Interaction of the replication terminator protein of Bacillus subtilis with DNA probed by NMR spectroscopy
Termination of DNA replication in Bacillus subtilis involves the polar arrest of replication forks by a specific complex formed between the dimeric 29 kDa replication terminator protein (RTP) and DNA terminator sites. We have used NMR spectroscopy to probe the changes in 1H– 15N correlation spectra...
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| Veröffentlicht in: | Biochemical and biophysical research communications 2005-09, Vol.335 (2), p.361-366 |
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| creator | Hastings, Adam F. Otting, Gottfried Folmer, Rutger H.A. Duggin, Iain G. Wake, R. Gerry Wilce, Matthew C.J. Wilce, Jacqueline A. |
| description | Termination of DNA replication in
Bacillus subtilis involves the polar arrest of replication forks by a specific complex formed between the dimeric 29
kDa replication terminator protein (RTP) and DNA terminator sites. We have used NMR spectroscopy to probe the changes in
1H–
15N correlation spectra of a
15N-labelled RTP.C110S mutant upon the addition of a 21 base pair symmetrical DNA binding site. Assignment of the
1H–
15N correlations was achieved using a suite of triple resonance NMR experiments with
15N,
13C,70%
2H enriched protein recorded at 800
MHz and using TROSY pulse sequences. Perturbations to
1H–
15N spectra revealed that the N-termini, α3-helices and several loops are affected by the binding interaction. An analysis of this data in light of the crystallographically determined apo- and DNA-bound forms of RTP.C110S revealed that the NMR spectral perturbations correlate more closely to protein structural changes upon complex formation rather than to interactions at the protein–DNA interface. |
| doi_str_mv | 10.1016/j.bbrc.2005.07.082 |
| format | Article |
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Bacillus subtilis involves the polar arrest of replication forks by a specific complex formed between the dimeric 29
kDa replication terminator protein (RTP) and DNA terminator sites. We have used NMR spectroscopy to probe the changes in
1H–
15N correlation spectra of a
15N-labelled RTP.C110S mutant upon the addition of a 21 base pair symmetrical DNA binding site. Assignment of the
1H–
15N correlations was achieved using a suite of triple resonance NMR experiments with
15N,
13C,70%
2H enriched protein recorded at 800
MHz and using TROSY pulse sequences. Perturbations to
1H–
15N spectra revealed that the N-termini, α3-helices and several loops are affected by the binding interaction. An analysis of this data in light of the crystallographically determined apo- and DNA-bound forms of RTP.C110S revealed that the NMR spectral perturbations correlate more closely to protein structural changes upon complex formation rather than to interactions at the protein–DNA interface.</description><identifier>ISSN: 0006-291X</identifier><identifier>EISSN: 1090-2104</identifier><identifier>DOI: 10.1016/j.bbrc.2005.07.082</identifier><identifier>PMID: 16061201</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>60 APPLIED LIFE SCIENCES ; BACILLUS SUBTILIS ; Bacillus subtilis - metabolism ; Bacterial Proteins - chemistry ; Binding Sites ; CARBON 13 ; Crystallography, X-Ray ; DEUTERIUM ; DISTURBANCES ; DNA ; DNA - chemistry ; DNA REPLICATION ; DNA-Binding Proteins - chemistry ; Duplex DNA ; HSQC titration ; Kinetics ; Magnetic Resonance Spectroscopy - methods ; Models, Molecular ; NITROGEN 15 ; NMR spectroscopy ; NUCLEAR MAGNETIC RESONANCE ; Oligonucleotides - chemistry ; Protein Binding ; Protein Conformation ; Protein Structure, Secondary ; Protein Structure, Tertiary ; PROTEINS ; Protein–DNA complex ; RTP ; SPECTROSCOPY ; Structural perturbation ; Terminator DNA ; TITRATION ; TROSY ; Winged-helix protein</subject><ispartof>Biochemical and biophysical research communications, 2005-09, Vol.335 (2), p.361-366</ispartof><rights>2005 Elsevier Inc.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c413t-dd5eca64d293d0c0898d00940a229dc93a24fad6adbd9a289fe52dccd7c481d43</citedby><cites>FETCH-LOGICAL-c413t-dd5eca64d293d0c0898d00940a229dc93a24fad6adbd9a289fe52dccd7c481d43</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/j.bbrc.2005.07.082$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>230,314,780,784,885,3550,27924,27925,45995</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/16061201$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink><backlink>$$Uhttps://www.osti.gov/biblio/20710977$$D View this record in Osti.gov$$Hfree_for_read</backlink></links><search><creatorcontrib>Hastings, Adam F.</creatorcontrib><creatorcontrib>Otting, Gottfried</creatorcontrib><creatorcontrib>Folmer, Rutger H.A.</creatorcontrib><creatorcontrib>Duggin, Iain G.</creatorcontrib><creatorcontrib>Wake, R. Gerry</creatorcontrib><creatorcontrib>Wilce, Matthew C.J.</creatorcontrib><creatorcontrib>Wilce, Jacqueline A.</creatorcontrib><title>Interaction of the replication terminator protein of Bacillus subtilis with DNA probed by NMR spectroscopy</title><title>Biochemical and biophysical research communications</title><addtitle>Biochem Biophys Res Commun</addtitle><description>Termination of DNA replication in
