, MOLECULAR CELL, 64(5), 951 - 966, Dec. 2016 , Refereed
    Summary:The Mre11-Rad50-Xrs2 (MRX) complex is related to SMC complexes that form rings capable of holding two distinct DNA strands together. MRX functions at stalled replication forks and double-strand breaks (DSBs). A mutation in the N-terminal OB fold of the 70 kDa subunit of yeast replication protein A, rfa1-t11, abrogates MRX recruitment to both types of DNA damage. The rfa1 mutation is functionally epistatic with loss of any of the MRX subunits for survival of replication fork stress or DSB recovery, although it does not compromise end-resection. High-resolution imaging shows that either the rfa1-t11 or the rad50D mutation lets stalled replication forks collapse and allows the separation not only of opposing ends but of sister chromatids at breaks. Given that cohesin loss does not provoke visible sister separation as long as the RPA-MRX contacts are intact, we conclude that MRX also serves as a structural linchpin holding sister chromatids together at breaks.
  • Rad61/Wpl1 (Wapl), a cohesin regulator, controls chromosome compaction during meiosis, Challa, Kiran, Lee, Min-Su, Shinohara, Miki, Kim, Keun P., Shinohara, Akira, NUCLEIC ACIDS RESEARCH, NUCLEIC ACIDS RESEARCH, 44(7), 3190 - 3203, Apr. 2016 , Refereed
    Summary:Meiosis-specific cohesin, required for the linking of the sister chromatids, plays a critical role in various chromosomal events during meiotic prophase I, such as chromosome morphogenesis and dynamics, as well as recombination. Rad61/Wpl1 (Wapl in other organisms) negatively regulates cohesin functions. In this study, we show that meiotic chromosome axes are shortened in the budding yeast rad61/wpl1 mutant, suggesting that Rad61/Wpl1 negatively regulates chromosome axis compaction. Rad61/Wpl1 is required for efficient resolution of telomere clustering during meiosis I, indicating a positive effect of Rad61/Wpl1 on the cohesin function required for telomere dynamics. Additionally, we demonstrate distinct activities of Rad61/Wpl1 during the meiotic recombination, including its effects on the efficient processing of intermediates. Thus, Rad61/Wpl1 both positively and negatively regulates various cohesin-mediated chromosomal processes during meiosis.
  • The Double-Strand Break Landscape of Meiotic Chromosomes Is Shaped by the Paf1 Transcription Elongation Complex in Saccharomyces cerevisiae., Santosh K Gothwal, Neem J Patel, Meaghan M Colletti, Hiroyuki Sasanuma, Miki Shinohara, Andreas Hochwagen, Akira Shinohara, Genetics, Genetics, 202(2), 497 - 512, Feb. 2016 , Refereed
    Summary:Histone modification is a critical determinant of the frequency and location of meiotic double-strand breaks (DSBs), and thus recombination. Set1-dependent histone H3K4 methylation and Dot1-dependent H3K79 methylation play important roles in this process in budding yeast. Given that the RNA polymerase II associated factor 1 complex, Paf1C, promotes both types of methylation, we addressed the role of the Paf1C component, Rtf1, in the regulation of meiotic DSB formation. Similar to a set1 mutation, disruption of RTF1 decreased the occurrence of DSBs in the genome. However, the rtf1 set1 double mutant exhibited a larger reduction in the levels of DSBs than either of the single mutants, indicating independent contributions of Rtf1 and Set1 to DSB formation. Importantly, the distribution of DSBs along chromosomes in the rtf1 mutant changed in a manner that was different from the distributions observed in both set1 and set1 dot1 mutants, including enhanced DSB formation at some DSB-cold regions that are occupied by nucleosomes in wild-type cells. These observations suggest that Rtf1, and by extension the Paf1C, modulate the genomic DSB landscape independently of H3K4 methylation.
  • Rad61/Wpl1 (Wapl), a cohesin regulator, controls chromosome compaction during meiosis, Kiran Challa, Min-Su Lee, Miki Shinohara, Keun P. Kim, Akira Shinohara, Nucleic Acids Research, Nucleic Acids Research, 44(7), 3190 - 3203, Jan. 28 2016 , Refereed
    Summary:Meiosis-specific cohesin, required for the linking of the sister chromatids, plays a critical role in various chromosomal events during meiotic prophase I, such as chromosome morphogenesis and dynamics, as well as recombination. Rad61/Wpl1 (Wapl in other organisms) negatively regulates cohesin functions. In this study, we show that meiotic chromosome axes are shortened in the budding yeast rad61/wpl1 mutant, suggesting that Rad61/Wpl1 negatively regulates chromosome axis compaction. Rad61/Wpl1 is required for efficient resolution of telomere clustering during meiosis I, indicating a positive effect of Rad61/Wpl1 on the cohesin function required for telomere dynamics. Additionally, we demonstrate distinct activities of Rad61/Wpl1 during the meiotic recombination, including its effects on the efficient processing of intermediates. Thus, Rad61/Wpl1 both positively and negatively regulates various cohesin-mediated chromosomal processes during meiosis.
  • DNA damage response clamp 9-1-1 promotes assembly of ZMM proteins for formation of crossovers and synaptonemal complex, Miki Shinohara, Kayoko Hayashihara, Jennifer T. Grubb, Douglas K. Bishop, Akira Shinohara, JOURNAL OF CELL SCIENCE, JOURNAL OF CELL SCIENCE, 128(8), 1494 - 1506, Apr. 2015 , Refereed
    Summary:Formation of crossovers between homologous chromosomes during meiosis is positively regulated by the ZMM proteins (also known as SIC proteins). DNA damage checkpoint proteins also promote efficient formation of interhomolog crossovers. Here, we examined, in budding yeast, the meiotic role of the heterotrimeric DNA damage response clamp composed of Rad17, Ddc1 and Mec3 (known as '9-1-1' in other organisms) and a component of the clamp loader, Rad24 (known as Rad17 in other organisms). Cytological analysis indicated that the 9-1-1 clamp and its loader are not required for the chromosomal loading of RecA homologs Rad51 or Dmc1, but are necessary for the efficient loading of ZMM proteins. Interestingly, the loading of ZMM proteins onto meiotic chromosomes was independent of the checkpoint kinase Mec1 (the homolog of ATR) as well as Rad51. Furthermore, the ZMM member Zip3 (also known as Cst9) bound to the 9-1-1 complex in a cell-free system. These data suggest that, in addition to promoting interhomolog bias mediated by Rad51-Dmc1, the 9-1-1 clamp promotes crossover formation through a specific role in the assembly of ZMM proteins. Thus, the 9-1-1 complex functions to promote two crucial meiotic recombination processes, the regulation of interhomolog recombination and crossover formation mediated by ZMM.
  • DNA damage response clamp 9-1-1 promotes assembly of ZMM proteins for formation of crossovers and synaptonemal complex, Miki Shinohara, Kayoko Hayashihara, Jennifer T. Grubb, Douglas K. Bishop, Akira Shinohara, Journal of Cell Science, Journal of Cell Science, 128(8), 1494 - 1506, 2015 , Refereed
    Summary:Formation of crossovers between homologous chromosomes during meiosis is positively regulated by the ZMM proteins (also known as SIC proteins). DNA damage checkpoint proteins also promote efficient formation of interhomolog crossovers. Here, we examined, in budding yeast, the meiotic role of the heterotrimeric DNA damage response clamp composed of Rad17, Ddc1 and Mec3 (known as '9-1-1' in other organisms) and a component of the clamp loader, Rad24 (known as Rad17 in other organisms). Cytological analysis indicated that the 9-1-1 clamp and its loader are not required for the chromosomal loading of RecA homologs Rad51 or Dmc1, but are necessary for the efficient loading of ZMM proteins. Interestingly, the loading of ZMM proteins onto meiotic chromosomes was independent of the checkpoint kinase Mec1 (the homolog of ATR) as well as Rad51. Furthermore, the ZMM member Zip3 (also known as Cst9) bound to the 9-1-1 complex in a cell-free system. These data suggest that, in addition to promoting interhomolog bias mediated by Rad51-Dmc1, the 9-1-1 clamp promotes crossover formation through a specific role in the assembly of ZMM proteins. Thus, the 9-1-1 complex functions to promote two crucial meiotic recombination processes, the regulation of interhomolog recombination and crossover formation mediated by ZMM.
