Crystal Structure of π Initiator Protein-Iteron Complex of Plasmid R6K: Implications for Initiation of Plasmid DNA Replication
We have determined the crystal structure of a monomeric biologically active form of the π initiator protein of plasmid R6K as a complex with a single copy of its cognate DNA-binding site (iteron) at 3.1-Å resolution. The initiator belongs to the family of winged helix type of proteins. The structure...
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| Veröffentlicht in: | Proceedings of the National Academy of Sciences - PNAS 2006-12, Vol.103 (49), p.18481-18486 |
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| container_title | Proceedings of the National Academy of Sciences - PNAS |
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| creator | Swan, Michael K. Bastia, Deepak Davies, Christopher |
| description | We have determined the crystal structure of a monomeric biologically active form of the π initiator protein of plasmid R6K as a complex with a single copy of its cognate DNA-binding site (iteron) at 3.1-Å resolution. The initiator belongs to the family of winged helix type of proteins. The structure reveals that the protein contacts the iteron DNA at two primary recognition helices, namely the C-terminal α4' and the N-terminal α4 helices, that recognize the 5' half and the 3' half of the 22-bp iteron, respectively. The base-amino acid contacts are all located in α4', whereas the α4 helix and its vicinity mainly contact the phosphate groups of the iteron. Mutational analyses show that the contacts of both recognition helices with DNA are necessary for iteron binding and replication initiation. Considerations of a large number of site-directed mutations reveal that two distinct regions, namely α2 and α5 and its vicinity, are required for DNA looping and initiator dimerization, respectively. Further analysis of mutant forms of π revealed the possible domain that interacts with the DnaB helicase. Thus, the structure-function analysis presented illuminates aspects of initiation mechanism of R6K and its control. |
| doi_str_mv | 10.1073/pnas.0609046103 |
| format | Article |
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The initiator belongs to the family of winged helix type of proteins. The structure reveals that the protein contacts the iteron DNA at two primary recognition helices, namely the C-terminal α4' and the N-terminal α4 helices, that recognize the 5' half and the 3' half of the 22-bp iteron, respectively. The base-amino acid contacts are all located in α4', whereas the α4 helix and its vicinity mainly contact the phosphate groups of the iteron. Mutational analyses show that the contacts of both recognition helices with DNA are necessary for iteron binding and replication initiation. Considerations of a large number of site-directed mutations reveal that two distinct regions, namely α2 and α5 and its vicinity, are required for DNA looping and initiator dimerization, respectively. Further analysis of mutant forms of π revealed the possible domain that interacts with the DnaB helicase. Thus, the structure-function analysis presented illuminates aspects of initiation mechanism of R6K and its control.</description><identifier>ISSN: 0027-8424</identifier><identifier>EISSN: 1091-6490</identifier><identifier>DOI: 10.1073/pnas.0609046103</identifier><identifier>PMID: 17124167</identifier><language>eng</language><publisher>United States: National Academy of Sciences</publisher><subject>Amino Acid Sequence ; Biochemistry ; Biological Sciences ; CONTROL ; CRYSTAL STRUCTURE ; Crystallography, X-Ray ; DIMERIZATION ; Dimers ; DNA ; DNA - chemical synthesis ; DNA - metabolism ; DNA Helicases - biosynthesis ; DNA Helicases - chemistry ; DNA REPLICATION ; DNA Replication - genetics ; DNA-Binding Proteins - biosynthesis ; DNA-Binding Proteins - chemistry ; Genetic mutation ; MATERIALS SCIENCE ; Molecular Sequence Data ; Monomers ; MUTANTS ; MUTATIONS ; national synchrotron light source ; PHOSPHATES ; PLASMIDS ; Plasmids - biosynthesis ; Plasmids - chemical synthesis ; PROTEINS ; RESOLUTION ; Structure-Activity Relationship ; Trans-Activators - biosynthesis ; Trans-Activators - chemistry</subject><ispartof>Proceedings of the National Academy of Sciences - PNAS, 2006-12, Vol.103 (49), p.18481-18486</ispartof><rights>Copyright 2006 National Academy of Sciences of the United States of America</rights><rights>2006 by The National Academy of Sciences of the USA 2006</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c529t-fa154a6f46cdc1aec524e67552649be88116310a3e12f4e9ed70709f5f5d295c3</citedby><cites>FETCH-LOGICAL-c529t-fa154a6f46cdc1aec524e67552649be88116310a3e12f4e9ed70709f5f5d295c3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Uhttp://www.pnas.org/content/103/49.cover.gif</thumbnail><linktopdf>$$Uhttps://www.jstor.org/stable/pdf/30051139$$EPDF$$P50$$Gjstor$$H</linktopdf><linktohtml>$$Uhttps://www.jstor.org/stable/30051139$$EHTML$$P50$$Gjstor$$H</linktohtml><link.rule.ids>230,314,727,780,784,803,885,27922,27923,53789,53791,58015,58248</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/17124167$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink><backlink>$$Uhttps://www.osti.gov/biblio/930236$$D View this record in Osti.gov$$Hfree_for_read</backlink></links><search><creatorcontrib>Swan, Michael K.