DnaA boxes in the P1 plasmid origin: the effect of their position on the directionality of replication and plasmid copy number
Identifieur interne : 000A06 ( Istex/Corpus ); précédent : 000A05; suivant : 000A07DnaA boxes in the P1 plasmid origin: the effect of their position on the directionality of replication and plasmid copy number
Auteurs : Kyusung Park ; Dhruba K. ChattorajSource :
- Journal of Molecular Biology [ 0022-2836 ] ; 2001.
English descriptors
- KwdEn :
- Teeft :
- Acad, Anking sequence, Appropriate antibiotics, Bacteriol, Bamhi, Band intensities, Beckman, Bidirectional, Bidirectional replication, Binding protein, Biol, Chattoraj, Chem, Chimeric plasmids, Coli, Consensus dnaa, Consensus dnaa boxes, Copy number, Copy numbers, Dnaa, Dnaa boxes, Dnaa functions, Dnaa protein, Ecori, Escherichia, Escherichia coli, Form molecules, Helicase, Helicase loading, Hindiii, Hindiii site, Initiation site, Iterons, Kmno4, Linearized, Migration patterns, Minip1, Minip1 plasmid, Minip1 plasmids, Natl, Natl acad, Open region, Opening signal, Oric, Origin opening, P1ori, Plasmid, Plasmid copy number, Plasmid origin, Plasmid replication, Plasmid replication figure, Proc, Psti fragment, Pvui, Pvui site, Repa, Replication, Replication forks, Replication initiation, Replication intermediates, Replication origin, Resultant plasmid, Styi, Transcription, Unpublished results.
Abstract
Abstract: The DnaA protein is essential for initiation of DNA replication in a wide variety of bacterial and plasmid replicons. The replication origin in these replicons invariably contains specific binding sites for the protein, called DnaA boxes. Plasmid P1 contains a set of DnaA boxes at each end of its origin but can function with either one of the sets. Here we report that the location of origin-opening, initiation site of replication forks and directionality of replication do not change whether the boxes are present at both or at one of the ends of the origin. Replication was bidirectional in all cases. These results imply that DnaA functions similarly from the two ends of the origin. However, origins with DnaA boxes proximal to the origin-opening location opened more efficiently and maintained plasmids at higher copy numbers. Origins with the distal set were inactive unless the adjacent P1 DNA sequences beyond the boxes were included. At either end, phasing of the boxes with respect to the remainder of the origin influenced the copy number. Thus, although the boxes can be at either end, their precise context is critical for efficient origin function.
Url:
DOI: 10.1006/jmbi.2001.4741
Links to Exploration step
ISTEX:911BED3F96E7A055BF967158D03F96F4DE6CEC34Le document en format XML
<record><TEI wicri:istexFullTextTei="biblStruct"><teiHeader><fileDesc><titleStmt><title xml:lang="en">DnaA boxes in the P1 plasmid origin: the effect of their position on the directionality of replication and plasmid copy number</title><author><name sortKey="Park, Kyusung" sort="Park, Kyusung" uniqKey="Park K" first="Kyusung" last="Park">Kyusung Park</name><affiliation><mods:affiliation>Laboratory of Biochemistry, Center for Cancer Research National Cancer Institute National Institutes of Health Bethesda, MD 20892-4255, USA</mods:affiliation></affiliation></author><author><name sortKey="Chattoraj, Dhruba K" sort="Chattoraj, Dhruba K" uniqKey="Chattoraj D" first="Dhruba K" last="Chattoraj">Dhruba K. Chattoraj</name><affiliation><mods:affiliation>E-mail: chattoraj@nih.gov</mods:affiliation></affiliation><affiliation><mods:affiliation>Laboratory of Biochemistry, Center for Cancer Research National Cancer Institute National Institutes of Health Bethesda, MD 20892-4255, USA</mods:affiliation></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">ISTEX</idno><idno type="RBID">ISTEX:911BED3F96E7A055BF967158D03F96F4DE6CEC34</idno><date when="2001" year="2001">2001</date><idno type="doi">10.1006/jmbi.2001.4741</idno><idno type="url">https://api.istex.fr/ark:/67375/6H6-2ZCNFMDK-W/fulltext.pdf</idno><idno type="wicri:Area/Istex/Corpus">000A06</idno><idno type="wicri:explorRef" wicri:stream="Istex" wicri:step="Corpus" wicri:corpus="ISTEX">000A06</idno></publicationStmt><sourceDesc><biblStruct><analytic><title level="a" type="main" xml:lang="en">DnaA boxes in the P1 plasmid origin: the effect of their position on the directionality of replication and