  • Double-strand break repair-adox: Restoration of suppressed double-strand break repair during mitosis induces genomic instability, Terasawa, Masahiro, Shinohara, Akira, Shinohara, Miki, CANCER SCIENCE, CANCER SCIENCE, 105(12), 1519 - 1525, Dec. 2014 , Refereed
    Summary:Double-strand breaks (DSBs) are one of the severest types of DNA damage. Unrepaired DSBs easily induce cell death and chromosome aberrations. To maintain genomic stability, cells have checkpoint and DSB repair systems to respond to DNA damage throughout most of the cell cycle. The failure of this process often results in apoptosis or genomic instability, such as aneuploidy, deletion, or translocation. Therefore, DSB repair is essential for maintenance of genomic stability. During mitosis, however, cells seem to suppress the DNA damage response and proceed to the next G(1) phase, even if there are unrepaired DSBs. The biological significance of this suppression is not known. In this review, we summarize recent studies of mitotic DSB repair and discuss the mechanisms of suppression of DSB repair during mitosis. DSB repair, which maintains genomic integrity in other phases of the cell cycle, is rather toxic to cells during mitosis, often resulting in chromosome missegregation and aberration. Cells have multiple safeguards to prevent genomic instability during mitosis: inhibition of 53BP1 or BRCA1 localization to DSB sites, which is important to promote non-homologous end joining or homologous recombination, respectively, and also modulation of the non-homologous end joining core complex to inhibit DSB repair. We discuss how DSBs during mitosis are toxic and the multiple safeguard systems that suppress genomic instability.
  • The small GTPase Rab5 homologue Ypt5 regulates cell morphology, sexual development, ion-stress response and vacuolar formation in fission yeast, Yuta Tsukamoto, Chisako Katayama, Miki Shinohara, Akira Shinohara, Shohei Maekawa, Masaaki Miyamoto, Biochemical and Biophysical Research Communications, Biochemical and Biophysical Research Communications, 441(4), 867 - 872, Nov. 29 2013 , Refereed
    Summary:Inner-membrane transport is critical to cell function. Rab family GTPases play an important role in vesicle transport. In mammalian cells, Rab5 is reported to be involved in the regulation of endosome formation, phagocytosis and chromosome alignment. Here, we examined the role of the fission yeast Rab5 homologue Ypt5 using a point mutant allele. Mutant cells displayed abnormal cell morphology, mating, sporulation, endocytosis, vacuole fusion and responses to ion stress. Our data strongly suggest that fission yeast Rab5 is involved in the regulation of various types of cellular functions. © 2013 Elsevier Inc.
  • Remodeling of the Rad51 DNA Strand-Exchange Protein by the Srs2 Helicase, Hiroyuki Sasanuma, Yuko Furihata, Miki Shinohara, Akira Shinohara, GENETICS, GENETICS, 194(4), 859 - +, Aug. 2013 , Refereed
    Summary:Homologous recombination is associated with the dynamic assembly and disassembly of DNA-protein complexes. Assembly of a nucleoprotein filament comprising ssDNA and the RecA homolog, Rad51, is a key step required for homology search during recombination. The budding yeast Srs2 DNA translocase is known to dismantle Rad51 filament in vitro. However, there is limited evidence to support the dismantling activity of Srs2 in vivo. Here, we show that Srs2 indeed disrupts Rad51-containing complexes from chromosomes during meiosis. Overexpression of Srs2 during the meiotic prophase impairs meiotic recombination and removes Rad51 from meiotic chromosomes. This dismantling activity is specific for Rad51, as Srs2 Overexpression does not remove Dmc1 (a meiosis-specific Rad51 homolog), Rad52 (a Rad51 mediator), or replication protein A (RPA; a single-stranded DNA-binding protein). Rather, RPA replaces Rad51 under these conditions. A mutant Srs2 lacking helicase activity cannot remove Rad51 from meiotic chromosomes. Interestingly, the Rad51-binding domain of Srs2, which is critical for Rad51-dismantling activity in vitro, is not essential for this activity in vivo. Our results suggest that a precise level of Srs2, in the form of the Srs2 translocase, is required to appropriately regulate the Rad51 nucleoprotein filament dynamics during meiosis.
  • A new protein complex promoting the assembly of Rad51 filaments, Hiroyuki Sasanuma, Maki S. Tawaramoto, Jessica P. Lao, Harumi Hosaka, Eri Sanda, Mamoru Suzuki, Eiki Yamashita, Neil Hunter, Miki Shinohara, Atsushi Nakagawa, Akira Shinohara, NATURE COMMUNICATIONS, NATURE COMMUNICATIONS, 4, Apr. 2013 , Refereed
    Summary:During homologous recombination, eukaryotic RecA homologue Rad51 assembles into a nucleoprotein filament on single-stranded DNA to catalyse homologous pairing and DNA-strand exchange with a homologous template. Rad51 nucleoprotein filaments are highly dynamic and regulated via the coordinated actions of various accessory proteins including Rad51 mediators. Here, we identify a new Rad51 mediator complex. The PCSS complex, comprising budding yeast Psy3, Csm2, Shu1 and Shu2 proteins, binds to recombination sites and is required for Rad51 assembly and function during meiosis. Within the hetero-tetramer, Psy3-Csm2 constitutes a core sub-complex with DNA-binding activity. In vitro, purified Psy3-Csm2 stabilizes the Rad51-single-stranded DNA complex independently of nucleotide cofactor. The mechanism of Rad51 stabilization is inferred by our high-resolution crystal structure, which reveals Psy3-Csm2 to be a structural mimic of the Rad51-dimer, a fundamental unit of the Rad51-filament. Together, these results reveal a novel molecular mechanism for this class of Rad51-mediators, which includes the human Rad51 paralogues.
  • Multiple Pathways Suppress Non-Allelic Homologous Recombination during Meiosis in Saccharomyces cerevisiae, Miki Shinohara, Akira Shinohara, PLOS ONE, PLOS ONE, 8(4), Apr. 2013 , Refereed
    Summary:Recombination during meiosis in the form of crossover events promotes the segregation of homologous chromosomes by providing the only physical linkage between these chromosomes. Recombination occurs not only between allelic sites but also between non-allelic (ectopic) sites. Ectopic recombination is often suppressed to prevent non-productive linkages. In this study, we examined the effects of various mutations in genes involved in meiotic recombination on ectopic recombination during meiosis. RAD24, a DNA damage checkpoint clamp-loader gene, suppressed ectopic recombination in wild type in the same pathway as RAD51. In the absence of RAD24, a meiosis-specific recA homolog, DMC1, suppressed the recombination. Homology search and strand exchange in ectopic recombination occurred when either the RAD51 or the DMC1 recA homolog was absent, but was promoted by RAD52. Unexpectedly, the zip1 mutant, which is defective in chromosome synapsis, showed a decrease, rather than an increase, in ectopic recombination. Our results provide evidence for two types of ectopic recombination: one that occurs in wild-type cells and a second that occurs predominantly when the checkpoint pathway is inactivated.
  • Cyclin-dependent kinase-dependent phosphorylation of Lif1 and Sae2 controls imprecise nonhomologous end joining accompanied by double-strand break resection, Matsuzaki, Kenichiro, Terasawa, Masahiro, Iwasaki, Daichi, Higashide, Mika, Shinohara, Miki, GENES TO CELLS, GENES TO CELLS, 17(6), 473 - 493, Jun. 2012 , Refereed
    Summary:DNA double-strand breaks (DSBs) are repaired by two distinct pathways, homologous recombination (HR) and nonhomologous end joining (NHEJ). NHEJ includes two pathways, that is, precise and imprecise end joining. We found that Lif1, a component of the DNA ligase IV complex in Saccharomyces cerevisiae, was phosphorylated by cyclin-dependent kinase (CDK) at Ser261 during the S to G2 phase but not during G1 phase. This phosphorylation was required for efficient NHEJ in G2/M cells, rather than in G1 cells. It also promotes the stable binding of Lif1 protein to DSBs, specifically in G2/M-arrested cells, which shows the resection of DSB ends. Thus, Lif1 phosphorylation plays a critical role in a certain type of imprecise NHEJ accompanied by DSB end resection and micro-homology. Lif1 phosphorylation at Ser261 is probably involved in micro-homology-dependent end joining associated with producing single-stranded DSB ends that are formed by Sae2 as early intermediates in the HR pathway. CDK-dependent modification of the NHEJ pathway might make DSB ends compatible for NHEJ and thus prevent competition between HR and NHEJ in hierarchy on the choice of DSB repair pathways.
