A Novel Role of Human Holliday Junction Resolvase GEN1 in the Maintenance of Centrosome Integrity
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{"title"=>"A Novel Role of Human Holliday Junction Resolvase GEN1 in the Maintenance of Centrosome Integrity", "type"=>"journal", "authors"=>[{"first_name"=>"Min", "last_name"=>"Gao", "scopus_author_id"=>"57199670382"}, {"first_name"=>"Jannie", "last_name"=>"Rendtlew Danielsen", "scopus_author_id"=>"57188955341"}, {"first_name"=>"Lei Zhen", "last_name"=>"Wei", "scopus_author_id"=>"25825795200"}, {"first_name"=>"Dong Ping", "last_name"=>"Zhou", "scopus_author_id"=>"55486719600"}, {"first_name"=>"Qian", "last_name"=>"Xu", "scopus_author_id"=>"55486841100"}, {"first_name"=>"Miao Miao", "last_name"=>"Li", "scopus_author_id"=>"55486830800"}, {"first_name"=>"Zhao Qi", "last_name"=>"Wang", "scopus_author_id"=>"55719649900"}, {"first_name"=>"Wei Min", "last_name"=>"Tong", "scopus_author_id"=>"7202449320"}, {"first_name"=>"Yun Gui", "last_name"=>"Yang", "scopus_author_id"=>"7409386081"}], "year"=>2012, "source"=>"PLoS ONE", "identifiers"=>{"pui"=>"366057760", "issn"=>"19326203", "isbn"=>"1932-6203 (Electronic)\\r1932-6203 (Linking)", "doi"=>"10.1371/journal.pone.0049687", "scopus"=>"2-s2.0-84869208744", "pmid"=>"23166748", "sgr"=>"84869208744"}, "id"=>"b9440c4a-f73b-3527-90e2-79fca2924df3", "abstract"=>"The maintenance of genomic stability requires accurate genome replication, repair of DNA damage, and the precise segregation of chromosomes in mitosis. GEN1 possesses Holliday junction resolvase activity in vitro and presumably functions in homology driven repair of DNA double strand breaks. However, little is currently known about the cellular functions of human GEN1. In the present study we demonstrate that GEN1 is a novel centrosome associated protein and we characterize the various phenotypes associated with GEN1 deficiency. We identify an N-terminal centrosome localization signal in GEN1, which is required and sufficient for centrosome localization. We report that GEN1 depletion results in aberrant centrosome numbers associated with the formation of multiple spindle poles in mitosis, an increased number of cells with multi-nuclei, increased apoptosis and an elevated level of spontaneous DNA damage. We find homologous recombination severely impaired in GEN1 deficient cells, suggesting that GEN1 functions as a Holliday junction resolvase in vivo as well as in vitro. Complementation of GEN1 depleted cells with various GEN1 constructs revealed that centrosome association but not catalytic activity of GEN1 is required for preventing centrosome hyper-amplification, formation of multiple mitotic spindles, and multi-nucleation. Our findings provide novel insight into the biological functions of GEN1 by uncovering an important role of GEN1 in the regulation of centrosome integrity.", "link"=>"http://www.mendeley.com/research/novel-role-human-holliday-junction-resolvase-gen1-maintenance-centrosome-integrity", "reader_count"=>28, "reader_count_by_academic_status"=>{"Unspecified"=>1, "Professor > Associate Professor"=>1, "Researcher"=>11, "Student > Doctoral Student"=>2, "Student > Ph. D. Student"=>9, "Other"=>1, "Student > Bachelor"=>3}, "reader_count_by_user_role"=>{"Unspecified"=>1, "Professor > Associate Professor"=>1, "Researcher"=>11, "Student > Doctoral Student"=>2, "Student > Ph. D. Student"=>9, "Other"=>1, "Student > Bachelor"=>3}, "reader_count_by_subject_area"=>{"Unspecified"=>2, "Biochemistry, Genetics and Molecular Biology"=>9, "Agricultural and Biological Sciences"=>15, "Medicine and Dentistry"=>2}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>2}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>15}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>9}, "Unspecified"=>{"Unspecified"=>2}}, "reader_count_by_country"=>{"United States"=>1, "Spain"=>1}, "group_count"=>0}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/289828", "https://ndownloader.figshare.com/files/289874", "https://ndownloader.figshare.com/files/289927", "https://ndownloader.figshare.com/files/289984"], "description"=>"<div><p>The