Replication Factor C from the Hyperthermophilic Archaeon Pyrococcus abyssi Does Not Need ATP Hydrolysis for Clamp-loading and Contains a Functionally Conserved RFC PCNA-binding Domain
The molecular organization of the replication complex in archaea is similar to that in eukaryotes. Only two proteins homologous to subunits of eukaryotic replication factor C (RFC) have been detected in Pyrococcus abyssi ( Pab). The genes encoding these two proteins are arranged in tandem. We cloned...
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| Veröffentlicht in: | Journal of molecular biology 2002-11, Vol.323 (5), p.795-810 |
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| creator | Henneke, Ghislaine Gueguen, Yannick Flament, Didier Azam, Philippe Querellou, Joël Dietrich, Jacques Hübscher, Ulrich Raffin, Jean-Paul |
| description | The molecular organization of the replication complex in archaea is similar to that in eukaryotes. Only two proteins homologous to subunits of eukaryotic replication factor C (RFC) have been detected in
Pyrococcus abyssi (
Pab). The genes encoding these two proteins are arranged in tandem. We cloned these two genes and co-expressed the corresponding recombinant proteins in
Escherichia coli. Two inteins present in the gene encoding the small subunit (
PabRFC-small) were removed during cloning. The recombinant protein complex was purified by anion-exchange and hydroxyapatite chromatography. Also, the
PabRFC-small subunit could be purified, while the large subunit (
PabRFC-large) alone was completely insoluble. The highly purified
PabRFC complex possessed an ATPase activity, which was not enhanced by DNA. The
Pab proliferating cell nuclear antigen (PCNA) activated the
PabRFC complex in a DNA-dependent manner, but the
PabRFC-small ATPase activity was neither DNA-dependent nor PCNA-dependent. The
PabRFC complex was able to stimulate
PabPCNA-dependent DNA synthesis by the
Pabfamily D heterodimeric DNA polymerase. Finally, (i) the
PabRFC-large fraction cross-reacted with anti-human-RFC PCNA-binding domain antibody, corroborating the conservation of the protein sequence, (ii) the human PCNA stimulated the
PabRFC complex ATPase activity in a DNA-dependent way and (iii) the
PabRFC complex could load human PCNA onto primed single-stranded circular DNA, suggesting that the PCNA-binding domain of RFC has been functionally conserved during evolution. In addition, ATP hydrolysis was not required either for DNA polymerase stimulation or PCNA-loading
in vitro. |
| doi_str_mv | 10.1016/S0022-2836(02)01028-8 |
| format | Article |
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Pyrococcus abyssi (
Pab). The genes encoding these two proteins are arranged in tandem. We cloned these two genes and co-expressed the corresponding recombinant proteins in
Escherichia coli. Two inteins present in the gene encoding the small subunit (
PabRFC-small) were removed during cloning. The recombinant protein complex was purified by anion-exchange and hydroxyapatite chromatography. Also, the
PabRFC-small subunit could be purified, while the large subunit (
PabRFC-large) alone was completely insoluble. The highly purified
PabRFC complex possessed an ATPase activity, which was not enhanced by DNA. The
Pab proliferating cell nuclear antigen (PCNA) activated the
PabRFC complex in a DNA-dependent manner, but the
PabRFC-small ATPase activity was neither DNA-dependent nor PCNA-dependent. The
PabRFC complex was able to stimulate
PabPCNA-dependent DNA synthesis by the
Pabfamily D heterodimeric DNA polymerase. Finally, (i) the
PabRFC-large fraction cross-reacted with anti-human-RFC PCNA-binding domain antibody, corroborating the conservation of the protein sequence, (ii) the human PCNA stimulated the
PabRFC complex ATPase activity in a DNA-dependent way and (iii) the
PabRFC complex could load human PCNA onto primed single-stranded circular DNA, suggesting that the PCNA-binding domain of RFC has been functionally conserved during evolution. In addition, ATP hydrolysis was not required either for DNA polymerase stimulation or PCNA-loading