plasmid copy number</title><author><name sortKey="Park, Kyusung" sort="Park, Kyusung" uniqKey="Park K" first="Kyusung" last="Park">Kyusung Park</name><affiliation><mods:affiliation>Laboratory of Biochemistry, Center for Cancer Research National Cancer Institute National Institutes of Health Bethesda, MD 20892-4255, USA</mods:affiliation></affiliation></author><author><name sortKey="Chattoraj, Dhruba K" sort="Chattoraj, Dhruba K" uniqKey="Chattoraj D" first="Dhruba K" last="Chattoraj">Dhruba K. Chattoraj</name><affiliation><mods:affiliation>E-mail: chattoraj@nih.gov</mods:affiliation></affiliation><affiliation><mods:affiliation>Laboratory of Biochemistry, Center for Cancer Research National Cancer Institute National Institutes of Health Bethesda, MD 20892-4255, USA</mods:affiliation></affiliation></author></analytic><monogr></monogr><series><title level="j">Journal of Molecular Biology</title><title level="j" type="abbrev">YJMBI</title><idno type="ISSN">0022-2836</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="2001">2001</date><biblScope unit="volume">310</biblScope><biblScope unit="issue">1</biblScope><biblScope unit="page" from="69">69</biblScope><biblScope unit="page" to="81">81</biblScope></imprint><idno type="ISSN">0022-2836</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0022-2836</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>2D</term><term>DnaA box</term><term>EtBr</term><term>RI</term><term>direction of replication</term><term>origin of replication</term><term>origin-opening</term><term>plasmid P1</term></keywords><keywords scheme="Teeft" xml:lang="en"><term>Acad</term><term>Anking sequence</term><term>Appropriate antibiotics</term><term>Bacteriol</term><term>Bamhi</term><term>Band intensities</term><term>Beckman</term><term>Bidirectional</term><term>Bidirectional replication</term><term>Binding protein</term><term>Biol</term><term>Chattoraj</term><term>Chem</term><term>Chimeric plasmids</term><term>Coli</term><term>Consensus dnaa</term><term>Consensus dnaa boxes</term><term>Copy number</term><term>Copy numbers</term><term>Dnaa</term><term>Dnaa boxes</term><term>Dnaa functions</term><term>Dnaa protein</term><term>Ecori</term><term>Escherichia</term><term>Escherichia coli</term><term>Form molecules</term><term>Helicase</term><term>Helicase loading</term><term>Hindiii</term><term>Hindiii site</term><term>Initiation site</term><term>Iterons</term><term>Kmno4</term><term>Linearized</term><term>Migration patterns</term><term>Minip1</term><term>Minip1 plasmid</term><term>Minip1 plasmids</term><term>Natl</term><term>Natl acad</term><term>Open region</term><term>Opening signal</term><term>Oric</term><term>Origin opening</term><term>P1ori</term><term>Plasmid</term><term>Plasmid copy number</term><term>Plasmid origin</term><term>Plasmid replication</term><term>Plasmid replication figure</term><term>Proc</term><term>Psti fragment</term><term>Pvui</term><term>Pvui site</term><term>Repa</term><term>Replication</term><term>Replication forks</term><term>Replication initiation</term><term>Replication intermediates</term><term>Replication origin</term><term>Resultant plasmid</term><term>Styi</term><term>Transcription</term><term>Unpublished results</term></keywords></textClass><langUsage><language ident="en">en</language></langUsage></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Abstract: The DnaA protein is essential for initiation of DNA replication in a wide variety of bacterial and plasmid replicons. The replication origin in these replicons invariably contains specific binding sites for the protein, called DnaA boxes. Plasmid P1 contains a set of DnaA boxes at each end of its origin but can function with either one of the sets. Here we report that the location of origin-opening, initiation site of replication forks and directionality of replication do not change whether the boxes are present at both or at one of the ends of the origin. Replication was bidirectional in all cases. These results imply that DnaA functions similarly from the two ends of the origin. However, origins with DnaA boxes proximal to the origin-opening location opened more efficiently and maintained plasmids at higher copy numbers. Origins with the distal set were inactive unless the adjacent P1 DNA sequences beyond the boxes were included. At either end, phasing of the boxes with respect to the remainder of the origin influenced the copy number. Thus, although the boxes can be at either end, their precise context is critical for efficient origin function.