  • Csm4-dependent telomere movement on nuclear envelope promotes meiotic recombination, Hiromichi Kosaka, Miki Shinohara, Akira Shinohara, PLoS Genetics, PLoS Genetics, 4(9), e1000196, Sep. 2008 , Refereed
    Summary:During meiotic prophase, chromosomes display rapid movement, and their telomeres attach to the nuclear envelope and cluster to form a "chromosomal bouquet." Little is known about the roles of the chromosome movement and telomere clustering in this phase. In budding yeast, telomere clustering is promoted by a meiosis-specific, telomere-binding protein, Ndj1. Here, we show that a meiosis-specific protein, Csm4, which forms a complex with Ndj1, facilitates bouquet formation. In the absence of Csm4, Ndj1-bound telomeres tether to nuclear envelopes but do not cluster, suggesting that telomere clustering in the meiotic prophase consists of at least two distinct steps: Ndj1-dependent tethering to the nuclear envelope and Csm4-dependent clustering/movement. Similar to Ndj1, Csm4 is required for several distinct steps during meiotic recombination. Our results suggest that Csm4 promotes efficient second-end capture of a double-strand break following a homology search, as well as resolution of the double-Holliday junction during crossover formation. We propose that chromosome movement and associated telomere dynamics at the nuclear envelope promotes the completion of key biochemical steps during meiotic recombination. © 2008 Kosaka et al.
  • Csm4-Dependent Telomere Movement on Nuclear Envelope Promotes Meiotic Recombination, Hiromichi Kosaka, Miki Shinohara, Akira Shinohara, PLOS GENETICS, PLOS GENETICS, 4(9), Sep. 2008 , Refereed
    Summary:During meiotic prophase, chromosomes display rapid movement, and their telomeres attach to the nuclear envelope and cluster to form a "chromosomal bouquet.'' Little is known about the roles of the chromosome movement and telomere clustering in this phase. In budding yeast, telomere clustering is promoted by a meiosis-specific, telomere-binding protein, Ndj1. Here, we show that a meiosis-specific protein, Csm4, which forms a complex with Ndj1, facilitates bouquet formation. In the absence of Csm4, Ndj1-bound telomeres tether to nuclear envelopes but do not cluster, suggesting that telomere clustering in the meiotic prophase consists of at least two distinct steps: Ndj1-dependent tethering to the nuclear envelope and Csm4-dependent clustering/movement. Similar to Ndj1, Csm4 is required for several distinct steps during meiotic recombination. Our results suggest that Csm4 promotes efficient second-end capture of a double-strand break following a homology search, as well as resolution of the double-Holliday junction during crossover formation. We propose that chromosome movement and associated telomere dynamics at the nuclear envelope promotes the completion of key biochemical steps during meiotic recombination.
  • Rapid telomere movement in meiotic prophase is promoted by NDJ1, MPS3, and CSM4 and is modulated by recombination, Michael N. Conrad, Chih-Ying Lee, Gene Chao, M. Shinohara, H. Kosaka, A. Shinohara, J. -A. Conchello, Michael E. Dresser, CELL, CELL, 133(7), 1175 - 1187, Jun. 2008 , Refereed
    Summary:Haploidization of the genome in meiosis requires that chromosomes be sorted exclusively into pairs stabilized by synaptonemal complexes (SCs) and crossovers. This sorting and pairing is accompanied by active chromosome positioning in meiotic prophase in which telomeres cluster near the spindle pole to form the bouquet before dispersing around the nuclear envelope. We now describe telomere-led rapid prophase movements (RPMs) that frequently exceed 1 mu m/s and persist throughout meiotic prophase. Bouquet formation and RPMs depend on NDJ1, MPS3, and a new member of this pathway, CSM4, which encodes a meiosis-specific nuclear envelope protein required specifically for telomere mobility. RPMs initiate independently of recombination but differ quantitatively in mutants that fail to complete recombination, suggesting that RPMs respond to recombination status. Together with recombination defects described for ndj1, our observations suggest that RPMs and SCs balance the disruption and stabilization of recombinational interactions, respectively, to regulate crossing over.
  • Forkhead-associated domain of yeast Xrs2, a homolog of human Nbs1, promotes nonhomologous end joining through interaction with a ligase IV partner protein, Lif1, Kenichiro Matsuzaki, Akira Shinohara, Miki Shinohara, GENETICS, GENETICS, 179(1), 213 - 225, May 2008 , Refereed
    Summary:DNA double-strand breaks (DSB) are repaired through two different pathways, homologous recombination (HR) and nonhomologous end joining (NHEJ). Yeast Xrs2 a homolog of human Nbs1, is a component of the Mre1 1-Rad50-Xrs2 (MRX) complex required for both HR and NHEJ. Previous studies showed that the N-terminal forkhead-associated (FHA) domain of Xrs2/Nbs1 in yeast is not involved in HR, but. is likely to be in NHEJ. In this study, we showed that the FHA domain of Xrs2 plays a critical role in efficient DSB repair by NHEJ. The FHA domain of Xrs2 specifically interacts with Lif1, a component of the ligase IV complex, Dn14-Nej1-Lif1 (DNL). Lif1, which is phosphorylated in vivo, contains two Xrs2-binding regions. Serine 383 of Lif1 plays an important role in the interaction with Xrs2 as well as in NHEJ. Interestingly, the phospho-mimetic substitutions of serine 383 enhance the NHEJ activity of Lif1. Our results suggest that the phosphorylation of Lif1 at serine 383 is recognized by the Xrs2 FHA domain, which in turn may promote recruitment of the DNL complex to DSB for NHEJ. The interaction between Xrs2 and Lif1 through the FHA domain is conserved in humans; the FHA domain Nbs1 interacts with Xrcc4, a Lif1 homolog of human.
  • Crossover assurance and crossover interference are distinctly regulated by the ZMM proteins during yeast meiosis, Miki Shinohara, Steve D. Oh, Neil Hunter, Akira Shinohara, NATURE GENETICS, NATURE GENETICS, 40(3), 299 - 309, Mar. 2008 , Refereed
    Summary:Meiotic crossing-over is highly regulated such that each homolog pair typically receives at least one crossover (assurance) and adjacent crossovers are widely spaced (interference). Here we provide evidence that interference and assurance are mechanistically distinct processes that are separated by mutations in a new ZMM (Zip, Msh, Mer) protein from Saccharomyces cerevisiae, Spo16. Like other zmm mutants, spo16 cells have defects in both crossing-over and synaptonemal complex formation. Unlike in previously characterized zmm mutants, the residual crossovers in spo16 cells show interference comparable to that in the wild type. Spo16 interacts with a second ZMM protein, Spo22 (also known as Zip4), and spo22 mutants also show normal interference. Notably, assembly of the MutS homologs Msh4 and Msh5 on chromosomes occurs in both spo16 and spo22, but not in other zmm mutants. We suggest that crossover interference requires the normal assembly of recombination complexes containing Msh4 and Msh5 but does not require Spo16- and Spo22-dependent extension of synaptonemal complexes. In contrast, crossover assurance requires all ZMM proteins and full-length synaptonemal complexes.
  • Rad52 promotes postinvasion steps of meiotic double-strand-break repair, Jessica P. Lao, Steve D. Oh, Miki Shinohara, Akira Shinohara, Neil Hunter, MOLECULAR CELL, MOLECULAR CELL, 29(4), 517 - 524, Feb. 2008 , Refereed
    Summary:During DNA double-strand-break (DSB) repair by recombination, the broken chromosome uses a homologous chromosome as a repair template. Early steps of recombination are well characterized: DSB ends assemble filaments of RecA-family proteins that catalyze homologous pairing and strand-invasion reactions. By contrast, the postinvasion steps of recombination are poorly characterized. Rad52 plays an essential role during early steps of recombination by mediating assembly of a RecA homolog, Rad51, into nucleoprotein filaments. The meiosis-specific RecA-homolog Dmc1 does not show this dependence, however. By exploiting the Rad52 independence of Dmcl, we reveal that Rac152 promotes postinvasion steps of both crossover and noncrossover pathways of meiotic recombination in Saccharomyces cerevisiae. This activity resides in the N-terminal region of Rad52, which can anneal complementary DNA strands, and is independent of its Rad51-assembly function. Our findings show that Rac152 functions in temporally and biochemically distinct reactions and suggest a general annealing mechanism for reuniting DSB ends during recombination.