maintenance of genomic stability requires accurate genome replication, repair of DNA damage, and the precise segregation of chromosomes in mitosis. GEN1 possesses Holliday junction resolvase activity <em>in vitro</em> and presumably functions in homology driven repair of DNA double strand breaks. However, little is currently known about the cellular functions of human GEN1. In the present study we demonstrate that GEN1 is a novel centrosome associated protein and we characterize the various phenotypes associated with GEN1 deficiency. We identify an N-terminal centrosome localization signal in GEN1, which is required and sufficient for centrosome localization. We report that GEN1 depletion results in aberrant centrosome numbers associated with the formation of multiple spindle poles in mitosis, an increased number of cells with multi-nuclei, increased apoptosis and an elevated level of spontaneous DNA damage. We find homologous recombination severely impaired in GEN1 deficient cells, suggesting that GEN1 functions as a Holliday junction resolvase <em>in vivo</em> as well as <em>in vitro</em>. Complementation of GEN1 depleted cells with various GEN1 constructs revealed that centrosome association but not catalytic activity of GEN1 is required for preventing centrosome hyper-amplification, formation of multiple mitotic spindles, and multi-nucleation. Our findings provide novel insight into the biological functions of GEN1 by uncovering an important role of GEN1 in the regulation of centrosome integrity.</p> </div>", "links"=>[], "tags"=>["holliday", "junction", "resolvase", "gen1", "centrosome", "integrity"], "article_id"=>117069, "categories"=>["Cancer", "Cell Biology", "Genetics"], "users"=>["Min Gao", "Jannie Rendtlew Danielsen", "Lei-Zhen Wei", "Dong-Ping Zhou", "Qian Xu", "Miao-Miao Li", "Zhao-Qi Wang", "Wei-Min Tong", "Yun-Gui Yang"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0049687.s001", "https://dx.doi.org/10.1371/journal.pone.0049687.s002", "https://dx.doi.org/10.1371/journal.pone.0049687.s003", "https://dx.doi.org/10.1371/journal.pone.0049687.s004"], "stats"=>{"downloads"=>6, "page_views"=>8, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/A_Novel_Role_of_Human_Holliday_Junction_Resolvase_GEN1_in_the_Maintenance_of_Centrosome_Integrity__/117069", "title"=>"A Novel Role of Human Holliday Junction Resolvase GEN1 in the Maintenance of Centrosome Integrity", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2012-11-16 01:57:49"}
  • {"files"=>["https://ndownloader.figshare.com/files/540615"], "description"=>"<p>(left) HeLa cells were treated with GEN1 or control (CTRL) siRNA for 48 h, methanol fixed and immunostained with the indicated antibodies. (right) Graph represents the mean of three independent experiments. (n = 300 cells/condition/experiment). Scale bar, 10 µm. <b>B.</b> HeLa cells treated with CTRL or GEN1 siRNA were methanol fixed and immunostained with anti-GEN1 (651–892aa) antibody and anti-γ-tubulin antibody. Scale bars, 10 µm.</p>", "links"=>[], "tags"=>["deficiency", "leads", "supernumerary", "centrosomes"], "article_id"=>211098, "categories"=>["Cancer", "Cell Biology", "Genetics"], "users"=>["Min Gao", "Jannie Rendtlew Danielsen", "Lei-Zhen Wei", "Dong-Ping Zhou", "Qian Xu", "Miao-Miao Li", "Zhao-Qi Wang", "Wei-Min Tong", "Yun-Gui Yang"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0049687.g002", "stats"=>{"downloads"=>1, "page_views"=>9, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_GEN1_deficiency_leads_to_supernumerary_centrosomes_in_mitosis_A_/211098", "title"=>"GEN1 deficiency leads to supernumerary centrosomes in mitosis. A.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-11-16 00:18:18"}