</div></front></TEI><istex><corpusName>elsevier</corpusName><keywords><teeft><json:string>plasmid</json:string><json:string>dnaa</json:string><json:string>replication</json:string><json:string>dnaa boxes</json:string><json:string>copy number</json:string><json:string>minip1</json:string><json:string>p1ori</json:string><json:string>coli</json:string><json:string>hindiii</json:string><json:string>bidirectional</json:string><json:string>repa</json:string><json:string>escherichia</json:string><json:string>pvui</json:string><json:string>biol</json:string><json:string>plasmid replication</json:string><json:string>linearized</json:string><json:string>bacteriol</json:string><json:string>styi</json:string><json:string>minip1 plasmids</json:string><json:string>bidirectional replication</json:string><json:string>oric</json:string><json:string>iterons</json:string><json:string>bamhi</json:string><json:string>escherichia coli</json:string><json:string>proc</json:string><json:string>pvui site</json:string><json:string>natl acad</json:string><json:string>chem</json:string><json:string>ecori</json:string><json:string>helicase</json:string><json:string>chattoraj</json:string><json:string>acad</json:string><json:string>natl</json:string><json:string>kmno4</json:string><json:string>plasmid copy number</json:string><json:string>replication origin</json:string><json:string>initiation site</json:string><json:string>consensus dnaa boxes</json:string><json:string>unpublished results</json:string><json:string>dnaa protein</json:string><json:string>opening signal</json:string><json:string>replication intermediates</json:string><json:string>band intensities</json:string><json:string>open region</json:string><json:string>chimeric plasmids</json:string><json:string>replication forks</json:string><json:string>psti fragment</json:string><json:string>copy numbers</json:string><json:string>transcription</json:string><json:string>beckman</json:string><json:string>consensus dnaa</json:string><json:string>anking sequence</json:string><json:string>helicase loading</json:string><json:string>dnaa functions</json:string><json:string>replication initiation</json:string><json:string>origin opening</json:string><json:string>appropriate antibiotics</json:string><json:string>plasmid replication figure</json:string><json:string>binding protein</json:string><json:string>migration patterns</json:string><json:string>resultant plasmid</json:string><json:string>minip1 plasmid</json:string><json:string>form molecules</json:string><json:string>plasmid origin</json:string><json:string>hindiii site</json:string></teeft></keywords><author><json:item><name>Kyusung Park</name><affiliations><json:string>Laboratory of Biochemistry, Center for Cancer Research National Cancer Institute National Institutes of Health Bethesda, MD 20892-4255, USA</json:string></affiliations></json:item><json:item><name>Dhruba K Chattoraj</name><affiliations><json:string>E-mail: chattoraj@nih.gov</json:string><json:string>Laboratory of Biochemistry, Center for Cancer Research National Cancer Institute National Institutes of Health Bethesda, MD 20892-4255, USA</json:string></affiliations></json:item></author><subject><json:item><lang><json:string>eng</json:string></lang><value>Regular article</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>origin of replication</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>plasmid P1</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>DnaA box</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>origin-opening</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>direction of replication</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>RI : replication intermediate</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>EtBr : ethidium bromide</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>2D : two-dimensional</value></json:item></subject><arkIstex>ark:/67375/6H6-2ZCNFMDK-W</arkIstex><language><json:string>eng</json:string></language><originalGenre><json:string>Full-length article</json:string></originalGenre><abstract>Abstract: The DnaA protein is essential for initiation of DNA replication in a wide variety of bacterial and plasmid replicons. The