Heteroduplex DNA Position Defines the Roles of the Sgs1, Srs2, and Mph1 Helicases in Promoting Distinct Recombination Outcomes
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{"title"=>"Heteroduplex DNA Position Defines the Roles of the Sgs1, Srs2, and Mph1 Helicases in Promoting Distinct Recombination Outcomes", "type"=>"journal", "authors"=>[{"first_name"=>"Katrina", "last_name"=>"Mitchel", "scopus_author_id"=>"57192410072"}, {"first_name"=>"Kevin", "last_name"=>"Lehner", "scopus_author_id"=>"23091242500"}, {"first_name"=>"Sue", "last_name"=>"Jinks-Robertson", "scopus_author_id"=>"7005305932"}], "year"=>2013, "source"=>"PLoS Genetics", "identifiers"=>{"issn"=>"15537390", "scopus"=>"2-s2.0-84875974599", "pui"=>"368694405", "doi"=>"10.1371/journal.pgen.1003340", "isbn"=>"1553-7404 (Electronic)\\n1553-7390 (Linking)", "sgr"=>"84875974599", "pmid"=>"23516370"}, "id"=>"97e7391a-b62d-3549-aa62-1b31344e7f00", "abstract"=>"The contributions of the Sgs1, Mph1, and Srs2 DNA helicases during mitotic double-strand break (DSB) repair in yeast were investigated using a gap-repair assay. A diverged chromosomal substrate was used as a repair template for the gapped plasmid, allowing mismatch-containing heteroduplex DNA (hDNA) formed during recombination to be monitored. Overall DSB repair efficiencies and the proportions of crossovers (COs) versus noncrossovers (NCOs) were determined in wild-type and helicase-defective strains, allowing the efficiency of CO and NCO production in each background to be calculated. In addition, the products of individual NCO events were sequenced to determine the location of hDNA. Because hDNA position is expected to differ depending on whether a NCO is produced by synthesis-dependent-strand-annealing (SDSA) or through a Holliday junction (HJ)-containing intermediate, its position allows the underlying molecular mechanism to be inferred. Results demonstrate that each helicase reduces the proportion of CO recombinants, but that each does so in a fundamentally different way. Mph1 does not affect the overall efficiency of gap repair, and its loss alters the CO-NCO by promoting SDSA at the expense of HJ-containing intermediates. By contrast, Sgs1 and Srs2 are each required for efficient gap repair, strongly promoting NCO formation and having little effect on CO efficiency. hDNA analyses suggest that all three helicases promote SDSA, and that Sgs1 and Srs2 additionally dismantle HJ-containing intermediates. The hDNA data are consistent with the proposed role of Sgs1 in the dissolution of double HJs, and we propose that Srs2 dismantles nicked HJs.", "link"=>"http://www.mendeley.com/research/heteroduplex-dna-position-defines-roles-sgs1-srs2-mph1-helicases-promoting-distinct-recombination-ou", "reader_count"=>44, "reader_count_by_academic_status"=>{"Unspecified"=>2, "Professor > Associate Professor"=>2, "Researcher"=>11, "Student > Doctoral Student"=>2, "Student > Ph. D. Student"=>20, "Student > Master"=>2, "Student > Bachelor"=>3, "Lecturer"=>1, "Professor"=>1}, "reader_count_by_user_role"=>{"Unspecified"=>2, "Professor > Associate Professor"=>2, "Researcher"=>11, "Student > Doctoral Student"=>2, "Student > Ph. D. Student"=>20, "Student > Master"=>2, "Student > Bachelor"=>3, "Lecturer"=>1, "Professor"=>1}, "reader_count_by_subject_area"=>{"Unspecified"=>2, "Environmental Science"=>1, "Biochemistry, Genetics and Molecular Biology"=>10, "Agricultural and Biological Sciences"=>30, "Physics and Astronomy"=>1}, "reader_count_by_subdiscipline"=>{"Physics and Astronomy"=>{"Physics and Astronomy"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>30}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>10}, "Unspecified"=>{"Unspecified"=>2}, "Environmental Science"=>{"Environmental Science"=>1}}, "reader_count_by_country"=>{"United States"=>3, "United Kingdom"=>1, "Portugal"=>1, "Spain"=>2}, "group_count"=>0}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/987792"], "description"=>"<p>Data presented in <a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003340#pgen-1003340-t001\" target=\"_blank\">Table 1</a>, <a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003340#pgen-1003340-t002\" target=\"_blank\">Table 2</a>, and <a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003340#pgen-1003340-t003\" target=\"_blank\">Table 3</a> are summarized. A. The height of each bar indicates the normalized mean of each type of product. COs are in red and NCOs are in blue. Asterisks indicate p<0.05. B. NCOs with bidirectional hDNA are in dark blue, and NCOs with unidirectional hDNA are in light blue. Asterisks indicate p<0.05.