  • {"files"=>["https://ndownloader.figshare.com/files/540995"], "description"=>"<p>48 h following siRNA treatment with the indicated siRNAs U20S/DR-GFP cells were transfected with I-SceI plasmid for 48 h. Cells were processed for flow cytometric analysis of GFP, and the extent of HR was scored as the number of GFP-positive cells. Graph represents the mean of three independent experiments. Error bars indicate S.E.M. <b>B.</b> HeLa cells treated with CTRL or GEN1 siRNA (48 h) were fixed in ice-cold acetone–methanol (1∶1) and immunostained with GEN1 and γ-H2AX. Scale bar, 100 µm. Graph represents the mean of three independent experiments. (n = 100 cells/condition/experiment). Error bars indicate S.E.M. <b>C.</b> HeLa cells were treated with CTRL or GEN1 siRNA for 48 h. After fixation in ice-cold acetone–methanol (1∶1), half the cells were immunostained with γ-H2AX and afterwards all cells were incubated with secondary antibody. The samples were then analyzed by flow cytometry and WinMDI software.</p>", "links"=>[], "tags"=>["hr", "dna"], "article_id"=>211482, "categories"=>["Cancer", "Cell Biology", "Genetics"], "users"=>["Min Gao", "Jannie Rendtlew Danielsen", "Lei-Zhen Wei", "Dong-Ping Zhou", "Qian Xu", "Miao-Miao Li", "Zhao-Qi Wang", "Wei-Min Tong", "Yun-Gui Yang"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0049687.g005", "stats"=>{"downloads"=>3, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_GEN1_is_required_for_HR_of_DNA_DSBs_A_/211482", "title"=>"GEN1 is required for HR of DNA DSBs. A.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-11-16 00:24:42"}
  • {"files"=>["https://ndownloader.figshare.com/files/541122"], "description"=>"<p>HeLa cells were simultaneously treated with GEN1 siRNA and the indicated siRNA insensitive GFP-GEN1 constructs for 48 h, methanol fixed and immunostained with the γ-tubulin and DAPI. Quantification was based on three independent experiments. (n = 100 cells/condition/experiment). The white box shows a 6.8 magnification of the centrosome identified by γ-tubulin. Error bars indicate S.E.M. Scale bars, 10 µm. <b>B.</b> HeLa cells were treated as in A. but immunostained with α-tubulin and DAPI. Quantification was based on three independent experiments. Error bars indicate S.E.M. (n = 100 cells/condition/experiment). Scale bars, 10 µm. <b>C.</b> HeLa cells were treated as in A. but immunostained with γ-H2AX and DAPI. Quantification was based on three independent experiments. Error bars indicate S.E.M. (n = 100 cells/condition/experiment). Scale bars, 10 µm.</p>", "links"=>[], "tags"=>["gen1", "maintaining", "centrosome", "appears", "separable", "hj", "resolvase"], "article_id"=>211601, "categories"=>["Cancer", "Cell Biology", "Genetics"], "users"=>["Min Gao", "Jannie Rendtlew Danielsen", "Lei-Zhen Wei", "Dong-Ping Zhou", "Qian Xu", "Miao-Miao Li", "Zhao-Qi Wang", "Wei-Min Tong", "Yun-Gui Yang"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0049687.g006", "stats"=>{"downloads"=>0, "page_views"=>12, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_The_role_of_GEN1_in_maintaining_centrosome_integrity_appears_separable_from_its_HJ_resolvase_function_A_/211601", "title"=>"The role of GEN1 in maintaining centrosome integrity appears separable from its HJ resolvase function. A.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-11-16 00:26:41"}
  • {"files"=>["https://ndownloader.figshare.com/files/540887"], "description"=>"<p>(left) 293T cells were treated with CTRL or GEN1 siRNA for 48 h stained with propidium iodine (PI) and analyzed by flow cytometry. (middle) G2/M cells were identified using Modfit software. (right) Mitotic cells were detected by phospho H3 antibody staining followed by flow cytometric analysis. Quantification is based on three independent experiments. Error bars indicate S.E.M. <b>B.</b> Hela cells were treated with CTRL or GEN1 siRNA for 48 h, labeled with or without 10 uM BrdU for 2 hours, stained with FITC conjugated anti-BrdU antibodies and propidium iodine (PI), and analyzed by flow cytometry. x-axis: propidium iodine (PI). y-axis FITC. <b>C.</b> CTRL or GEN1 siRNA treated Hela cells were incubated with 10 uM BrdU for 2 hours, and return to BrdU-free medium for the indicated times. Cell cycle progression of BrdU labeled cells were analyzed by flow cytometry. <b>D.