replication origin in these replicons invariably contains specific binding sites for the protein, called DnaA boxes. Plasmid P1 contains a set of DnaA boxes at each end of its origin but can function with either one of the sets. Here we report that the location of origin-opening, initiation site of replication forks and directionality of replication do not change whether the boxes are present at both or at one of the ends of the origin. Replication was bidirectional in all cases. These results imply that DnaA functions similarly from the two ends of the origin. However, origins with DnaA boxes proximal to the origin-opening location opened more efficiently and maintained plasmids at higher copy numbers. Origins with the distal set were inactive unless the adjacent P1 DNA sequences beyond the boxes were included. At either end, phasing of the boxes with respect to the remainder of the origin influenced the copy number. Thus, although the boxes can be at either end, their precise context is critical for efficient origin function.</abstract><qualityIndicators><refBibsNative>true</refBibsNative><abstractWordCount>191</abstractWordCount><abstractCharCount>1175</abstractCharCount><keywordCount>9</keywordCount><score>9.292</score><pdfWordCount>8262</pdfWordCount><pdfCharCount>48252</pdfCharCount><pdfVersion>1.3</pdfVersion><pdfPageCount>13</pdfPageCount><pdfPageSize>595 x 842 pts (A4)</pdfPageSize></qualityIndicators><title>DnaA boxes in the P1 plasmid origin: the effect of their position on the directionality of replication and plasmid copy number</title><pmid><json:string>11419937</json:string></pmid><pii><json:string>S0022-2836(01)94741-2</json:string></pii><genre><json:string>research-article</json:string></genre><serie><title>Escherichia coli and Salmonella</title><language><json:string>unknown</json:string></language><volume>vol. 2</volume><pages><first>1579</first><last>1601</last></pages></serie><host><title>Journal of Molecular Biology</title><language><json:string>unknown</json:string></language><publicationDate>2001</publicationDate><issn><json:string>0022-2836</json:string></issn><pii><json:string>S0022-2836(00)X0232-X</json:string></pii><volume>310</volume><issue>1</issue><pages><first>69</first><last>81</last></pages><genre><json:string>journal</json:string></genre></host><namedEntities><unitex><date><json:string>2001</json:string></date><geogName></geogName><orgName><json:string>Biotech</json:string></orgName><orgName_funder></orgName_funder><orgName_provider></orgName_provider><persName><json:string>Health Bethesda</json:string><json:string>T.M. Hill</json:string><json:string>Tom Hill</json:string><json:string>M. Valjavec-Gratian</json:string><json:string>T.A. Henderson</json:string><json:string>Majda Valjavec-Gratian</json:string><json:string>Michael Lichten</json:string><json:string>K. Park</json:string><json:string>M. ValjavecGratian</json:string><json:string>Plasmids</json:string><json:string>K. Chattoraj</json:string></persName><placeName></placeName><ref_url><json:string>http://www.idealibrary.com</json:string></ref_url><ref_bibl><json:string>Invitrogen Corporation, 1620</json:string></ref_bibl><bibl></bibl></unitex></namedEntities><ark><json:string>ark:/67375/6H6-2ZCNFMDK-W</json:string></ark><categories><wos><json:string>1 - science</json:string><json:string>2 - biochemistry & molecular biology</json:string></wos><scienceMetrix><json:string>1 - health sciences</json:string><json:string>2 - biomedical research</json:string><json:string>3 - biochemistry & molecular biology</json:string></scienceMetrix><scopus><json:string>1 - Life Sciences</json:string><json:string>2 - Biochemistry, Genetics and Molecular Biology</json:string><json:string>3 - Molecular Biology</json:string></scopus><inist><json:string>1 - sciences appliquees, technologies et medecines</json:string><json:string>2 - sciences biologiques et medicales</json:string><json:string>3 - sciences biologiques fondamentales et appliquees. psychologie</json:string><json:string>4 - microbiologie</json:string></inist></categories><publicationDate>2001</publicationDate><copyrightDate>2001</copyrightDate><doi><json:string>10.1006/jmbi.2001.4741</json:string></doi><id>911BED3F96E7A055BF967158D03F96F4DE6CEC34</id><score>1</score><fulltext><json:item><extension>pdf</extension><original>true</original><mimetype>application/pdf</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-2ZCNFMDK-W/fulltext.pdf</uri></json:item><json:item><extension>zip</extension><original>false</original><mimetype>application/zip</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-2ZCNFMDK-W/bundle.zip</uri></json:item><istex:fulltextTEI