  • Distinct functions of the two specificity determinants in replication initiation of plasmids ColE2-P9 and ColE3-CA38, Kazuteru Aoki, Miki Shinohara, Tateo Itoh, JOURNAL OF BACTERIOLOGY, JOURNAL OF BACTERIOLOGY, 189(6), 2392 - 2400, Mar. 2007 , Refereed
    Summary:The plasmid ColE2-P9 Rep protein specifically binds to the cognate replication origin to initiate DNA replication. The replicons of the plasmids ColE2-P9 and ColE3-CA38 are closely related, although the actions of the Rep proteins on the origins are specific to the plasmids. The previous chimera analysis identified two regions, regions A and B, in the Rep proteins and two sites, alpha and P, in the origins as specificity determinants and showed that when each component of the region A-site alpha pair and the region B-site beta pair is derived from the same plasmid, plasmid DNA replication is efficient. It is also indicated that the replication specificity is mainly determined by region A and site alpha. By using an electrophoretic mobility shift assay, we demonstrated that region B and site beta play a critical role for stable Rep protein-origin binding and, furthermore, that 284-Thr in this region of the ColE2 Rep protein and the corresponding 293-Trp of the ColE3 Rep protein mainly determine the Rep-origin binding specificity. On the other hand, region A and site alpha were involved in the efficient unwinding of several nucleotide residues around site ut, although they were not involved in the stable binding of the Rep protein to the origin. Finally, we discussed how the action of the Rep protein on the origin involving these specificity determinants leads to the plasmid-specific replication initiation.
  • Isolation and characterization of novel xrs2 mutations in Saccharomyces cerevisiae, H Shima, M Suzuki, M Shinohara, GENETICS, GENETICS, 170(1), 71 - 85, May 2005 , Refereed
    Summary:The Mre11/Rad50/Xrs2 (MRX) complex is involved in DNA damage repair, DNA damage response, telomere control, and meiotic recombination. Here, we constructed and characterized novel mutant alleles of XRS2. The alleles with mutations in the C-terminal conserved domain of Xrs2 were grouped into the same class. Mutant Xrs2 in this class lacked Mre11 interaction ability. The second class, lacking a C-terminal end, showed defects only in telomere control. A previous study showed that this C-terminal end contains a Tell-association domain. These results indicate that Xrs2 contains two functional domains, Mre11- and Tell-binding domains. While the Mre11-binding domain is essential for Xrs2 function, the Tell-binding domain maybe essential only for Te11 function in telomere maintenance. The third class, despite containing a large deletion in the N-terminal region, showed no defects in DNA damage repair. However, some mutants, which showed a reduced level of Xrs2 protein, were partially defective in formation of meiotic DSBs and telomere maintenance. These defects were suppressed by overexpression of the mutant Xrs2 protein. This result suggests that the total amount of Xrs2 protein is a critical determinant for the function of the MRX complex especially with regard to telomere maintenance and meiotic DSB formation.
  • A protein complex containing Mei5 and Sae3 promotes the assembly of the meiosis-specific RecA homolog Dmc1, Atsuko Hayase, Misato Takagi, Toshiko Miyazaki, Hiroyuki Oshiumi, Miki Shinohara, Akira Shinohara, Cell, Cell, 119(7), 927 - 940, Dec. 29 2004 , Refereed
    Summary:Meiotic recombination requires the meiosis-specific RecA homolog Dmc1 as well as the mitotic RecA homolog Rad51. Here, we show that the two meiosis-specific proteins Mei5 and Sae3 are necessary for the assembly of Dmc1, but not for Rad51, on chromosomes including the association of Dmc1 with a recombination hot spot. Mei5, Sae3, and Dmc1 form a ternary and evolutionary conserved complex that requires Rad51 for recruitment to chromosomes. Mei5, Sae3, and Dmc1 are mutually dependent for their chromosome association, and their absence prevents the disassembly of Rad51 filaments. Our results suggest that Mei5 and Sae3 are loading factors for the Dmc1 recombinase and that the Dmc1-Mei5-Sae3 complex is integrated onto Rad51 ensembles and, together with Rad51, plays both catalytic and structural roles in interhomolog recombination during meiosis.
  • A protein complex containing Mei5 and Sae3 promotes the assembly of the meiosis-specific RecA homolog Dmc1, A Hayase, M Takagi, T Miyazaki, H Oshiumi, M Shinohara, A Shinohara, CELL, CELL, 119(7), 927 - 940, Dec. 2004 , Refereed
    Summary:Meiotic recombination requires the meiosis-specific RecA homolog Dmc1 as well as the mitotic RecA homolog Rad51. Here, we show that the two meiosisspecific proteins Mei5 and Sae3 are necessary for the assembly of Dmc1, but not for Rad51, on chromosomes including the association of Dmc1 with a recombination hot spot. Mei5, Sae3, and Dmc1 form a ternary and evolutionary conserved complex that requires Rad51 for recruitment to chromosomes. Mei5, Sae3, and Dmc1 are mutually dependent for their chromosome association, and their absence prevents the disassembly of Rad51 filaments. Our results suggest that Mei5 and Sae3 are loading factors for the Dmc1 recombinase and that the Dmc1-Mei5-Sae3 complex is integrated onto Rad51 ensembles and, together with Rad51, plays both catalytic and structural roles in interhomolog recombination during meiosis.
  • Rad6-Bre1-mediated histone H2B ubiquitylation modulates the formation of double-strand breaks during meiosis, K Yamashita, M Shinohara, A Shinohara, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 101(31), 11380 - 11385, Aug. 2004 , Refereed
    Summary:An E2 ubiquitin-conjugating enzyme, Rad6, working with an E3 ubiquitin ligase Bre1, catalyzes monoubiquitylation of histone H2B on a C-terminal lysine residue. The rad6 mutant of Saccharomyces cerevisiae shows a meiotic prophase arrest. Here, we analyzed meiotic defects of a rad6 null mutant of budding yeast. The rad6 mutant exhibits pleiotropic phenotypes during meiosis. RAD6 is required for efficient formation of double-strand breaks (DSBs) at meiotic recombination hotspots, which is catalyzed by Spo11. The mutation decreases overall frequencies of DSBs in a cell. The effect of the rad6 mutation is local along chromosomes; levels of DSBs at stronger hotspots are particularly reduced in the mutant. The absence of RAD6 has little effect on the formation of ectopic DSBs targeted by Spo11 fusion protein with a Ga14 DNA-binding domain. Furthermore, the disruption of the BRE1 as well as substitution of the ubiquitylation site of histone H2B also reduces some DSB formation similar to the rad6. These results suggest that Rad6-Bre1, through ubiquitylation of histone H2B, is necessary for efficient recruitment and/or stabilization of a DSB-forming machinery containing Spoil. Histone tail modifications might play a role in DSB formation during meiosis.
  • In vivo assembly and disassembly of Rad51 and Rad52 complexes during double-strand break repair, T Miyazaki, DA Bressan, M Shinohara, JE Haber, A Shinohara, EMBO JOURNAL, EMBO JOURNAL, 23(4), 939 - 949, Feb. 2004 , Refereed
    Summary:Assembly and disassembly of Rad51 and Rad52 complexes were monitored by immunofluorescence during homologous recombination initiated by an HO endonuclease-induced double-strand break (DSB) at the MAT locus. DSB-induced Rad51 and Rad52 foci colocalize with a TetR-GFP focus at tetO sequences adjacent to MAT. In strains in which HO cleaves three sites on chromosome III, we observe three distinct foci that colocalize with adjacent GFP chromosome marks. We compared the kinetics of focus formation with recombination intermediates and products when HO-cleaved MATalpha recombines with the donor, MATalpha. Rad51 assembly occurs 1 h after HO cleavage. Rad51 disassembly occurs at the same time that new DNA synthesis is initiated after single-stranded (ss) MAT DNA invades MATalpha. We present evidence for three distinct roles for Rad52 in recombination: a presynaptic role necessary for Rad51 assembly, a synaptic role with Rad51 filaments, and a postsynaptic role after Rad51 dissociates. Additional biochemical studies suggest the presence of an ssDNA complex containing both Rad51 and Rad52.