</p>", "links"=>[], "tags"=>["gap-repair", "wt", "helicase-deficient"], "article_id"=>652449, "categories"=>["Genetics"], "users"=>["Katrina Mitchel", "Kevin Lehner", "Sue Jinks-Robertson"], "doi"=>"https://dx.doi.org/10.1371/journal.pgen.1003340.g004", "stats"=>{"downloads"=>1, "page_views"=>5, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Distribution_of_gap_repair_products_in_WT_and_helicase_deficient_strains_/652449", "title"=>"Distribution of gap-repair products in WT and helicase-deficient strains.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-03-15 11:37:35"}
  • {"files"=>["https://ndownloader.figshare.com/files/987794"], "description"=>"<p>Sgs1, Srs2 and Mph1 are represented by green, yellow and pink symbols, respectively. A. Whereas all three helicases may be able to displace the invading end from the D-loop, Sgs1 and Srs2 are postulated to have additional activities during SDSA. Sgs1 may be required to rewind the invaded duplex, while the strippase activity of Srs2 may be important for 2<sup>nd</sup>-end annealing. Gray ovals represent Rad51, and its removal by Srs2 is indicated. B. If not dismantled by Mph1, the D-loop can be captured by the other end of the DSB and form an HJ-containing intermediate. A completely ligated dHJ is likely a substrate for Sgs1 complex, while a nicked sHJ or nicked dHJ may be a substrate for Srs2. The pattern of hDNA predicted by HJ cleavage is extremely rare in this system and does not contribute significantly to NCO events.</p>", "links"=>[], "tags"=>["helicase-mediated", "nco"], "article_id"=>652451, "categories"=>["Genetics"], "users"=>["Katrina Mitchel", "Kevin Lehner", "Sue Jinks-Robertson"], "doi"=>"https://dx.doi.org/10.1371/journal.pgen.1003340.g005", "stats"=>{"downloads"=>1, "page_views"=>10, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Models_for_helicase_mediated_regulation_of_NCO_formation_/652451", "title"=>"Models for helicase-mediated regulation of NCO formation.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-03-15 11:38:01"}
  • {"files"=>["https://ndownloader.figshare.com/files/987796", "https://ndownloader.figshare.com/files/987798", "https://ndownloader.figshare.com/files/987800", "https://ndownloader.figshare.com/files/987801"], "description"=>"<div><p>The contributions of the Sgs1, Mph1, and Srs2 DNA helicases during mitotic double-strand break (DSB) repair in yeast were investigated using a gap-repair assay. A diverged chromosomal substrate was used as a repair template for the gapped plasmid, allowing mismatch-containing heteroduplex DNA (hDNA) formed during recombination to be monitored. Overall DSB repair efficiencies and the proportions of crossovers (COs) versus noncrossovers (NCOs) were determined in wild-type and helicase-defective strains, allowing the efficiency of CO and NCO production in each background to be calculated. In addition, the products of individual NCO events were sequenced to determine the location of hDNA. Because hDNA position is expected to differ depending on whether a NCO is produced by synthesis-dependent-strand-annealing (SDSA) or through a Holliday junction (HJ)–containing intermediate, its position allows the underlying molecular mechanism to be inferred. Results demonstrate that each helicase reduces the proportion of CO recombinants, but that each does so in a fundamentally different way. Mph1 does not affect the overall efficiency of gap repair, and its loss alters the CO-NCO by promoting SDSA at the expense of HJ–containing intermediates. By contrast, Sgs1 and Srs2 are each required for efficient gap repair, strongly promoting NCO formation and having little effect on CO efficiency. hDNA analyses suggest that all three helicases promote SDSA, and that Sgs1 and Srs2 additionally dismantle HJ–containing intermediates. The hDNA data are consistent with the proposed role of Sgs1 in the dissolution of double HJs, and we propose that Srs2 dismantles nicked HJs.