uri="https://api.istex.fr/ark:/67375/6H6-2ZCNFMDK-W/fulltext.tei"><teiHeader><fileDesc><titleStmt><title level="a" type="main" xml:lang="en">DnaA boxes in the P1 plasmid origin: the effect of their position on the directionality of replication and plasmid copy number</title></titleStmt><publicationStmt><authority>ISTEX</authority><publisher scheme="https://scientific-publisher.data.istex.fr">ELSEVIER</publisher><availability><licence><p>©2001 Academic Press</p></licence><p scheme="https://loaded-corpus.data.istex.fr/ark:/67375/XBH-HKKZVM7B-M">elsevier</p></availability><date>2001</date></publicationStmt><notesStmt><note type="research-article" scheme="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</note><note type="journal" scheme="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</note><note>Edited by M. Gottesman</note><note type="content">Section title: Regular article</note><note type="content">Figure 1: 2D gel analysis of replication intermediates (RIs) of miniP1 plasmids, pSP102 (a) and pKP176 (b). pKP176 is identical to pSP102 except for the orientation of the PstI fragment containing the cat gene. The top line shows relevant features of pSP102: DnaA box ( ), GATC (G) and iteron (→)of the origin; and repA and cat genes. Both the genes are transcribed in the left to right direction. The schematic in the top left corner shows the expected gel running patterns of RIs with bubble, simple-Y and double-Y forms. The gray circle and the gray arc represent linear molecules of 4.3 kb (length of a plasmid monomer) and longer lengths, respectively. The gels show the RIs after linearization with PvuI (P), BamHI (B), HindIII(H) and StyI (S). The RIs expected from symmetric (first three rows) and asymmetric (last row) bidirectional replication, initiating near the PvuI site and after linearization with different enzymes, are also shown schematically for pSP102. Asymmetric bidirectional replication is supported here, since the gel patterns differed after digestion with HindIII and StyI, whose sites are symmetrically disposed with respect to the PvuI site. The asymmetry is attributed to cat transcriptions since inverting the gene in pKP176 makes the HindIII pattern appear as StyI and vice versa.</note><note type="content">Figure 2: 2D gel analysis of plasmids containing the replication terminator, ter. The ter binding protein, Tus, was supplied by induction from pBAD30tus. pTH102 ((a) and (b)) is used here as a control plasmid to monitor effective Tus induction. The schematics are as for Figure 1 except that they also show the insertion site and the blocking orientation of ter (the gray arrows). RIs of pTH102 were digested with AvaI, and those of miniP1 plasmids either with PvuI (pKP157 and pKP158) ((c) and (d)) or with BamHI (pKP155 and pKP156) ((e) and (f)). Note that simple-Y forms are seen only when the RIs were linearized close to ter in the blocking orientation.</note><note type="content">Figure 3: 2D gel analysis of RIs of miniP1 with varied location of DnaA boxes. The RIs were isolated from DH5Δlac(λrepA) and linearized at the NruI site (Figure 1). The gel patterns are similar to each other and resemble that of the HindIII digested pSP102 (Figure 1) in that they contain predominantly double-Y form molecules. The triangular smear under the double-Y line may be indicative of branch migration and/or near fully replicated intermediates with variable branch points due to asymmetric progression of the two forks.