  • Roles of RecA homologues Rad51 and Dmc1 during meiotic recombination, A Shinohara, M Shinohara, CYTOGENETIC AND GENOME RESEARCH, CYTOGENETIC AND GENOME RESEARCH, 107(3-4), 201 - 207, 2004 , Refereed
    Summary:RecA protein is involved in homology search and strand exchange processes during recombination. Mitotic cells in eukaryotes express one RecA, Rad51, which is essential for the repair of double-strand breaks (DSBs). Additionally, meiotic cells induce the second RecA, Dmc1. Both Rad51 and Dmc1 are necessary to generate a crossover between homologous chromosomes, which ensures the segregation of the chromosomes at meiotic division I. It is largely unknown how the two RecAs cooperate during meiotic recombination. In this review, recent advances on our knowledge about the roles of Rad51 and Dmc1 during meiosis are summarized and discussed. Copyright (C) 2004 S. Karger AG, Basel.
  • The N-terminal DNA-binding domain of Rad52 promotes RAD51-independent recombination in Saccharomyces cerevisiae, M Tsukamoto, K Yamashita, T Miyazaki, M Shinohara, A Shinohara, GENETICS, GENETICS, 165(4), 1703 - 1715, Dec. 2003 , Refereed
    Summary:In Saccharomyces cerevisiae, the Rad52 protein plays a role in both RAD51-dependent and RAD51-independent recombination pathways. We characterized a rad52 mutant, rad52-329, which lacks the C-terminal Rad51-interacting domain, and studied its role in RAD51-independent recombination. The rad52-329 mutant is completely defective in mating-type switching, but partially proficient in recombination between inverted repeats. We also analyzed the effect of the rad52-329 mutant on telomere recombination. Yeast cells lacking telomerase maintain telomere length by recombination. The rad52-329 mutant is deficient in RAD51-dependent telomere recombination, but is proficient in RAD51-independent telomere recombination. In addition, we examined the roles of other recombination genes in the telomere recombination. The RAD51-independent recombination in the rad52-329 mutant is promoted by a paralogue of Rad52, Rad59. All components of the Rad50-Mre11-Xrs2 complex are also important, but not essential, for RAD51-independent telomere recombination. Interestingly, RAD51 inhibits the RAD51-independent, RAD52-dependent telomere recombination. These findings indicate that Rad52 itself, and more precisely its N-terminal DNA-binding domain, promote an essential reaction in recombination in the absence of RAD51.
  • The mitotic DNA damage checkpoint proteins Rad17 and Rad24 are required for repair of double-strand breaks during meiosis in yeast, M Shinohara, K Sakai, T Ogawa, A Shinohara, GENETICS, GENETICS, 164(3), 855 - 865, Jul. 2003 , Refereed
    Summary:We show here that deletion of the DNA damage checkpoint genes RAD17 and RAD24 in Saccharomyces cerevisiae delays repair of meiotic double-strand breaks (DSBs) and results in an altered ratio of crossover-to-noncrossover products. These mutations also decrease the colocalization of immunostaining foci of the RecA homologs Rad51 and Dmc1 and cause a delay in the disappearance of Rad51 foci, but not of Dmc1. These observations imply that RAD17 and RAD24 promote efficient repair of meiotic DSBs by facilitating proper assembly of the meiotic recombination complex containing Rad51. Consistent with this proposal, extra copies of RAD51 and RAD54 substantially suppress not only the spore inviability of the rad24 Mutant, but also the gamma-ray sensitivity of the mutant. Unexpectedly, the entry into meiosis I (metaphase I) is delayed in the checkpoint single mutants compared to wild type. The control of the cell cycle in response to meiotic DSBs is also discussed.
  • Crossover interference in Saccharomyces cerevisiae requires a TID1/RDH54- and DMC1-dependent pathway, M Shinohara, K Sakai, A Shinohara, DY Bishop, GENETICS, GENETICS, 163(4), 1273 - 1286, Apr. 2003 , Refereed
    Summary:Two RecA-like rccombinases, Rad51 and Dmc1, function together during double-strand break (DSB)mediated meiotic recombination to promote homologous strand invasion in the budding yeast Saccharomyces cerevisiae. Two partially redundant proteins, Rad54 and Tid1/Rdh54, act as recombinase accessory factors. Here, tetrad analysis shows that mutants lacking Tid1 form four-viable-spore tetrads with levels of interhomolog crossover (CO) and noncrossover recombination similar to, or slightly greater than, those in wild type. Importantly, tid1 mutants show a marked defect in crossover interference, a mechanism that distributes crossover events nonrandomly along chromosomes during meiosis. Previous work showed that dmc1Delta mutants are strongly defective in strand invasion and meiotic progression and that these defects can be partially suppressed by increasing the copy number of RAD54. Tetrad analysis is used to show that meiotic recombination in RAD54-suppressed dmc1Delta cells is similar to that in tid1; the frequency of COs and gene conversions is near normal, but crossover interference is defective. These results support the proposal that crossover interference acts at the strand invasion stage of recombination.
  • Tid1/Rdh54 promotes colocalization of Rad51 and Dmc1 during meiotic recombination, M Shinohara, SL Gasior, DK Bishop, A Shinohara, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 97(20), 10814 - 10819, Sep. 2000 , Refereed
    Summary:Two RecA homologs, Rad51 and Dmc1, assemble as cytologically visible complexes (foci) at the same sites on meiotic chromosomes. Time course analysis confirms that co-foci appear and disappear as the single predominant form. A large fraction of co-foci are eliminated in a red1 mutant, which is expected as a characteristic of the interhomolog-specific recombination pathway. Previous studies suggested that normal Dmc1 loading depends on Rad51. We show here that a mutation in TID1/RDH54, encoding a RAD54 homolog, reduces Rad51-Dmc1 colocalization relative to WT. A rad54, mutation, in contrast, has relatively little effect on RecA homolog foci except when strains also contain a tid1/rdh54 mutation the role of Tid1/Rdh54 in coordinating RecA homolog assembly may be very direct, because Tid1/Rdh54 is known to physically bind both Dmc1 and Rad51. Also, Dmc1 foci appear early in a tid1/rdh54 mutant. Thus, Tid1 may normally act with Rad51 to promote ordered RecA homolog assembly by blocking Dmc1 until Rad51 is present. Finally, whereas double-staining foci predominate in WT nuclei, a subset of nuclei with expanded chromatin exhibit individual Rad51 and Dmc1 foci side-by-side, suggesting that a Rad51 homo-oligomer and a Dmc1 homo-oligomer assemble next tb one another at the site of a single double-strand break (DSB) recombination intermediate.
  • High copy number suppression of the meiotic arrest caused by a dmc1 mutation: REC114 imposes an early recombination block and RAD54 promotes a DMC1-independent DSB repair pathway, DK Bishop, Y Nikolski, J Oshiro, J Chon, M Shinohara, Chen, X, GENES TO CELLS, GENES TO CELLS, 4(8), 425 - 443, Aug. 1999 , Refereed
    Summary:Background: DMC1, the meiosis-specific eukaryotic homologue of bacterial recA, is required for completion of meiotic recombination and cell cycle progression past prophase. In a dmc1 mutant, double strand break recombination intermediates accumulate and cells arrest in prophase. We isolated genes which, when present at high copy numbers, suppress the meiotic arrest phenotype conferred by dmc1 mutations. Results: Among the genes isolated were two which suppress arrest by altering the recombination process. REC114 suppresses formation of double strand break (DSB) recombination intermediates. The low viability of spores produced by dmc1 mutants carrying high copy numbers of REC114 is rescued when reductional segregation is bypassed by mutation of spo13. High copy numbers of RAD54 suppress dmc1 arrest, promote DSB repair, and allow formation of viable spores following reductional segregation. Analysis of the combined effects of a null mutation in RED1, a gene required for meiotic chromosome structure, with null mutations in RAD54 and DMC1 shows that RAD54, while not normally important for repair of DSBs during meiosis, is required for efficient repair of breaks by the intersister recombination pathway that operates in red1 dmc1 double mutants. Conclusions: Over-expression of REC114 suppresses meiotic arrest by preventing formation of DSBs. High copy numbers of RAD54 activate a DMC1-independent mechanism that promotes repair of DSBs by homology-mediated recombination. The ability of RAD54 to promote DMC1-independent recombination is proposed to involve suppression of a constraint that normally promotes recombination between homologous chromatids rather than sisters.
