Reconstitution of Saccharomyces cerevisiae prereplicative complex assembly in vitro
The assembly of the prereplicative complex (pre‐RC) at the origin of replication in eukaryotes is a highly regulated and highly conserved process that plays a critical role in preventing multiple rounds of DNA replication per cell division cycle. This study analyzes the molecular dynamics of the ass...
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| Veröffentlicht in: | Genes to cells : devoted to molecular & cellular mechanisms 2006-07, Vol.11 (7), p.745-756 |
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| creator | Kawasaki, Yasuo Kim, Hee‐Dai Kojima, Akihiro Seki, Takashi Sugino, Akio |
| description | The assembly of the prereplicative complex (pre‐RC) at the origin of replication in eukaryotes is a highly regulated and highly conserved process that plays a critical role in preventing multiple rounds of DNA replication per cell division cycle. This study analyzes the molecular dynamics of the assembly of Saccharomyces cerevisiae pre‐RC in vitro using ARS1 plasmid DNA and yeast whole cell extracts. In addition, pre‐RC assembly was reconstituted in vitro using ARS1 DNA and purified origin‐recognition complex (ORC), Cdc6p and Cdt1p‐Mcm2‐7p. The results reveal sequential recruitment of ORC, Cdc6p, Cdt1p and Mcm2‐7p on to ARS1 DNA. When Mcm2‐7p is maximally loaded, Cdc6p and Cdt1p are released, suggesting that these two proteins are co‐ordinately regulated during pre‐RC assembly. In extracts from sid2‐21 mutant cells that are deficient in CDT1, ORC and Cdc6p bind to ARS1 but Cdt1p and Mcm2‐7p do not. However, Mcm2‐7p does bind in the presence of exogenous Cdt1p or Cdt1p‐Mcm2‐7p complex. Cdt1p‐Mcm2‐7p complex, which was purified from G1‐, early S or G2/M‐arrested cells, exhibits structure‐specific DNA binding, interacting only with bubble‐ or Y‐shape‐DNA, but the biological significance of this observation is not yet known. |
| doi_str_mv | 10.1111/j.1365-2443.2006.00975.x |
| format | Article |
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This study analyzes the molecular dynamics of the assembly of Saccharomyces cerevisiae pre‐RC in vitro using ARS1 plasmid DNA and yeast whole cell extracts. In addition, pre‐RC assembly was reconstituted in vitro using ARS1 DNA and purified origin‐recognition complex (ORC), Cdc6p and Cdt1p‐Mcm2‐7p. The results reveal sequential recruitment of ORC, Cdc6p, Cdt1p and Mcm2‐7p on to ARS1 DNA. When Mcm2‐7p is maximally loaded, Cdc6p and Cdt1p are released, suggesting that these two proteins are co‐ordinately regulated during pre‐RC assembly. In extracts from sid2‐21 mutant cells that are deficient in CDT1, ORC and Cdc6p bind to ARS1 but Cdt1p and Mcm2‐7p do not. However, Mcm2‐7p does bind in the presence of exogenous Cdt1p or Cdt1p‐Mcm2‐7p complex. Cdt1p‐Mcm2‐7p complex, which was purified from G1‐, early S or G2/M‐arrested cells, exhibits structure‐specific DNA binding, interacting only with bubble‐ or Y‐shape‐DNA, but the biological significance of this observation is not yet known.</description><identifier>ISSN: 1356-9597</identifier><identifier>EISSN: 1365-2443</identifier><identifier>DOI: 10.1111/j.1365-2443.2006.00975.x</identifier><identifier>PMID: 16824194</identifier><language>eng</language><publisher>Malden, USA: Blackwell Publishing Inc</publisher><subject>Cell Cycle Proteins - genetics ; Cell Division - genetics ; DNA Replication - genetics ; DNA Replication - physiology ; DNA-Binding Proteins - genetics ; Electrophoretic Mobility Shift Assay ; G1 Phase - genetics ; G2 Phase - genetics ; Mass Spectrometry ; Origin Recognition Complex - genetics ; Replication Origin - genetics ; S Phase - genetics ; Saccharomyces cerevisiae ; Saccharomyces cerevisiae - genetics ; Saccharomyces cerevisiae - physiology ; Saccharomyces cerevisiae Proteins - genetics ; Saccharomyces cerevisiae Proteins - metabolism ; Transcription Factors - genetics</subject><ispartof>Genes to cells : devoted to molecular & cellular mechanisms, 2006-07, Vol.11 (7), p.745-756</ispartof><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c3975-9f95ed0de65efa4944101ede70c3467d830f7602819e768f7c3cf6035504f2db3</citedby><cites>FETCH-LOGICAL-c3975-9f95ed0de65efa4944101ede70c3467d830f7602819e768f7c3cf6035504f2db3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://onlinelibrary.wiley.com/doi/pdf/10.1111%2Fj.1365-2443.2006.00975.x$$EPDF$$P50$$Gwiley$$H</linktopdf><linktohtml>$$Uhttps://onlinelibrary.wiley.com/doi/full/10.1111%2Fj.1365-2443.2006.00975.x$$EHTML$$P50$$Gwiley$$H</linktohtml><link.rule.ids>315,781,785,1418,1434,27929,27930,45579,45580,46414,46838</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/16824194$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Kawasaki, Yasuo</creatorcontrib><creatorcontrib>Kim, Hee‐Dai</creatorcontrib><creatorcontrib>Kojima, Akihiro</creatorcontrib><creatorcontrib>Seki, Takashi</creatorcontrib><creatorcontrib>Sugino, Akio</creatorcontrib><title>Reconstitution of Saccharomyces cerevisiae prereplicative complex assembly in vitro</title><title>Genes to cells : devoted to molecular & cellular mechanisms</title><addtitle>Genes Cells</addtitle><description>The assembly of the prereplicative complex (pre‐RC) at the origin of replication in eukaryotes is a highly regulated and highly conserved process that plays a critical role in preventing multiple rounds of DNA replication per cell division cycle. 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Cdt1p‐Mcm2‐7p complex, which was purified from G1‐, early S or G2/M‐arrested cells, exhibits structure‐specific DNA binding, interacting only with bubble‐ or Y‐shape‐DNA, but the biological significance of this observation is not yet known.</abstract><cop>Malden, USA</cop><pub>Blackwell Publishing Inc</pub><pmid>16824194</pmid><doi>10.1111/j.1365-2443.2006.00975.x</doi><tpages>12</tpages></addata></record> |
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| source | MEDLINE; Access via Wiley Online Library; Open Access Titles of Japan; EZB-FREE-00999 freely available EZB journals; Wiley Online Library (Open Access Collection) |
| subjects | Cell Cycle Proteins - genetics Cell Division - genetics DNA Replication - genetics DNA Replication - physiology DNA-Binding Proteins - genetics Electrophoretic Mobility Shift Assay G1 Phase - genetics G2 Phase - genetics Mass Spectrometry Origin Recognition Complex - genetics Replication Origin - genetics S Phase - genetics Saccharomyces cerevisiae Saccharomyces cerevisiae - genetics Saccharomyces cerevisiae - physiology Saccharomyces cerevisiae Proteins - genetics Saccharomyces cerevisiae Proteins - metabolism Transcription Factors - genetics |
| title | Reconstitution of Saccharomyces cerevisiae prereplicative complex assembly in vitro |
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