</note><note type="content">Figure 4: (a) Probing of miniP1 origin-opening in vivo by reactivity to KMnO4. The host was dnaC(ts), and contained a miniP1 plasmid (pDKC412-418) and a second compatible plasmid (pALA177) that supplied RepA. The KMnO4-modified bases in the origin region were visualized by primer extension. No particular region of hypersensitivity to KMnO4 was apparent at 30°C with the exception of the A+T region of the origin containing the GATC sequences (marked as G) (lanes 1, 3, 5, 7, 9 and 11). At 42°C, the sensitivity increased in the region (bracket) containing the GATC sequence proximal to the DnaA boxes on the left ( ) and decreased in the remainder of the lane (lanes 2, 4, 6, 8, 10 and 12). The pattern was essentially identical when the only DnaA boxes present were next to the iterons (lane 12). (b) The efficiency of origin-opening and copy numbers of the plasmids used in (a). The schematics show the region of P1 DNA (white bar) present in the plasmids. Additionally, the plasmids had the Ω-Cm cassette in all cases (not shown). The efficiency of origin-opening was estimated as described in Materials and Methods. The copy numbers are averages of at least three independent measurements and relative to pSP102 copy number of eight.56</note><note type="content">Figure 5: (a) Origin-opening in miniP1 with consensus DnaA boxes. Experiments were done as for Figure 4(a). No particular region of hypersensitivity to KMnO4 is apparent at the permissive temperature (lanes 1, 3, 5, 7, 9 and 11). Upon increase in temperature, the opening signal, when present, is at the same position as seen in Figure 4(a) (lanes 2, 8, 10 and 12). Opening was undetectable in origins with a single consensus DnaA box (lanes 4 and 6). Increasing the number of boxes to two affected opening in a position sensitive manner. When the boxes flanked the origin (lane 8) or when both the boxes were on the left (lanes 10) opening was enhanced. In contrast, a plasmid with two boxes at the downstream position could be constructed only when the boxes were also present on the left; even then the opening was marginal (lanes 12). (b) Efficiency of origin-opening and the copy number of miniP1 plasmids with consensus DnaA boxes (shaded gray). The plasmids had the Ω-Cm cassette as in Figure 4, all oriented identically.</note><note type="content">Figure 6: Copy number of pUC19-P1ori chimeric plasmids containing a consensus DnaA box and a 9 or 5 bp spacer. The schematics are as for Figure 4(b). The 9 or 5 bp spacer is indicated with an open rectangle at the site of insertion. The ori fragments were present between the HindIII and EcoRI sites of the vector, pUC19. The copy number was measured in a polAts host at the non-permissive temperature, 42°C. RepA was provided in trans from an integrated λ phage.</note><note type="content">Figure 7: Efficiency of origin-opening and copy number of pUC19-P1ori chimeric plasmids containing natural DnaA boxes. A P1ori is cloned in the same manner as for Figure 6 except for pKP133 and pKP145 where the 225 bp HindIII fragment containing P1ori was inverted with respect to plasmids, pALA631 and pKP131, respectively. Origin-opening and copy number were determined as for Figures 4 and 6, respectively.</note></notesStmt><sourceDesc><biblStruct type="inbook"><analytic><title level="a" type="main" xml:lang="en">DnaA boxes in the P1 plasmid origin: the effect of their position on the directionality of replication and plasmid copy number</title><author xml:id="author-0000"><persName><forename type="first">Kyusung</forename><surname>Park</surname></persName><note type="biography">K. Park, Invitrogen Corporation, 1620 Faraday Ave., Carlsbad, CA 90028, USA.</note><affiliation>K. Park, Invitrogen Corporation, 1620 Faraday Ave., Carlsbad, CA 90028, USA.</affiliation><affiliation>Laboratory of Biochemistry, Center for Cancer Research National Cancer Institute National Institutes of Health Bethesda, MD 20892-4255, USA</affiliation></author><author xml:id="author-0001"><persName><forename type="first">Dhruba K</forename><surname>Chattoraj</surname></persName><email>chattoraj@nih.gov</email><note type="correspondence"><p>Corresponding author</p></note><affiliation>Laboratory of Biochemistry, Center for Cancer Research National Cancer Institute National Institutes of Health Bethesda, MD 20892-4255, USA</affiliation></author><idno type="istex">911BED3F96E7A055BF967158D03F96F4DE6CEC34</idno><idno type="ark">ark:/67375/6H6-2ZCNFMDK-W</idno><idno type="DOI">10.1006/jmbi.2001.4741</idno><idno type="PII">S0022-2836(01)94741-2</idno></analytic><monogr><title level="j">Journal of Molecular Biology</title><title level="j" type="abbrev">YJMBI</title><idno type="pISSN">0022-2836</idno><idno type="PII">S0022-2836(00)X0232-X</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="2001"></date><biblScope unit="volume">310</biblScope><biblScope unit="issue">1</biblScope><biblScope unit="page" from="69">69</biblScope><biblScope