</creatorcontrib><creatorcontrib>Bastia, Deepak</creatorcontrib><creatorcontrib>Davies, Christopher</creatorcontrib><creatorcontrib>Brookhaven National Laboratory (BNL) National Synchrotron Light Source</creatorcontrib><title>Crystal Structure of π Initiator Protein-Iteron Complex of Plasmid R6K: Implications for Initiation of Plasmid DNA Replication</title><title>Proceedings of the National Academy of Sciences - PNAS</title><addtitle>Proc Natl Acad Sci U S A</addtitle><description>We have determined the crystal structure of a monomeric biologically active form of the π initiator protein of plasmid R6K as a complex with a single copy of its cognate DNA-binding site (iteron) at 3.1-Å resolution. The initiator belongs to the family of winged helix type of proteins. The structure reveals that the protein contacts the iteron DNA at two primary recognition helices, namely the C-terminal α4' and the N-terminal α4 helices, that recognize the 5' half and the 3' half of the 22-bp iteron, respectively. The base-amino acid contacts are all located in α4', whereas the α4 helix and its vicinity mainly contact the phosphate groups of the iteron. Mutational analyses show that the contacts of both recognition helices with DNA are necessary for iteron binding and replication initiation. Considerations of a large number of site-directed mutations reveal that two distinct regions, namely α2 and α5 and its vicinity, are required for DNA looping and initiator dimerization, respectively. Further analysis of mutant forms of π revealed the possible domain that interacts with the DnaB helicase. Thus, the structure-function analysis presented illuminates aspects of initiation mechanism of R6K and its control.</description><subject>Amino Acid Sequence</subject><subject>Biochemistry</subject><subject>Biological Sciences</subject><subject>CONTROL</subject><subject>CRYSTAL STRUCTURE</subject><subject>Crystallography, X-Ray</subject><subject>DIMERIZATION</subject><subject>Dimers</subject><subject>DNA</subject><subject>DNA - chemical synthesis</subject><subject>DNA - metabolism</subject><subject>DNA Helicases - biosynthesis</subject><subject>DNA Helicases - chemistry</subject><subject>DNA REPLICATION</subject><subject>DNA Replication - genetics</subject><subject>DNA-Binding Proteins - biosynthesis</subject><subject>DNA-Binding Proteins - chemistry</subject><subject>Genetic mutation</subject><subject>MATERIALS SCIENCE</subject><subject>Molecular Sequence Data</subject><subject>Monomers</subject><subject>MUTANTS</subject><subject>MUTATIONS</subject><subject>national synchrotron light source</subject><subject>PHOSPHATES</subject><subject>PLASMIDS</subject><subject>Plasmids - biosynthesis</subject><subject>Plasmids - chemical synthesis</subject><subject>PROTEINS</subject><subject>RESOLUTION</subject><subject>Structure-Activity Relationship</subject><subject>Trans-Activators - biosynthesis</subject><subject>Trans-Activators - chemistry</subject><issn>0027-8424</issn><issn>1091-6490</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2006</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFks1u1DAUhSMEokNhzQpkNkgs0t4bO07MAqka_kZUUBVYW67nhrrKxIPtoHYFb8gr4dFEM2XVleXr7xz7yKconiIcITT8eD2YeAQSFAiJwO8VMwSFpRQK7hczgKopW1GJg-JRjFcAoOoWHhYH2GAlUDaz4vc83MRkevY1hdGmMRDzHfv7hy0Gl5xJPrCz4BO5oVwkCn5gc79a93S9wc56E1duyc7lp9dskcfOmuT8EFmXdZND3t9m334-Yee0Qx8XDzrTR3oyrYfF9_fvvs0_lqdfPizmJ6elrSuVys5gLYzshLRLi4byVJBs6rrKUS-obRElRzCcsOoEKVo20IDq6q5eVqq2_LB4s_VdjxcrWloaUjC9Xge3MuFGe-P0_yeDu9Q__C-NUnHZttngxdbAx-R0tC6RvbR-GMgmrThUXGbm5XRJ8D9HikmvXLTU92YgP0Yt26pqFao7QVQ5mKo3jsdb0AYfY6Bu92IEvWmA3jRA7xuQFc9vB93z05dngE3ARrm341ooja1oMSOv7kB0N_Z9ouuU2Wdb9irmsuxgDlAjcsX_ASPY0Kk</recordid><startdate>20061205</startdate><enddate>20061205</enddate><creator>Swan, Michael K.