</b> HeLa cells treated with CTRL or GEN1 siRNA (48 h) were methanol fixed and immunostained with GEN1(anti-GEN1 (651–892aa) antibody) and α-tubulin. DNA was visualized by DAPI. (Right) Graph represents the mean of three independent experiments. (n = 100 cells/condition/experiment). Error bars indicate S.E.M. <b>E.</b> HeLa cells were treated with CTRL or GEN1 siRNA for 48 h fixed, stained with annexin V and analyzed by flow cytometry. Quantification was based on three independent experiments. Error bars indicate S.E.M.</p>", "links"=>[], "tags"=>["depletion", "interferes", "progression", "multi-nucleation"], "article_id"=>211375, "categories"=>["Cancer", "Cell Biology", "Genetics"], "users"=>["Min Gao", "Jannie Rendtlew Danielsen", "Lei-Zhen Wei", "Dong-Ping Zhou", "Qian Xu", "Miao-Miao Li", "Zhao-Qi Wang", "Wei-Min Tong", "Yun-Gui Yang"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0049687.g004", "stats"=>{"downloads"=>1, "page_views"=>8, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_GEN1_depletion_interferes_with_cell_cycle_progression_and_results_in_multi_nucleation_and_apoptosis_A_/211375", "title"=>"GEN1 depletion interferes with cell cycle progression and results in multi-nucleation and apoptosis. A.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-11-16 00:22:55"}
  • {"files"=>["https://ndownloader.figshare.com/files/540444"], "description"=>"<p>(top) MRC5 cells were fixed with methanol and immunostained with GEN1 antibody (GST-GEN1 651–892 antibody) and DAPI. Scale bars, 10 µm. (bottom) Cells were lysed and separated by SDS-PAGE and immunoblotted using the indicated antibodies. <b>B.</b> HeLa cells were fixed with methanol and immunostained with anti-GEN1 (651–892aa) antibody, anti-γ-tubulin antibody and DAPI. Scale bars, 10 µm. <b>C.</b> Hela cells at different phases of the cell cycle were fixed with methanol and co-immunostained with the indicated antibodies and DAPI. Scale bars, 10 µm. <b>D.</b> Centrosomes were isolated from HeLa cells and purified fractions from a discontinuous sucrose gradient were separated by SDS-PAGE and immunoblotted using the indicated antibodies. <b>E.</b> Schematic showing GEN1 domains and alignment of the Cyclin E CLS and the putative CLS in GEN1. Highly conserved amino acids are shown in red. In black are shown three of the key amino acids within the endonuclease domain of GEN1. XPG (-N: N-terminal, -I, internal) nuclease domain domain very similar to the nuclease domain first identified in XPG. <b>F.</b> HeLa cells were transfected with the indicated plasmids and 24 h later methanol fixed and immonostained with γ-tubulin and DAPI. GEN1 was detected by GFP fluorescence. Scale bars, 10 µm. Images to the left are zoomed 27.5×. CLS-4A: centrosome localization defective mutant, Ci-3A: catalytic inactive mutant.</p>", "links"=>[], "tags"=>["gen1", "localizes"], "article_id"=>210935, "categories"=>["Cancer", "Cell Biology", "Genetics"], "users"=>["Min Gao", "Jannie Rendtlew Danielsen", "Lei-Zhen Wei", "Dong-Ping Zhou", "Qian Xu", "Miao-Miao Li", "Zhao-Qi Wang", "Wei-Min Tong", "Yun-Gui Yang"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0049687.g001", "stats"=>{"downloads"=>0, "page_views"=>8, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Human_GEN1_localizes_on_the_centrosome_A_/210935", "title"=>"Human GEN1 localizes on the centrosome. A.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-11-16 00:15:35"}
  • {"files"=>["https://ndownloader.figshare.com/files/540783"], "description"=>"<p>HeLa cells were treated with GEN1 or control (CTRL) siRNA for 48 h, methanol fixed and immunostained with the indicated antibodies. Identification of centrosomes and centrioles was based on immunostaining with γ-tubulin and Centrin 2 antibodies, respectively. Scale bar, 10 µm. <b>B.</b> Quantification of centrosomes with 1, 2 or more than 2 centrioles in CTRL or GEN1 depleted interphase cells. Graph represents the mean of three independent experiments. (n = 300 centrosomes/condition/experiment). Error bars indicate S.E.M. <b>C.