  • Rad52 forms ring structures and co-operates with RPA in single-strand DNA annealing, A Shinohara, M Shinohara, T Ohta, S Matsuda, T Ogawa, GENES TO CELLS, GENES TO CELLS, 3(3), 145 - 156, Mar. 1998 , Refereed
    Summary:Background: The RAD52 epistasis group in Saccharomyces cerevisiae is involved in various types of homologous recombination including recombinational double-strand break (DSB) repair and meiotic recombination. A RecA homologue, Rad51, plays a pivotal role in homology search and strand exchange. Genetic analysis has shown that among members of its epistasis group, RAD52 alone is required for recombination between direct repeats yielding deletions. Very little has been discovered about the biochemical roles and structure of the Rad52 protein. Results: Purified Rad52 protein binds to both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA). Electron microscope observations revealed that Rad52 molecules form multimeric rings. An increase in the intensity of fluorescence when Rad52 is bound to epsilon DNA showed an alteration of the structure of ssDNA. RPA was binding to Rad52 and enhanced the annealing of complementary ssDNA molecules. This enhancement was not observed in Escherichia coli SSB protein of T4 phage gp32 protein. Conclusion: Rad52 forms a ring-like structure and binds to ssDNA. Its structure and DNA binding properties are different from those of Rad51. The interaction of Rad52 with RPA plays an important role in the enhancement of annealing of complementary ssDNAs. We therefore propose that Rad52 mediates the RAD51-independent recombination through an ssDNA annealing, assisted by RPA.
  • Characterization of the roles of the Saccharomyces cerevisiae RAD54 gene and a homologue of RAD54, RDH54/TIL1, in mitosis and meiosis, M Shinohara, E ShitaYamaguchi, JM Buerstedde, H Shinagawa, H Ogawa, A Shinohara, GENETICS, GENETICS, 147(4), 1545 - 1556, Dec. 1997 , Refereed
    Summary:The RAD54 gene, which encodes a protein in the SW12/SNF2 family, plays an important role in recombination and DNA repair in Saccharomyces cerevisiae. The yeast genome project revealed a homologue of RAD54, RDD54/TLD1. Properties of the rdh54/tid1 mutant and the rad54 rdh54/tid1 double mutant are shown for mitosis and meiosis. The rad54 mutant is sensitive to the aIkylating agent, methyl methanesulfonate (MMS), and is defective in interchromosomal and intrachromosomal gene conversion. The rdh54/tid1 single mutant, on the other hand, does not show any significant deficiency in mitosis. However, the rad54 rdh54/tid1 mutant is more sensitive to MMS and more defective in interchromosomal gene conversion than is the rad54 mutant, but shows the same frequency of intrachromosomal gene conversion as the rad54 mutant. These results suggest that RDH54/TID1 is involved in a minor pathway of mitotic recombination in the absence of RAD54. In meiosis, both single mutants produce viable spores at slightly reduced frequency. However, only the rdh54/tid1 mutant, but not the rad54 mutant, shows significant defects in recombination: retardation of the repair of meiosis-specific double-strand breaks (DSBs) and delayed formation of physical recombinants. Furthermore, the rad54 rdh54/tid1 double mutant is completely defective in meiosis, accumulating DSBs with more recessed ends than the wild type and producing fewer physical recombinants than the wild type. These results suggest that one of the differences between the late stages of mitotic recombination and meiotic recombination might be specified by differential dependency on the Rad54 and Rdh54/Tid1 proteins.
  • Specificity determinants in interaction of the initiator (Rep) proteins with the origins in the plasmids ColE2-P9 and ColE3-CA38 identified by chimera analysis, M Shinohara, T Itoh, JOURNAL OF MOLECULAR BIOLOGY, JOURNAL OF MOLECULAR BIOLOGY, 257(2), 290 - 300, Mar. 1996 , Refereed
    Summary:The ColE2-P9 Rep protein specifically binds to the origin and initiates DNA synthesis. Interaction of the Rep proteins with the origins of plasmids ColE2-P9 and ColE3-CA38 (one of the close relatives of ColE2-P9) is plasmid-specific. By using chimeric rep genes and chimeric origins we showed that the two region, A and B, in the C-terminal regions of the Rep proteins and the two sites, alpha and beta, in the origins are important for the determination of specificity. When each of the A/alpha and B/beta Fairs is from the same plasmids, the plasmid replication is efficient. On the other hand, if only the A/alpha pair is from the same plasmids, the plasmid replication is inefficient. For the region A, the plasmid-specificity is mainly determined by the presence or absence of a nine-amino acid sequence. For the region B, the specificity is probably determined by several amino acids. The region B contains a segment of amino acid sequence which shows significant homology with the DNA recognition helices of various DNA binding proteins. At the site alpha, the single additional base-pair in the ColE3-CA38 origin can be either A/T or T/A. At the site beta, however, the single additional base-pair in the ColE2-P9 origin must be G/C. Among other possibilities we propose that the region A is a linker connecting the two domains in the Rep protein involved in DNA-binding and that the region B is apart of the sequence-specific DNA-binding domain. (C) 1996 Academic Press Limited

Conference Activities & Talks

  • Synapsis-dependent meiotic CO control in long-sized chromosome, SHINOHARA Miki, HIGASHIDE Mika, EMBO conference Meiosis,   2017 08 26
  • Eviction of Ku from single-stranded DSB ends through Tel1/ATM activity is essential to ensure NHEJ fidelity., SHINOHARA Miki,   2016 11 30 , 招待有り
  • Eviction of Ku from single-stranded DSB ends through Tel1/ATM activity is essential to ensure NHEJ fidelity., SHINOHARA Miki, The 10th International 3R Symposium,   2016 11 15 , 招待有り
  • Double-strand break repair-adox: restoration of suppressed double-strand break repair during mitosis induces genomic instability in human cells, SHINOHARA Miki, International Symposium on Structure and Folding of Disease Related Proteins,   2015 12 04 , 招待有り
  • The MRX/N complex ensures DSB repair fidelity through multiple pathways including Xrs2-FHA–dependent Tel1/ATM activation, SHINOHARA Miki, RSC-IPR joint Symposium, Protein Structure and Function,   2015 11 14 , 招待有り
  • Mitosis-specific phosphorylation of XRCC4 maintains genome stability by suppression of DNA damage repair, Terasawa, M, Shinohara, M,   2015 10 08 , 招待有り
  • Double-strand break repair-adox: restoration of suppressed double-strand break repair during mitosis induces genomic instability, SHINOHARA Miki, The 15th International Congress of Radiation Research,   2015 05 29
  • Canonical non-homologous end joining in mitosis induces genome instability and is suppressed by M-phase-specific phosphorylation of XRCC4 via CDK1 and PLK1, SHINOHARA Miki, The 9th 3R International symposium,   2014 11 17 , 招待有り
  • Canonical NHEJ on mitotic chromosome induces genome instability and is suppressed by XRCC4 through M-phase phosphorylation by CDK, SHINOHARA Miki, The 9th International Symposium of the Institute Network,   2014 06 20 , 招待有り
  • A coordination mechanism between chromosome segregation and regulations of DSB repair pathways during mitosis, SHINOHARA Miki, TERASAWA Masahiro,   2013 10 04

Misc

  • [Molecular mechanism of meiotic crossover formation]., Shinohara M, Shinohara A, Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme, 54, 4 Suppl, 466, 471,   2009 03 , Refereed
  • [Mechanism of DNA recombination ad chromosomal morphogenesis in the meiotic phase]., Shinohara A, Shinohara M, Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme, 53, 11, 1315, 1325,   2008 09 , Refereed
  • DNA exchage on chromosomes: Molecular mechanisms of homologous recombinaion and its cellular functions, Akira Shinohara, Miki Shinohara, Cell Technology,   2003 03
  • 多機能性スカフォールド蛋白質Slx4はセントロメア近傍領域特異的なクロスオーバー(CO)組換えの抑制および染色体全体の二重鎖切断(DSB)分布に寄与する(Slx4, multifunctional scaffold protein, functions for negative regulation of CO formation specific in centromere-proximal region and DSB distribution on entire chromosomes), Higashide Mika, Shinohara Miki, Genes & Genetic Systems, 91, 6, 355, 355,   2016 12