</p> </div>", "links"=>[], "tags"=>["heteroduplex", "dna", "defines", "mph1", "helicases", "promoting", "recombination", "outcomes"], "article_id"=>652453, "categories"=>["Genetics"], "users"=>["Katrina Mitchel", "Kevin Lehner", "Sue Jinks-Robertson"], "doi"=>["https://dx.doi.org/10.1371/journal.pgen.1003340.s001", "https://dx.doi.org/10.1371/journal.pgen.1003340.s002", "https://dx.doi.org/10.1371/journal.pgen.1003340.s003", "https://dx.doi.org/10.1371/journal.pgen.1003340.s004"], "stats"=>{"downloads"=>36, "page_views"=>23, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Heteroduplex_DNA_Position_Defines_the_Roles_of_the_Sgs1_Srs2_and_Mph1_Helicases_in_Promoting_Distinct_Recombination_Outcomes__/652453", "title"=>"Heteroduplex DNA Position Defines the Roles of the Sgs1, Srs2, and Mph1 Helicases in Promoting Distinct Recombination Outcomes", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2013-03-15 11:38:36"}
  • {"files"=>["https://ndownloader.figshare.com/files/1007162"], "description"=>"<p>All strains are <i>mlh1</i>Δ. Mean plasmid-repair, CO and NCO efficiencies were determined as in <a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003340#pgen-1003340-t001\" target=\"_blank\">Table 1</a>. The position of hDNA was determined by sequencing individual NCO events; proportions of unidirectional and bidirectional hDNA were derived using only those transformants in which hDNA was detected. One NCO event with the pattern of hDNA consistent with HJ cleavage was detected in the <i>mph1</i>Δ <i>sgs1</i>Δ background. Asterisks indicate a significant difference when compared to WT (p<0.05).</p>", "links"=>[], "tags"=>["nco", "unidirectional", "bidirectional", "hdna", "repaired", "plasmid"], "article_id"=>667788, "categories"=>["Genetics"], "users"=>["Katrina Mitchel", "Kevin Lehner", "Sue Jinks-Robertson"], "doi"=>"https://dx.doi.org/10.1371/journal.pgen.1003340.t002", "stats"=>{"downloads"=>0, "page_views"=>16, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Efficiency_of_NCO_events_with_unidirectional_versus_bidirectional_hDNA_in_the_repaired_plasmid_allele_/667788", "title"=>"Efficiency of NCO events with unidirectional versus bidirectional hDNA in the repaired plasmid allele.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2013-03-14 02:09:48"}
  • {"files"=>["https://ndownloader.figshare.com/files/1007144"], "description"=>"<p>All strains are <i>mlh1</i>Δ. Mean plasmid-repair, CO and NCO efficiencies were determined as in <a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003340#pgen-1003340-t001\" target=\"_blank\">Table 1</a>. The position of hDNA was determined by sequencing individual NCO events; proportions of unidirectional and bidirectional hDNA were derived using only those transformants in which hDNA was detected. Asterisks indicate a significant difference when compared to WT (p<0.05).</p>", "links"=>[], "tags"=>["nco", "unidirectional", "bidirectional", "hdna", "repaired", "plasmid"], "article_id"=>667766, "categories"=>["Genetics"], "users"=>["Katrina Mitchel", "Kevin Lehner", "Sue Jinks-Robertson"], "doi"=>"https://dx.doi.org/10.1371/journal.pgen.1003340.t003", "stats"=>{"downloads"=>1, "page_views"=>10, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Efficiency_of_NCO_events_with_unidirectional_versus_bidirectional_hDNA_in_the_repaired_plasmid_allele_/667766", "title"=>"Efficiency of NCO events with unidirectional versus bidirectional hDNA in the repaired plasmid allele.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2013-03-14 02:09:26"}