in vitro.</description><identifier>ISSN: 0022-2836</identifier><identifier>EISSN: 1089-8638</identifier><identifier>DOI: 10.1016/S0022-2836(02)01028-8</identifier><identifier>PMID: 12417194</identifier><language>eng</language><publisher>England: Elsevier Ltd</publisher><subject>Adenosine Triphosphatases - metabolism ; Adenosine Triphosphate - metabolism ; Amino Acid Sequence ; archaea ; Base Sequence ; Binding Sites ; Conserved Sequence ; Cross Reactions ; DNA Polymerase II - metabolism ; DNA Replication ; DNA, Archaeal - genetics ; DNA-Binding Proteins - chemistry ; DNA-Binding Proteins - genetics ; DNA-Binding Proteins - metabolism ; Escherichia coli - genetics ; Gene Expression ; Genes, Archaeal ; Humans ; Hydrolysis ; hyperthermophile ; Macromolecular Substances ; Molecular Sequence Data ; PCNA-binding domain ; Proliferating Cell Nuclear Antigen - metabolism ; Protein Structure, Tertiary ; Protein Subunits ; Pyrococcus - genetics ; Pyrococcus - metabolism ; Pyrococcus abyssi ; Recombinant Proteins - chemistry ; Recombinant Proteins - genetics ; Recombinant Proteins - metabolism ; replication factor C ; Replication Protein C ; Sequence Homology, Amino Acid</subject><ispartof>Journal of molecular biology, 2002-11, Vol.323 (5), p.795-810</ispartof><rights>2002 Elsevier Science Ltd</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c439t-a501cbf8cb76bb19b4bdc06285f425e429badcd1baa3120d033a8921bf69db333</citedby><cites>FETCH-LOGICAL-c439t-a501cbf8cb76bb19b4bdc06285f425e429badcd1baa3120d033a8921bf69db333</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://www.sciencedirect.com/science/article/pii/S0022283602010288$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,776,780,3536,27903,27904,65309</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/12417194$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Henneke, Ghislaine</creatorcontrib><creatorcontrib>Gueguen, Yannick</creatorcontrib><creatorcontrib>Flament, Didier</creatorcontrib><creatorcontrib>Azam, Philippe</creatorcontrib><creatorcontrib>Querellou, Joël</creatorcontrib><creatorcontrib>Dietrich, Jacques</creatorcontrib><creatorcontrib>Hübscher, Ulrich</creatorcontrib><creatorcontrib>Raffin, Jean-Paul</creatorcontrib><title>Replication Factor C from the Hyperthermophilic Archaeon Pyrococcus abyssi Does Not Need ATP Hydrolysis for Clamp-loading and Contains a Functionally Conserved RFC PCNA-binding Domain</title><title>Journal of molecular biology</title><addtitle>J Mol Biol</addtitle><description>The molecular organization of the replication complex in archaea is similar to that in eukaryotes. Only two proteins homologous to subunits of eukaryotic replication factor C (RFC) have been detected in
Pyrococcus abyssi (
Pab). The genes encoding these two proteins are arranged in tandem. We cloned these two genes and co-expressed the corresponding recombinant proteins in
Escherichia coli. Two inteins present in the gene encoding the small subunit (
PabRFC-small) were removed during cloning. The recombinant protein complex was purified by anion-exchange and hydroxyapatite chromatography. Also, the
PabRFC-small subunit could be purified, while the large subunit (
PabRFC-large) alone was completely insoluble. The highly purified
PabRFC complex possessed an ATPase activity, which was not enhanced by DNA. The
Pab proliferating cell nuclear antigen (PCNA) activated the
PabRFC complex in a DNA-dependent manner, but the
PabRFC-small ATPase activity was neither DNA-dependent nor PCNA-dependent. The
PabRFC complex was able to stimulate
PabPCNA-dependent DNA synthesis by the
Pabfamily D heterodimeric DNA polymerase. Finally, (i) the
PabRFC-large fraction cross-reacted with anti-human-RFC PCNA-binding domain antibody, corroborating the conservation of the protein sequence, (ii) the human PCNA stimulated the
PabRFC complex ATPase activity in a DNA-dependent way and (iii) the
PabRFC complex could load human PCNA onto primed single-stranded circular DNA, suggesting that the PCNA-binding domain of RFC has been functionally conserved during evolution. In addition, ATP hydrolysis was not required either for DNA polymerase stimulation or PCNA-loading