  • 出芽酵母の減数分裂組換えにおけるPP4ホスファターゼ機能の解析(Characterization of PP4 Phosphatase Function During Meiotic Recombination in Budding Yeast), Ke Li, Shinohara Miki, Genes & Genetic Systems, 91, 6, 355, 355,   2016 12
  • MRX複合体は、Xrs2 FHAドメイン依存的なTel1活性化を介してNHEJの正確性を保証する(The MRX complex ensures NHEJ fidelity through Xrs2-FHA? dependent Tell activation), Iwasaki Daichi, Hayashihara Kayak, Shinohara Miki, Genes & Genetic Systems, 91, 6, 356, 356,   2016 12
  • MRX複合体はXrs2-FHA依存的なTel1活性化を含む複数の経路を介してNHEJの正確性を保証する(The MRX complex ensures NHEJ fidelity through multiple pathways including Xrs2-FHA-dependent Tell activation), Shinohara Miki, Iwasaki Daichi, Genes & Genetic Systems, 90, 6, 387, 387,   2015 12
  • 減数分裂期のシナプトネマ複合体の伸長は染色体サイズ依存的に交叉型組換えの数を制御する(Elongation of synaptonemal complex in meiosis regulates crossover numbers between homologs dependent on chromosome-size), Higashide Mika, Shinohara Miki, Genes & Genetic Systems, 90, 6, 389, 389,   2015 12
  • 非相同末端結合因子XRCC4のM期特異的リン酸化はDNA損傷修復抑制を介してゲノム安定性を維持する, 寺澤 匡博, 篠原 美紀, 日本癌学会総会記事, 74回, IS1, 4,   2015 10
  • 不正確なDNA二重鎖切断修復経路の抑制メカニズムとゲノム安定維持, 篠原 美紀, 谷郷 花圭, 寺澤 匡博, 日本癌学会総会記事, 74回, E, 1326,   2015 10
  • DNA二重鎖切断修復機構 有糸分裂中のDNA二重鎖切断修復制御の回復はゲノム不安定性を誘発する(Double-strand Break Repair-adox: Restoration of Suppressed Doublestrand Break Repair During Mitosis Induces Genomic Instability), Shinohara Miki, Terasawa Masahiro, Tanigo Haruka, 日本放射線影響学会大会講演要旨集, 58回, 5, 02,   2015 05
  • Prophase pathway of arm-cohesin removal in budding yeast meiosis, Challa Kiran, Miki Shinohara, Akira Shinohara, GENES & GENETIC SYSTEMS, 89, 6, 278, 278,   2014 12
  • ゲノム再配列の分子メカニズムとゲノム安定性の維持 分裂期染色体上のcanonicalな非相同末端結合はゲノム不安定性を誘導し、CDKおよびPLK1に依存的なXRCC4のM期特異的リン酸化により抑制される(Molecular mechanism of genome rearrangement and maintenance of genome stability Canonical Non-homologous End Joining on Mitotic Chromosome Induces Genome Instability and is Suppressed by XRCC4 through M-phase Specific Phosphorylation by CDK and PLK1), Terasawa Masahiro, Shinohara Miki, Genes & Genetic Systems, 89, 6, 284, 284,   2014 12
  • シナプトネマ複合体を構成する新規分子Gmc2は減数分裂期の相同染色体間の交叉数を制御する(Gmc2, a novel synaptonemal complex component, regulates the number of crossovers between homologous chromosomes in meiosis), Higashide Mika, Shinohara Miki, Genes & Genetic Systems, 89, 6, 324, 324,   2014 12
  • The small GTPase Rab5 controls cell morphology, sexual development, ion-stress response and vacuolar formation in fission yeast, Masaaki Miyamoto, Yuta Tsukamoto, Chisako Katayama, Miki Shinohara, Akira Shinohara, Shohei Maekawa, FASEB JOURNAL, 28, 1,   2014 04
  • Roles of Pal1 complex in Meiosis of Budding Yeast, Santosh Kumar Gothwal, Takehiko Usui, Miki Shinohara, Akira Shinohara, GENES & GENETIC SYSTEMS, 88, 6, 379, 379,   2013 12
  • Regulatory mechanism of nuclear envelope remodelling during budding yeast meiosis, Challa Kiran, Hbd Prasad Rao, Miki Shinohara, Akira Shinohara, GENES & GENETIC SYSTEMS, 88, 6, 379, 379,   2013 12
  • 構造特異的エンドヌクレアーゼ制御サブユニットSlx4の減数分裂前期における新しい機能(A novel function of Slx4, structure-specific endonuclease regulator subunit, in meiotic prophase), Higashide Mika, Shinohara Akira, Shinohara Miki, Genes & Genetic Systems, 88, 6, 375, 375,   2013 12
  • M期における染色体分配とDNA二重鎖切断修復経路制御の連携機構の解析(A coordination mechanisms between chromosome segregation and regulations of DSB repair pathways during mitosis), 篠原 美紀, 寺澤 匡博, 日本癌学会総会記事, 72回, 87, 87,   2013 10
  • 減数分裂性組換えにおけるSrs2ヘリカーゼの機能(The function of Srs2 helicase in meiotic recombination), Furihta Yuko, Sasanuma Hiroyuki, Shinohara Miki, Shinohara Akira, Genes & Genetic Systems, 87, 6, 403, 403,   2012 12
  • Saccharomyces cerevisiaeの減数分裂組換えにおける、数種DNAの構造特異的エンドヌクレアーゼの調節サブユニットであるSlx4の新たな機能(A novel function of Slx4, a regulational subunit of several DNA structure specific endonucleases, in meiotic recombination in Saccharomyces cerevisiae), Higashide Mika, Shinohara Miki, Genes & Genetic Systems, 87, 6, 437, 437,   2012 12
  • 酵母Xrs2のFHAドメインはTel1キナーゼ活性の維持に関与し、NHEJのfidelityを制御する(FHA domain of yeast Xrs2 is involved in maintenance of Tel1 kinase activity and controls NHEJ fidelity), Shinohara Miki, Iwasaki Daichi, Nasada Yu, Genes & Genetic Systems, 87, 6, 437, 437,   2012 12
  • Saccharomyces cerevisiaeにおける減数分裂特異的DNA二重鎖切断に反応した、DNA damage checkpoint蛋白質活性化抑制の分子機序(Molecular mechanisms to silence activation of DNA damage checkpoint proteins in response to meiosis-specific DNA double-strand breaks in Saccharomyces cerevisiae), Usui Takehiko, Shinohara Miki, Shinohara Akira, Genes & Genetic Systems, 87, 6, 437, 437,   2012 12
  • Meiosis-specific DNA double-strand breaks escape detection by DNA damage checkpoint mediator, Rad9, to avoid activation of Rad53 kinase in Saccharomyces cerevisiae, Takehiko Usui, Miki Shinohara, Akira Shinohara, GENES & GENETIC SYSTEMS, 86, 6, 401, 401,   2011 12
  • Cyclin-dependent kinase (CDK) regulates the NHEJ activity during S-G2 phase in budding yeast, Miki Shinohara, Kenichiro Matsuzaki, GENES & GENETIC SYSTEMS, 85, 6, 404, 404,   2010 12
  • CDKによる非相同末端結合の新規制御メカニズムの解析(Cyclin-dependent protein kinase (CDK) regulates the repair mode of non-homologous end joining in budding yeast), 松嵜 健一郎, 篠原 彰, 篠原 美紀, 日本生化学会大会・日本分子生物学会年会合同大会講演要旨集, 83回・33回, 2T2, 4,   2010 12
  • Analysis of a novel NHEJ regulatory mechanism by CDK, Kenichiro Matsuzaki, Akira Shinohara, Miki Shinohara, GENES & GENETIC SYSTEMS, 84, 6, 490, 490,   2009 12
  • Functional analysis of the PCSS complex involved in homologous recombination in yeast, Maki Tawaramoto, Hiroyuki Sasanuma, Miki Shinohara, Akira Shinohara, GENES & GENETIC SYSTEMS, 84, 6, 437, 437,   2009 12 , Refereed
  • 細胞骨格、核膜と核機能の連係 CDKによる核膜タンパク質Mps3の局在変化の制御(Interactions of nuclear functions with nuclear envelopes and cytoskeltons CDK-dependent control of dynamics of a nuclear envelope protein, Mps3, during meiosis), Rao H.B.D. Prasada, 篠原 美紀, 篠原 彰, 日本細胞生物学会大会講演要旨集, 61回, 113, 113,   2009 05
  • Actin dependent regulation of meiotic recombination, Saori Mori, Miki Shinohara, Akira Shinohara, GENES & GENETIC SYSTEMS, 83, 6, 512, 512,   2008 12
  • Regulation mechanism of non-homologous end joining through the Lifl phosphorylation, Miki Shinohara, Seikagaku, 80, 11, 1029, 1033,   2008
  • The role of the CSM2 gene in recombination during meiosis of S. cerevisiae, Eri Sanda, Akira Shinohara, Miki Shinohara, GENES & GENETIC SYSTEMS, 82, 6, 544, 544,   2007 12
  • Molecular mechanism of ZMM complex in homologous recombination during meiosis, Shinohara, Miki, Shinohara, Akira, GENES & GENETIC SYSTEMS, 82, 6, 505, 505,   2007 12 , Refereed
  • Functional analysis of the CSM4 gene involved in meiosis recombination, Hiromichi Kosaka, Hiroyuki Oshiumi, Miki Shinohara, Akira Shinohara, GENES & GENETIC SYSTEMS, 81, 6, 409, 409,   2006 12
  • Functional analysis of Spo16, a new synaptonemal complex component, in meiotic recombination, Shinohara, Miki, Oshiumi, Hiroyuki, Shinohara, Akira, GENES & GENETIC SYSTEMS, 81, 6, 462, 462,   2006 12 , Refereed
  • Crossover interference in Saccharomyces cerevisiae requires a TID1/RDH54- and DMC1-dependent pathway (vol 163, pg 1273, 2004), M Shinohara, K Sakai, A Shinohara, DK Bishop, GENETICS, 169, 4, 2389, 2390,   2005 04

Awards & Honors

  •   1998 , HFSPO, HFSP Long-term fellow awards

Research Grants & Projects

  • Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Challenging Research (Exploratory), Identification of the minimalized essential unit for genetic inheritance of artificial mini-chromosomes
  • Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area), Control of chromosome functions by axis-loop structure (chromosome 3D) during meiosis
  • Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Priority Areas, Molecular mechanism of the connection between chromosome networks and recombination, Genetic change in meiosis is driven by homologous recombination. The recombination not only generates diversity in the genome, but also plays an essential role in chromosome segregation at meiosis I. Meiotic recombination is mechanistically different from that in mitosis with more complex regulation. From this study, we have identified several key proteins/genes in the recombination which tell us a new idea on the mechanisms of meiotic recombination. Particularly, we have identified an active role of chromosome dynamics and structure in the control of the recombination.
  • Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B), Studies for regulation mechanism of meiotic crossover formation through the protein-chromosome structure
  • Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B), Molecular cloning of the gene for cancer-prone syndrome characterized by abnormal mitotic spindle checkpoint., Cancer-prone syndrome of premature chromatid separation with mosaic variegated aneuploidy (PCS syndrome) is a rare autosomal recessive disorder characterised by growth retardation, microcephaly, childhood cancer, premature chromatid separation of all chromosomes and mosaicism for various trisomies and monosomies. Biallelic BUB1B mutations were recently reported in five of eight families with MVA syndrome (probably identical to the PCS syndrome). We here describe molecular analysis of BUB1B (encoding BubR1) in seven Japanese families with the PCS syndrome. Monoallelic BUB1B mutations were found in all seven families studied : a single base deletion (1833delT) in four families ; and a splice site mutation, a nonsense mutation, and a missense mutation in one family each. Transcripts derived from the patients with the 1833delT mutation and the splice site mutation were significantly reduced due to nonsense-mediated mRNA decay. No mutation was found in the second alleles in the seven families studied, but RT-PCR of BUB1B and Western blot and haplotype analysis of BubR1 indicated a modest decrease of their transcripts. BubR1 in the cells from two patients showed both reduced protein expression and diminished kinetochore localization. Their expression level of p55cdc, a specific activator of anaphase-promoting complex, was normal but its kinetochore association was abolished. Microcell-mediated transfer of chromosome 15 (containing BUB1B) into the cells restored normal BubR1 levels, kinetochore localization of p55cdc, and the normal responses to colcemid treatment. These findings indicate the involvement of BubR1 in p55cdc-mediated mitotic checkpoint signaling, and suggest that >50% decrease in expression (or activity) of BubR1 is involved in the PCS syndrome.
  • Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Priority Areas, Cancersusceptibility disease Nijmegen Breakage Syndrome and the function of underlying gene., Nijmegen breakage syndrome (NBS) is a human hereditary disease, characterized by high sensitivity to radiation, chromosomal instability and predisposition to cancer, and the underlying gene, NBS1, reveals to code a repair protein, which lacks both nuclease activity and DNA-binding region. We presented here that NBS1 binds Mre11/Ras50 complex, a crucial nuclease for homologous recombination, at the C-terminus. Moreover, based on the evidence that histon H2AX is phosphorylated immediately after irradiation, we showed NBS1 binds to the phosphorylated histon through FHA/BRCT domains at the N-terminus. This binding was confirmed by both IP-western and in vitro binding assay. As a result, we proposed a damage response model, in which NBS1 recognizes damaged sites and initiates homologous recombination by recruitment of Mre11/Rad50 nuclease. In fact, analysis of homologous recombination using SCneo reporter gene showed significantly decreased homologous recombination in patient cells and mouse NBS cells. Furthermore, cohesin SMC1 binding to NBS1 and consequently SMC1 phosphorylation, a dispensable modification for S-checkpoint, were repressed in the clone lacking the C-terminus and N-terminus of NBS1. Similarly, NBS1 revealed to form the multi-protein complex including BRCA1 and WRN, mutated in Werner Syndrome, after irradiation. Since the disruption of complex formation results in chromosomal instability, it must be involved in maintenance of repair fidelity and cross-talk with checkpoint.
  • Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Specially Promoted Research, Molecular mechanisms of generation of pleiotropic effects of recombination genes, A recombination function is required for repair of DNA breakages, overcome of DNA replication-arrest at a DNA lesion and maintenance of telomere length. We are interested in how multiple functions are produced by a single recombination protein. We selected a Mre11/rad50/Xrs2 complex (MRX), Rad51 and Rad52 as representatives, and investigated mechanisms that give full play to their multiple functioning ability. The followings are main results obtained during this research term. We analyzed domains of Xrs2 which control the functions of MRX, and found (1)Xrs2 binds to Mre11 with a 32 amino-acid domain (MBX) near the C-terminus, and transports Mre11 into the nucleus. (2)Xrs2 is no more required for DNA damage repair if Mre11 has been transported into the nucleus. (3)For telomere elongation and meiotic recombination, in addition to the MBX, its C-terminal adjacent 104, and its N-terminal adjacent 49-, amino-acid domain are needed, respectively. I.We found a new checkpoint pathway, Te11-Mre11, that is specific to DNA double-strand breakage (DSB), In mitotic cells, Rad53 and Rad9 and in meiotic cells, Mre4/Mek1, are required, respectively. The MRX plays a sensor against the DSB, activates the pathway, and proceeds recombination under the guidance of the activated pathway. II.It is a new finding that not only rad51but also rad52 are necessary for DNA homology search. Rad51-Rad52-single-stranded DNA is the complex to do it. Rad52 is also required at the latest stage of recombination, production of a final recombinant molecule. III.A new helicase gene MER3 was found which is specific to meiotic recombination. As a frequency of meiotic crossover specifically decreases in this mutant, determination of recombinant type, crossover type or gene conversion type, is probably carried out during a process of formation of recombination intermediate, not at the resolution stage of the recombination intermediate as assumed.
  • Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C), Molecular mechanism for chromosome stability using Nbs1 knockout mice, The gene mutated in Nijmegen breakage syndrome, NBS1, encodes a 95-kD protein that forms a complex with RAD50 and MRE11, and the complex is involved in the initiation process of DNA double strand break repair. In mouse, complete loss of Nbs1 gene results in early embryonic lethality and ES cells inviality. Therefore, it has been suggested that human NBS patients are not null mutants but hypomorphic mutants. Consistent with this prediction, C-terminal truncated NBS1 protein was identified in NBS patients. In this study, we targeted for disruption of Nbs1 gene by replacing 3'-half of exon 3 and all exons of 4 and 5 into LacZ gene and PGKneo cassette. Our Nbs1 mutants also exhibited embryonic lethality, but the phenotypes were much milder than those reported previously. The timing of embrynic death delayed to the post-implantation stage of 8.5-9.5 dpc and the embryonic fibroblast cells were virtually viable. Analysis of embryonic fibroblasts revealed that the Nbs1 mutant allele expressed an unexpected small quantity of C-terminal Nbs1 protein, which may be analogous to the human C-terminal protein. Consistent with this result, the absence of C-terminal Nbs1 protein, by removing the PGKneo cassete, advanced the timing of embrynic death. We, therefore, concluded that the C-terminal Nbs1 protein diminished severity of lethal phenotype in mouse Nbs1 null mutants.