</b> Quantification of interphase and mitotic cells (<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049687#pone-0049687-g003\" target=\"_blank\"><b>Fig. 3A</b></a>) with extra centrosomes (interphase>1 centrosome (G1) or>1 linked centrosome pair (S-G2), mitosis>2 separated centrosomes). Graph represents the mean of three independent experiments (n = 300 cells/condition/experiment). Error bars indicate S.E.M. <b>D.</b> The percent of supernumerary centrosomes with 1, 2 or more than 2 centrioles after GEN1 depletion. Graph represents the mean of three independent experiments. (n = 300 centrosomes/condition/experiment). Error bars indicate S.E.M. <b>E.</b> Quantification of mitotic cells with>2 mitotic spindles 48 h after treatment with CTRL, GEN1 and/or ATR siRNA followed by incubation with or without ATM inhibitor(10 uM Ku55933. Mitotic spindles were visualized by GEN1 (anti-GEN1 (651–892aa) antibody) and γ-tubulin immunostaining. Graph represents the mean of three independent experiments. (n = 300 cells/condition/experiment). Error bars indicate S.E.M.</p>", "links"=>[], "tags"=>["depletion", "supernumerary", "centrosomes", "g2"], "article_id"=>211268, "categories"=>["Cancer", "Cell Biology", "Genetics"], "users"=>["Min Gao", "Jannie Rendtlew Danielsen", "Lei-Zhen Wei", "Dong-Ping Zhou", "Qian Xu", "Miao-Miao Li", "Zhao-Qi Wang", "Wei-Min Tong", "Yun-Gui Yang"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0049687.g003", "stats"=>{"downloads"=>1, "page_views"=>9, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_GEN1_depletion_results_in_supernumerary_centrosomes_in_late_G2_or_mitosis_A_/211268", "title"=>"GEN1 depletion results in supernumerary centrosomes in late G2 or mitosis. A.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-11-16 00:21:08"}

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  • {"unique-ip"=>"2", "full-text"=>"2", "pdf"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2020", "month"=>"4"}
  • {"unique-ip"=>"6", "full-text"=>"4", "pdf"=>"5", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2020", "month"=>"5"}
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Relative Metric

{"start_date"=>"2012-01-01T00:00:00Z", "end_date"=>"2012-12-31T00:00:00Z", "subject_areas"=>[{"subject_area"=>"/Biology and life sciences/Anatomy and physiology", "average_usage"=>[307, 540, 660, 756, 847, 936, 1025, 1111, 1199, 1282, 1361, 1432]}, {"subject_area"=>"/Biology and life sciences/Cell biology", "average_usage"=>[319, 556, 679, 785, 881, 970, 1062, 1149, 1236, 1323, 1402, 1474, 1545, 1617, 1681, 1754, 1822, 1892, 1963, 2031, 2099, 2165, 2233, 2299, 2359]}, {"subject_area"=>"/Biology and life sciences/Physiology", "average_usage"=>[307, 536, 653, 753, 839, 926, 1017, 1103, 1189, 1268, 1348, 1425, 1492, 1557, 1620, 1686, 1759, 1825, 1891, 1950, 2014, 2079, 2141, 2200, 2255]}, {"subject_area"=>"/Medicine and health sciences", "average_usage"=>[312, 547, 668, 769, 861, 953, 1046, 1135, 1224, 1307, 1388, 1464, 1536, 1606, 1676, 1744, 1812, 1882, 1954, 2020, 2089, 2155, 2218, 2282, 2344]}, {"subject_area"=>"/Medicine and health sciences/Immunology", "average_usage"=>[307, 545, 668, 765, 855, 943, 1029, 1113, 1202, 1285, 1361, 1439, 1506, 1571, 1636, 1706, 1772, 1837, 1901, 1969, 2031, 2096, 2160, 2228, 2287]}, {"subject_area"=>"/Medicine and health sciences/Physiology", "average_usage"=>[308, 535, 653, 754, 847, 930, 1020, 1103, 1188, 1268, 1351, 1420, 1487, 1550, 1614, 1679, 1757, 1830, 1898, 1961, 2024, 2086, 2150, 2216, 2266]}]}
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Net::HTTPTooManyRequests

Source
Scopus
Time
2019-08-27 21:08:54 UTC
Target URL
https://api.elsevier.com/content/search/index:SCOPUS?query=DOI(10.1371%2Fjournal.pone.0049687)
Trace

/app/models/concerns/networkable.rb:21:in `get_result'
/app/models/source.rb:165:in `get_data'
/app/models/retrieval_status.rb:47:in `perform_get_data'
/app/jobs/source_job.rb:52:in `block (2 levels) in perform'
/app/jobs/source_job.rb:51:in `block in perform'
/app/jobs/source_job.rb:35:in `each'
/app/jobs/source_job.rb:35:in `perform'