Bacillus subtilis involves the polar arrest of replication forks by a specific complex formed between the dimeric 29
kDa replication terminator protein (RTP) and DNA terminator sites. We have used NMR spectroscopy to probe the changes in
1H–
15N correlation spectra of a
15N-labelled RTP.C110S mutant upon the addition of a 21 base pair symmetrical DNA binding site. Assignment of the
1H–
15N correlations was achieved using a suite of triple resonance NMR experiments with
15N,
13C,70%
2H enriched protein recorded at 800
MHz and using TROSY pulse sequences. Perturbations to
1H–
15N spectra revealed that the N-termini, α3-helices and several loops are affected by the binding interaction. An analysis of this data in light of the crystallographically determined apo- and DNA-bound forms of RTP.C110S revealed that the NMR spectral perturbations correlate more closely to protein structural changes upon complex formation rather than to interactions at the protein–DNA interface.</description><subject>60 APPLIED LIFE SCIENCES</subject><subject>BACILLUS SUBTILIS</subject><subject>Bacillus subtilis - metabolism</subject><subject>Bacterial Proteins - chemistry</subject><subject>Binding Sites</subject><subject>CARBON 13</subject><subject>Crystallography, X-Ray</subject><subject>DEUTERIUM</subject><subject>DISTURBANCES</subject><subject>DNA</subject><subject>DNA - chemistry</subject><subject>DNA REPLICATION</subject><subject>DNA-Binding Proteins - chemistry</subject><subject>Duplex DNA</subject><subject>HSQC titration</subject><subject>Kinetics</subject><subject>Magnetic Resonance Spectroscopy - methods</subject><subject>Models, Molecular</subject><subject>NITROGEN 15</subject><subject>NMR spectroscopy</subject><subject>NUCLEAR MAGNETIC RESONANCE</subject><subject>Oligonucleotides - chemistry</subject><subject>Protein Binding</subject><subject>Protein Conformation</subject><subject>Protein Structure, Secondary</subject><subject>Protein Structure, Tertiary</subject><subject>PROTEINS</subject><subject>Protein–DNA complex</subject><subject>RTP</subject><subject>SPECTROSCOPY</subject><subject>Structural perturbation</subject><subject>Terminator DNA</subject><subject>TITRATION</subject><subject>TROSY</subject><subject>Winged-helix protein</subject><issn>0006-291X</issn><issn>1090-2104</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2005</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkVGL1DAUhYMo7uzqH_BBAoJvrTdpp23Al3VddWFdQRR8C-nNLZOh04xJqsy_N50Z8E2fLuR-OZxzD2MvBJQCRPNmW_Z9wFICrEtoS-jkI7YSoKCQAurHbAUATSGV-HHBLmPcAghRN-opuxANNEKCWLHt3ZQoGEzOT9wPPG2IB9qPDs3xKS93bjLJB74PPpE7Uu8MunGcI49zn9zoIv_t0oa_f7heqJ4s7w_84fNXHveEKfiIfn94xp4MZoz0_Dyv2PcPt99uPhX3Xz7e3VzfF1iLKhXWrglNU1upKgsIneosgKrBSKksqsrIejC2Mba3yshODbSWFtG2WHfC1tUVe3XS9TE5HdElwg36acpWtIQ2X6htM_X6RGXDP2eKSe9cRBpHM5Gfo266ul03nfwvKNqqy86qDMoTiDlwDDTofXA7Ew5agF4K01u9FKaXwjS0Go7qL8_qc78j-_fLuaEMvD0BlE_2y1FYEtGEZF1YAlnv_qX_B-a2qAA</recordid><startdate>20050923</startdate><enddate>20050923</enddate><creator>Hastings, Adam F.</creator><creator>Otting, Gottfried</creator><creator>Folmer, Rutger H.A.</creator><creator>Duggin, Iain G.</creator><creator>Wake, R. Gerry</creator><creator>Wilce, Matthew C.J.</creator><creator>Wilce, Jacqueline A.</creator><general>Elsevier Inc</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><scope>OTOTI</scope></search><sort><creationdate>20050923</creationdate><title>Interaction of the replication terminator protein of Bacillus subtilis with DNA probed by NMR spectroscopy</title><author>Hastings, Adam F. ; Otting, Gottfried ; Folmer, Rutger H.A. ; Duggin, Iain G. ; Wake, R. Gerry ; Wilce, Matthew C.J. ; Wilce, Jacqueline A.