</creator><creator>Bastia, Deepak</creator><creator>Davies, Christopher</creator><general>National Academy of Sciences</general><general>National Acad Sciences</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>7TM</scope><scope>7X8</scope><scope>OTOTI</scope><scope>5PM</scope></search><sort><creationdate>20061205</creationdate><title>Crystal Structure of π Initiator Protein-Iteron Complex of Plasmid R6K: Implications for Initiation of Plasmid DNA Replication</title><author>Swan, Michael K. ; Bastia, Deepak ; Davies, Christopher</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c529t-fa154a6f46cdc1aec524e67552649be88116310a3e12f4e9ed70709f5f5d295c3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2006</creationdate><topic>Amino Acid Sequence</topic><topic>Biochemistry</topic><topic>Biological Sciences</topic><topic>CONTROL</topic><topic>CRYSTAL STRUCTURE</topic><topic>Crystallography, X-Ray</topic><topic>DIMERIZATION</topic><topic>Dimers</topic><topic>DNA</topic><topic>DNA - chemical synthesis</topic><topic>DNA - metabolism</topic><topic>DNA Helicases - biosynthesis</topic><topic>DNA Helicases - chemistry</topic><topic>DNA REPLICATION</topic><topic>DNA Replication - genetics</topic><topic>DNA-Binding Proteins - biosynthesis</topic><topic>DNA-Binding Proteins - chemistry</topic><topic>Genetic mutation</topic><topic>MATERIALS SCIENCE</topic><topic>Molecular Sequence Data</topic><topic>Monomers</topic><topic>MUTANTS</topic><topic>MUTATIONS</topic><topic>national synchrotron light source</topic><topic>PHOSPHATES</topic><topic>PLASMIDS</topic><topic>Plasmids - biosynthesis</topic><topic>Plasmids - chemical synthesis</topic><topic>PROTEINS</topic><topic>RESOLUTION</topic><topic>Structure-Activity Relationship</topic><topic>Trans-Activators - biosynthesis</topic><topic>Trans-Activators - chemistry</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Swan, Michael K.</creatorcontrib><creatorcontrib>Bastia, Deepak</creatorcontrib><creatorcontrib>Davies, Christopher</creatorcontrib><creatorcontrib>Brookhaven National Laboratory (BNL) National Synchrotron Light Source</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Nucleic Acids Abstracts</collection><collection>MEDLINE - Academic</collection><collection>OSTI.GOV</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Proceedings of the National Academy of Sciences - PNAS</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Swan, Michael K.</au><au>Bastia, Deepak</au><au>Davies, Christopher</au><aucorp>Brookhaven National Laboratory (BNL) National Synchrotron Light Source</aucorp><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Crystal Structure of π Initiator Protein-Iteron Complex of Plasmid R6K: Implications for Initiation of Plasmid DNA Replication</atitle><jtitle>Proceedings of the National Academy of Sciences - PNAS</jtitle><addtitle>Proc Natl Acad Sci U S A</addtitle><date>2006-12-05</date><risdate>2006</risdate><volume>103</volume><issue>49</issue><spage>18481</spage><epage>18486</epage><pages>18481-18486</pages><issn>0027-8424</issn><eissn>1091-6490</eissn><abstract>We have determined the crystal structure of a monomeric biologically active form of the π initiator protein of plasmid R6K as a complex with a single copy of its cognate DNA-binding site (iteron) at 3.1-Å resolution. The initiator belongs to the family of winged helix type of proteins. The structure reveals that the protein contacts the iteron DNA at two primary recognition helices, namely the C-terminal α4' and the N-terminal α4 helices, that recognize the 5' half and the 3' half of the 22-bp iteron, respectively. The base-amino acid contacts are all located in α4', whereas the α4 helix and its vicinity mainly contact the phosphate groups of the iteron. Mutational analyses show that the contacts of both recognition helices with DNA are necessary for iteron binding and replication initiation. Considerations of a large number of site-directed mutations reveal that two distinct regions, namely α2 and α5 and its vicinity, are required for DNA looping and initiator dimerization, respectively. Further analysis of mutant forms of π revealed the possible domain that interacts with the DnaB helicase. Thus, the structure-function analysis presented illuminates aspects of initiation mechanism of R6K and its control.</abstract><cop>United States</cop><pub>National Academy of Sciences</pub><pmid>17124167</pmid><doi>10.1073/pnas.0609046103</doi><tpages>6</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Amino Acid Sequence Biochemistry Biological Sciences CONTROL CRYSTAL STRUCTURE Crystallography, X-Ray DIMERIZATION Dimers DNA DNA - chemical synthesis DNA - metabolism DNA Helicases - biosynthesis DNA Helicases - chemistry DNA REPLICATION DNA Replication - genetics DNA-Binding Proteins - biosynthesis DNA-Binding Proteins - chemistry Genetic mutation MATERIALS SCIENCE Molecular Sequence Data Monomers MUTANTS MUTATIONS national synchrotron light source PHOSPHATES PLASMIDS Plasmids - biosynthesis Plasmids - chemical synthesis PROTEINS RESOLUTION Structure-Activity Relationship Trans-Activators - biosynthesis Trans-Activators - chemistry |
| title | Crystal Structure of π Initiator Protein-Iteron Complex of Plasmid R6K: Implications for Initiation of Plasmid DNA Replication |
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