Stimulation of fission yeast and mouse Hop2-Mnd1 of the Dmc1 and Rad51 recombinases.

Genome Stability Laboratory, Laval University Cancer Research Center, Hôtel-Dieu de Québec, 9 McMahon, Quebec city, QC, Canada G1R 2J6.
Nucleic Acids Research (Impact Factor: 8.81). 03/2007; 35(8):2719-33. DOI: 10.1093/nar/gkm174
Source: PubMed

ABSTRACT Genetic analysis of fission yeast suggests a role for the spHop2-Mnd1 proteins in the Rad51 and Dmc1-dependent meiotic recombination pathways. In order to gain biochemical insights into this process, we purified Schizosaccharomyces pombe Hop2-Mnd1 to homogeneity. spHop2 and spMnd1 interact by co-immunoprecipitation and two-hybrid analysis. Electron microscopy reveals that S. pombe Hop2-Mnd1 binds single-strand DNA ends of 3'-tailed DNA. Interestingly, spHop2-Mnd1 promotes the renaturation of complementary single-strand DNA and catalyses strand exchange reactions with short oligonucleotides. Importantly, we show that spHop2-Mnd1 stimulates spDmc1-dependent strand exchange and strand invasion. Ca(2+) alleviate the requirement for the order of addition of the proteins on DNA. We also demonstrate that while spHop2-Mnd1 affects spDmc1 specifically, mHop2 or mHop2-Mnd1 stimulates both the hRad51 and hDmc1 recombinases in strand exchange assays. Thus, our results suggest a crucial role for S. pombe and mouse Hop2-Mnd1 in homologous pairing and strand exchange and reveal evolutionary divergence in their specificity for the Dmc1 and Rad51 recombinases.

  • [Show abstract] [Hide abstract]
    ABSTRACT: Hypoxia-inducible transcription factor-1 (HIF-1) plays a decisive role in cell survival and adaptation to hypoxic stress by controlling the expression of genes involved in oxygen homeostasis. HIF-1 activity is fine-tuned through specific post-translational modifications of its essential HIF-1α subunit. Amongst these modifications, phosphorylation is important for HIF-1 transcriptional activity. Studies have shown that the mitogen-activated protein kinases, p42/p44 MAPK, directly phosphorylate HIF-1α and increase HIF-1-mediated transcription. Pin1, a peptidyl-prolyl cis/trans isomerase, targets a number of proteins containing a phosphorylated Ser/Thr-Pro motif. Pin1 isomerization causes a change in target protein conformation which can modify their activity. Here, we identify Pin1 as an important HIF-1α partner. Immunoprecipitation and pull-down studies show that Pin1 interacts with HIF-1α. We demonstrate that the interaction between Pin1 and HIF-1α is regulated through p42/p44 MAPK pathway activation. By performing proteolysis studies, our results indicate that Pin1 catalytic activity generates a conformational change in HIF-1α. Finally, our work shows that Pin1 is required for gene-specific HIF-1 transcriptional activity. Our results indicate that the prolyl isomerase Pin1 regulates HIF-1 transcriptional activity by interacting with HIF-1α and promoting conformational changes in a p42/p44 MAPK phosphorylation-dependent manner.
    Cellular Signalling 04/2014; DOI:10.1016/j.cellsig.2014.04.005 · 4.47 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: During meiosis in Saccharomyces cerevisiae, the HOP2 and MND1 genes are essential for recombination. A previous biochemical study showed budding yeast Hop2-Mnd1 stimulated the activity of the meiosis-specific strand exchange protein ScDmc1 only 3-fold, whereas analogous studies using mammalian homologs showed >30-fold stimulation. The HOP2 gene was recently discovered to contain a second intron that lies near the 3 prime end. We show both HOP2 introns are efficiently spliced during meiosis, forming a predominant transcript that codes for a protein with a C-terminal sequence different from that of the previously studied version of the protein. Using the newly identified HOP2 open reading frame to direct synthesis of wild type Hop2 protein, we show that the Hop2-Mnd1 heterodimer stimulates Dmc1 D-loop activity up to 30-fold, similar to the activity of mammalian Hop2-Mnd1. ScHop2-Mnd1 stimulates ScDmc1 activity in the presence of physiological (micromolar) concentrations of Ca2+ ions, as long as Mg2+ is also present at physiological concentrations, leading us to hypothesize that ScDmc1 protomers bind both cations in the active Dmc1 filament. Co-factor requirements and order of addition experiments suggest that Hop2-Mnd1-mediated stimulation of Dmc1 involves a process that follows formation of functional Dmc1-ssDNA filaments. In dramatic contrast to mammalian orthologs, the stimulatory activity of budding yeast Hop2-Mnd1 appears to be specific to Dmc1; we observed no Hop2-Mnd1-mediated stimulation of the other budding yeast strand exchange protein Rad51. Together, these results support previous genetic experiments indicating that Hop2-Mnd1 specifically stimulates Dmc1 during meiotic recombination in budding yeast.
    Journal of Biological Chemistry 05/2014; 289(26). DOI:10.1074/jbc.M114.558601 · 4.60 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: In meiotic DNA recombination, the Hop2-Mnd1 complex promotes Dmc1-mediated single-stranded DNA (ssDNA) invasion into homologous chromosomes to form a synaptic complex by a yet-unclear mechanism. Here, the crystal structure of Hop2-Mnd1 reveals that it forms a curved rod-like structure consisting of three leucine zippers and two kinked junctions. One end of the rod is linked to two juxtaposed winged-helix domains, and the other end is capped by extra α-helices to form a helical bundle-like structure. Deletion analysis shows that the helical bundle-like structure is sufficient for interacting with the Dmc1-ssDNA nucleofilament, and molecular modeling suggests that the curved rod could be accommodated into the helical groove of the nucleofilament. Remarkably, the winged-helix domains are juxtaposed at fixed relative orientation, and their binding to DNA is likely to perturb the base pairing according to molecular simulations. These findings allow us to propose a model explaining how Hop2-Mnd1 juxtaposes Dmc1-bound ssDNA with distorted recipient double-stranded DNA and thus facilitates strand invasion. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.
    Nucleic Acids Research 03/2015; DOI:10.1093/nar/gkv172 · 8.81 Impact Factor

Full-text (3 Sources)

Available from
Jun 3, 2014