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c413t-dd5eca64d293d0c0898d00940a229dc93a24fad6adbd9a289fe52dccd7c481d43</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2005</creationdate><topic>60 APPLIED LIFE SCIENCES</topic><topic>BACILLUS SUBTILIS</topic><topic>Bacillus subtilis - metabolism</topic><topic>Bacterial Proteins - chemistry</topic><topic>Binding Sites</topic><topic>CARBON 13</topic><topic>Crystallography, X-Ray</topic><topic>DEUTERIUM</topic><topic>DISTURBANCES</topic><topic>DNA</topic><topic>DNA - chemistry</topic><topic>DNA REPLICATION</topic><topic>DNA-Binding Proteins - chemistry</topic><topic>Duplex DNA</topic><topic>HSQC titration</topic><topic>Kinetics</topic><topic>Magnetic Resonance Spectroscopy - methods</topic><topic>Models, Molecular</topic><topic>NITROGEN 15</topic><topic>NMR spectroscopy</topic><topic>NUCLEAR MAGNETIC RESONANCE</topic><topic>Oligonucleotides - chemistry</topic><topic>Protein Binding</topic><topic>Protein Conformation</topic><topic>Protein Structure, Secondary</topic><topic>Protein Structure, Tertiary</topic><topic>PROTEINS</topic><topic>Protein–DNA complex</topic><topic>RTP</topic><topic>SPECTROSCOPY</topic><topic>Structural perturbation</topic><topic>Terminator DNA</topic><topic>TITRATION</topic><topic>TROSY</topic><topic>Winged-helix protein</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Hastings, Adam F.</creatorcontrib><creatorcontrib>Otting, Gottfried</creatorcontrib><creatorcontrib>Folmer, Rutger H.A.</creatorcontrib><creatorcontrib>Duggin, Iain G.</creatorcontrib><creatorcontrib>Wake, R. Gerry</creatorcontrib><creatorcontrib>Wilce, Matthew C.J.</creatorcontrib><creatorcontrib>Wilce, Jacqueline A.</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><collection>OSTI.GOV</collection><jtitle>Biochemical and biophysical research communications</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Hastings, Adam F.</au><au>Otting, Gottfried</au><au>Folmer, Rutger H.A.</au><au>Duggin, Iain G.</au><au>Wake, R. Gerry</au><au>Wilce, Matthew C.J.</au><au>Wilce, Jacqueline A.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Interaction of the replication terminator protein of Bacillus subtilis with DNA probed by NMR spectroscopy</atitle><jtitle>Biochemical and biophysical research communications</jtitle><addtitle>Biochem Biophys Res Commun</addtitle><date>2005-09-23</date><risdate>2005</risdate><volume>335</volume><issue>2</issue><spage>361</spage><epage>366</epage><pages>361-366</pages><issn>0006-291X</issn><eissn>1090-2104</eissn><abstract>Termination of DNA replication in
Bacillus subtilis involves the polar arrest of replication forks by a specific complex formed between the dimeric 29
kDa replication terminator protein (RTP) and DNA terminator sites. We have used NMR spectroscopy to probe the changes in
1H–
15N correlation spectra of a
15N-labelled RTP.C110S mutant upon the addition of a 21 base pair symmetrical DNA binding site. Assignment of the
1H–
15N correlations was achieved using a suite of triple resonance NMR experiments with
15N,
13C,70%
2H enriched protein recorded at 800
MHz and using TROSY pulse sequences. Perturbations to
1H–
15N spectra revealed that the N-termini, α3-helices and several loops are affected by the binding interaction. An analysis of this data in light of the crystallographically determined apo- and DNA-bound forms of RTP.C110S revealed that the NMR spectral perturbations correlate more closely to protein structural changes upon complex formation rather than to interactions at the protein–DNA interface.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>16061201</pmid><doi>10.1016/j.bbrc.2005.07.082</doi><tpages>6</tpages></addata></record> |
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| ispartof | Biochemical and biophysical research communications, 2005-09, Vol.335 (2), p.361-366 |
| issn | 0006-291X 1090-2104 |
| language | eng |
| recordid | cdi_osti_scitechconnect_20710977 |
| source | MEDLINE; Access via ScienceDirect (Elsevier) |
| subjects | 60 APPLIED LIFE SCIENCES BACILLUS SUBTILIS Bacillus subtilis - metabolism Bacterial Proteins - chemistry Binding Sites CARBON 13 Crystallography, X-Ray DEUTERIUM DISTURBANCES DNA DNA - chemistry DNA REPLICATION DNA-Binding Proteins - chemistry Duplex DNA HSQC titration Kinetics Magnetic Resonance Spectroscopy - methods Models, Molecular NITROGEN 15 NMR spectroscopy NUCLEAR MAGNETIC RESONANCE Oligonucleotides - chemistry Protein Binding Protein Conformation Protein Structure, Secondary Protein Structure, Tertiary PROTEINS Protein–DNA complex RTP SPECTROSCOPY Structural perturbation Terminator DNA TITRATION TROSY Winged-helix protein |
| title | Interaction of the replication terminator protein of Bacillus subtilis with DNA probed by NMR spectroscopy |
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