in vitro.</description><subject>Adenosine Triphosphatases - metabolism</subject><subject>Adenosine Triphosphate - metabolism</subject><subject>Amino Acid Sequence</subject><subject>archaea</subject><subject>Base Sequence</subject><subject>Binding Sites</subject><subject>Conserved Sequence</subject><subject>Cross Reactions</subject><subject>DNA Polymerase II - metabolism</subject><subject>DNA Replication</subject><subject>DNA, Archaeal - genetics</subject><subject>DNA-Binding Proteins - chemistry</subject><subject>DNA-Binding Proteins - genetics</subject><subject>DNA-Binding Proteins - metabolism</subject><subject>Escherichia coli - genetics</subject><subject>Gene Expression</subject><subject>Genes, Archaeal</subject><subject>Humans</subject><subject>Hydrolysis</subject><subject>hyperthermophile</subject><subject>Macromolecular Substances</subject><subject>Molecular Sequence Data</subject><subject>PCNA-binding domain</subject><subject>Proliferating Cell Nuclear Antigen - metabolism</subject><subject>Protein Structure, Tertiary</subject><subject>Protein Subunits</subject><subject>Pyrococcus - genetics</subject><subject>Pyrococcus - metabolism</subject><subject>Pyrococcus abyssi</subject><subject>Recombinant Proteins - chemistry</subject><subject>Recombinant Proteins - genetics</subject><subject>Recombinant Proteins - metabolism</subject><subject>replication factor C</subject><subject>Replication Protein C</subject><subject>Sequence Homology, Amino Acid</subject><issn>0022-2836</issn><issn>1089-8638</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2002</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqNkc1u1DAUhS0EokPhEUBeIVgE_JNknBUapUyLVA2jUtaWf24YoyQOdlIpT8br4cyM6BJW17r6zjlXPgi9puQDJbT8-I0QxjImePmOsPeEEiYy8QStKBFVJkounqLVX-QCvYjxJyGk4Ll4ji4oy-maVvkK_b6DoXVGjc73eKvM6AOucRN8h8cD4Jt5gJAeofPDwSUQb4I5KEjwfg7eeGOmiJWeY3T4ykPEOz_iHYDFm_t9ktvg2zm6iJvFuFXdkLVeWdf_wKq3uPb9qFyfLPB26s1yhWrbedlHCA_J5m5b432922Ta9UfZle-S4iV61qg2wqvzvETft5_v65vs9uv1l3pzm5mcV2OmCkKNboTR61JrWulcW0NKJoomZwXkrNLKGku1UpwyYgnnSlSM6qasrOacX6K3J98h-F8TxFF2LhpoW9WDn6Jcs7Kg-X-AVJR5Cl7A4gSa4GMM0MghuE6FWVIil2rlsVq59CYJk8dqpUi6N-eASXdgH1XnLhPw6QRA-o8HB0FG46A3YF0AM0rr3T8i_gAvB7XD</recordid><startdate>20021108</startdate><enddate>20021108</enddate><creator>Henneke, Ghislaine</creator><creator>Gueguen, Yannick</creator><creator>Flament, Didier</creator><creator>Azam, Philippe</creator><creator>Querellou, Joël</creator><creator>Dietrich, Jacques</creator><creator>Hübscher, Ulrich</creator><creator>Raffin, Jean-Paul</creator><general>Elsevier Ltd</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QL</scope><scope>7TM</scope><scope>C1K</scope><scope>7X8</scope></search><sort><creationdate>20021108</creationdate><title>Replication Factor C from the Hyperthermophilic Archaeon Pyrococcus abyssi Does Not Need ATP Hydrolysis for Clamp-loading and Contains a Functionally Conserved RFC PCNA-binding Domain</title><author>Henneke, Ghislaine ; Gueguen, Yannick ; Flament, Didier ; Azam, Philippe ; Querellou, Joël ; Dietrich, Jacques ; Hübscher, Ulrich ; Raffin, Jean-Paul</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c439t-a501cbf8cb76bb19b4bdc06285f425e429badcd1baa3120d033a8921bf69db333</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2002</creationdate><topic>Adenosine Triphosphatases - metabolism</topic><topic>Adenosine Triphosphate - metabolism</topic><topic>Amino Acid Sequence</topic><topic>archaea</topic><topic>Base Sequence</topic><topic>Binding Sites</topic><topic>Conserved Sequence</topic><topic>Cross Reactions</topic><topic>DNA Polymerase II - metabolism</topic><topic>DNA Replication</topic><topic>DNA, Archaeal - genetics</topic><topic>DNA-Binding Proteins - chemistry</topic><topic>DNA-Binding Proteins - genetics</topic><topic>DNA-Binding Proteins - metabolism</topic><topic>Escherichia coli - genetics</topic><topic>Gene Expression</topic><topic>Genes, Archaeal</topic><topic>Humans</topic><topic>Hydrolysis</topic><topic>hyperthermophile</topic><topic>Macromolecular Substances</topic><topic>Molecular Sequence Data</topic><topic>PCNA-binding domain</topic><topic>Proliferating Cell Nuclear Antigen - metabolism</topic><topic>Protein Structure, Tertiary</topic><topic>Protein Subunits</topic><topic>Pyrococcus - genetics</topic><topic>Pyrococcus - metabolism</topic><topic>Pyrococcus abyssi</topic><topic>Recombinant Proteins - chemistry</topic><topic>Recombinant Proteins - genetics</topic><topic>Recombinant Proteins - metabolism</topic><topic>replication factor C</topic><topic>Replication Protein C</topic><topic>Sequence Homology, Amino Acid</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Henneke, Ghislaine</creatorcontrib><creatorcontrib>Gueguen, Yannick</creatorcontrib><creatorcontrib>Flament, Didier</creatorcontrib><creatorcontrib>Azam, Philippe</creatorcontrib><creatorcontrib>Querellou, Joël</creatorcontrib><creatorcontrib>Dietrich, Jacques</creatorcontrib><creatorcontrib>Hübscher, Ulrich</creatorcontrib><creatorcontrib>Raffin, Jean-Paul</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Nucleic