  • {"files"=>["https://ndownloader.figshare.com/files/987785"], "description"=>"<p>Single strands of DNA are represented by orange and black lines, and arrowheads indicate 3′ ends. Regions of hDNA are boxed, and newly synthesized DNA is depicted as dashed lines in the same color as the template allele. Additional detail is provided in the text.</p>", "links"=>[], "tags"=>["genetics and genomics"], "article_id"=>652442, "categories"=>["Genetics"], "users"=>["Katrina Mitchel", "Kevin Lehner", "Sue Jinks-Robertson"], "doi"=>"https://dx.doi.org/10.1371/journal.pgen.1003340.g001", "stats"=>{"downloads"=>0, "page_views"=>3, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Gap_repair_pathways_/652442", "title"=>"Gap-repair pathways.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-03-15 11:35:39"}
  • {"files"=>["https://ndownloader.figshare.com/files/987787"], "description"=>"<p>A. Schematic of the gap-repair system, which detects both CO and NCO events. B. The efficiency of gap repair in WT and helicase-deficient strains, all of which are MMR-defective (<i>mlh1Δ</i>). Normalized ratios of His<sup>+</sup> to Leu<sup>+</sup> transformants in individual transformations are depicted by filled circles; ratios obtained with the first transformation mix are labeled in blue, and grey indicates ratios obtained with the second transformation mix. The mean is indicated with a black bar.</p>", "links"=>[], "tags"=>["gap-repair"], "article_id"=>652444, "categories"=>["Genetics"], "users"=>["Katrina Mitchel", "Kevin Lehner", "Sue Jinks-Robertson"], "doi"=>"https://dx.doi.org/10.1371/journal.pgen.1003340.g002", "stats"=>{"downloads"=>2, "page_views"=>25, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Gap_repair_system_and_gap_repair_efficiency_in_the_absence_of_Mph1_Sgs1_or_Srs2_/652444", "title"=>"Gap-repair system and gap-repair efficiency in the absence of Mph1, Sgs1, or Srs2.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-03-15 11:36:08"}
  • {"files"=>["https://ndownloader.figshare.com/files/1007181"], "description"=>"<p>All strains were MMR-defective (<i>mlh1</i>Δ). Asterisks indicate a significant difference when compared to WT using a Student's t-test (p<0.05).</p>1<p>Plasmid repair efficiency reflects the mean His<sup>+</sup>∶Leu<sup>+</sup> ratio normalized to that obtained in the WT strain. N is number of independent transformations used to determine the mean.</p>2<p>Mean CO and NCO efficiencies were determined by multiplying the repair efficiency by the proportion of transformants that were categorized as CO and NCO events, respectively, based on plasmid stability.</p>", "links"=>[], "tags"=>["co", "wt", "helicase-deficient"], "article_id"=>667805, "categories"=>["Genetics"], "users"=>["Katrina Mitchel", "Kevin Lehner", "Sue Jinks-Robertson"], "doi"=>"https://dx.doi.org/10.1371/journal.pgen.1003340.t001", "stats"=>{"downloads"=>0, "page_views"=>9, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Gap_repair_efficiency_and_CO_production_in_WT_and_helicase_deficient_strains_/667805", "title"=>"Gap-repair efficiency and CO production in WT and helicase-deficient strains.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2013-03-14 02:10:05"}
  • {"files"=>["https://ndownloader.figshare.com/files/987789"], "description"=>"<p>Each line represents the plasmid allele of a single NCO event; plasmid sequence is depicted in yellow, chromosomal sequence in blue and hDNA in green. The positions of the SNPs are indicated to scale within the chromosomal allele. Only those NCOs with hDNA detected on the plasmid allele are shown, as they were the samples used for statistical analysis. Samples are grouped by strain background (all strains were <i>mlh1Δ</i>) and arranged based on the position of hDNA.</p>", "links"=>[], "tags"=>["hdna", "nco", "wt", "helicase-deficient"], "article_id"=>652446, "categories"=>["Genetics"], "users"=>["Katrina Mitchel", "Kevin Lehner", "Sue Jinks-Robertson"], "doi"=>"https://dx.doi.org/10.1371/journal.pgen.1003340.g003", "stats"=>{"downloads"=>1, "page_views"=>11, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Position_of_hDNA_in_NCO_products_of_WT_and_helicase_deficient_strains_/652446", "title"=>"Position of hDNA in NCO products of WT and helicase-deficient strains.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-03-15 11:36:42"}

PMC Usage Stats | Further Information

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Relative Metric

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