Acids Abstracts</collection><collection>Environmental Sciences and Pollution Management</collection><collection>MEDLINE - Academic</collection><jtitle>Journal of molecular biology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Henneke, Ghislaine</au><au>Gueguen, Yannick</au><au>Flament, Didier</au><au>Azam, Philippe</au><au>Querellou, Joël</au><au>Dietrich, Jacques</au><au>Hübscher, Ulrich</au><au>Raffin, Jean-Paul</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Replication Factor C from the Hyperthermophilic Archaeon Pyrococcus abyssi Does Not Need ATP Hydrolysis for Clamp-loading and Contains a Functionally Conserved RFC PCNA-binding Domain</atitle><jtitle>Journal of molecular biology</jtitle><addtitle>J Mol Biol</addtitle><date>2002-11-08</date><risdate>2002</risdate><volume>323</volume><issue>5</issue><spage>795</spage><epage>810</epage><pages>795-810</pages><issn>0022-2836</issn><eissn>1089-8638</eissn><abstract>The molecular organization of the replication complex in archaea is similar to that in eukaryotes. Only two proteins homologous to subunits of eukaryotic replication factor C (RFC) have been detected in
Pyrococcus abyssi (
Pab). The genes encoding these two proteins are arranged in tandem. We cloned these two genes and co-expressed the corresponding recombinant proteins in
Escherichia coli. Two inteins present in the gene encoding the small subunit (
PabRFC-small) were removed during cloning. The recombinant protein complex was purified by anion-exchange and hydroxyapatite chromatography. Also, the
PabRFC-small subunit could be purified, while the large subunit (
PabRFC-large) alone was completely insoluble. The highly purified
PabRFC complex possessed an ATPase activity, which was not enhanced by DNA. The
Pab proliferating cell nuclear antigen (PCNA) activated the
PabRFC complex in a DNA-dependent manner, but the
PabRFC-small ATPase activity was neither DNA-dependent nor PCNA-dependent. The
PabRFC complex was able to stimulate
PabPCNA-dependent DNA synthesis by the
Pabfamily D heterodimeric DNA polymerase. Finally, (i) the
PabRFC-large fraction cross-reacted with anti-human-RFC PCNA-binding domain antibody, corroborating the conservation of the protein sequence, (ii) the human PCNA stimulated the
PabRFC complex ATPase activity in a DNA-dependent way and (iii) the
PabRFC complex could load human PCNA onto primed single-stranded circular DNA, suggesting that the PCNA-binding domain of RFC has been functionally conserved during evolution. In addition, ATP hydrolysis was not required either for DNA polymerase stimulation or PCNA-loading
in vitro.</abstract><cop>England</cop><pub>Elsevier Ltd</pub><pmid>12417194</pmid><doi>10.1016/S0022-2836(02)01028-8</doi><tpages>16</tpages><oa>free_for_read</oa></addata></record> |
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| ispartof | Journal of molecular biology, 2002-11, Vol.323 (5), p.795-810 |
| issn | 0022-2836 1089-8638 |
| language | eng |
| recordid | cdi_proquest_miscellaneous_72651433 |
| source | MEDLINE; Elsevier ScienceDirect Journals |
| subjects | Adenosine Triphosphatases - metabolism Adenosine Triphosphate - metabolism Amino Acid Sequence archaea Base Sequence Binding Sites Conserved Sequence Cross Reactions DNA Polymerase II - metabolism DNA Replication DNA, Archaeal - genetics DNA-Binding Proteins - chemistry DNA-Binding Proteins - genetics DNA-Binding Proteins - metabolism Escherichia coli - genetics Gene Expression Genes, Archaeal Humans Hydrolysis hyperthermophile Macromolecular Substances Molecular Sequence Data PCNA-binding domain Proliferating Cell Nuclear Antigen - metabolism Protein Structure, Tertiary Protein Subunits Pyrococcus - genetics Pyrococcus - metabolism Pyrococcus abyssi Recombinant Proteins - chemistry Recombinant Proteins - genetics Recombinant Proteins - metabolism replication factor C Replication Protein C Sequence Homology, Amino Acid |
| title | Replication Factor C from the Hyperthermophilic Archaeon Pyrococcus abyssi Does Not Need ATP Hydrolysis for Clamp-loading and Contains a Functionally Conserved RFC PCNA-binding Domain |
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