unit="page" to="81">81</biblScope></imprint></monogr></biblStruct></sourceDesc></fileDesc><profileDesc><creation><date>2001</date></creation><langUsage><language ident="en">en</language></langUsage><abstract xml:lang="en"><p>Abstract: The DnaA protein is essential for initiation of DNA replication in a wide variety of bacterial and plasmid replicons. The replication origin in these replicons invariably contains specific binding sites for the protein, called DnaA boxes. Plasmid P1 contains a set of DnaA boxes at each end of its origin but can function with either one of the sets. Here we report that the location of origin-opening, initiation site of replication forks and directionality of replication do not change whether the boxes are present at both or at one of the ends of the origin. Replication was bidirectional in all cases. These results imply that DnaA functions similarly from the two ends of the origin. However, origins with DnaA boxes proximal to the origin-opening location opened more efficiently and maintained plasmids at higher copy numbers. Origins with the distal set were inactive unless the adjacent P1 DNA sequences beyond the boxes were included. At either end, phasing of the boxes with respect to the remainder of the origin influenced the copy number. Thus, although the boxes can be at either end, their precise context is critical for efficient origin function.</p></abstract><textClass><keywords scheme="keyword"><list><head>article-category</head><item><term>Regular article</term></item></list></keywords></textClass><textClass xml:lang="en"><keywords scheme="keyword"><list><head>Keywords</head><item><term>origin of replication</term></item><item><term>plasmid P1</term></item><item><term>DnaA box</term></item><item><term>origin-opening</term></item><item><term>direction of replication</term></item></list></keywords></textClass><textClass xml:lang="en"><keywords scheme="keyword"><list><head>Abbreviations</head><item><term>RI</term><term>replication intermediate</term></item><item><term>EtBr</term><term>ethidium bromide</term></item><item><term>2D</term><term>two-dimensional</term></item></list></keywords></textClass></profileDesc><revisionDesc><change when="2001-04-30">Modified</change><change when="2001">Published</change></revisionDesc></teiHeader></istex:fulltextTEI><json:item><extension>txt</extension><original>false</original><mimetype>text/plain</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-2ZCNFMDK-W/fulltext.txt</uri></json:item></fulltext><metadata><istex:metadataXml wicri:clean="Elsevier, elements deleted: ce:floats; body; tail"><istex:xmlDeclaration>version="1.0" encoding="utf-8"</istex:xmlDeclaration><istex:docType PUBLIC="-//ES//DTD journal article DTD version 4.5.2//EN//XML" URI="art452.dtd" name="istex:docType"><istex:entity SYSTEM="gr1" NDATA="IMAGE" name="GR1"></istex:entity><istex:entity SYSTEM="gr2" NDATA="IMAGE" name="GR2"></istex:entity><istex:entity SYSTEM="gr3" NDATA="IMAGE" name="GR3"></istex:entity><istex:entity SYSTEM="gr4a" NDATA="IMAGE" name="GR4a"></istex:entity><istex:entity SYSTEM="gr4b" NDATA="IMAGE" name="GR4b"></istex:entity><istex:entity SYSTEM="gr5a" NDATA="IMAGE" name="GR5a"></istex:entity><istex:entity SYSTEM="gr5b" NDATA="IMAGE" name="GR5b"></istex:entity><istex:entity SYSTEM="gr6" NDATA="IMAGE" name="GR6"></istex:entity><istex:entity SYSTEM="gr7" NDATA="IMAGE" name="GR7"></istex:entity><istex:entity SYSTEM="fx1" NDATA="IMAGE" name="FX1"></istex:entity><istex:entity SYSTEM="fx2" NDATA="IMAGE" name="FX2"></istex:entity></istex:docType><istex:document><converted-article version="4.5.2" docsubtype="fla" xml:lang="en"><item-info><jid>YJMBI</jid><aid>94741</aid><ce:pii>S0022-2836(01)94741-2</ce:pii><ce:doi>10.1006/jmbi.2001.4741</ce:doi><ce:copyright type="full-transfer" year="2001">Academic Press</ce:copyright><ce:doctopics><ce:doctopic><ce:text>Regular article</ce:text></ce:doctopic></ce:doctopics></item-info><head><ce:dochead><ce:textfn>Regular article</ce:textfn></ce:dochead><ce:title>DnaA boxes in the P1 plasmid origin: the effect of their position on the directionality of replication and plasmid copy number<ce:cross-ref refid="FN1"><ce:sup>1</ce:sup></ce:cross-ref><ce:footnote id="FN1"><ce:label>1</ce:label><ce:note-para><ce:bold><ce:italic>Edited by M. Gottesman</ce:italic></ce:bold></ce:note-para></ce:footnote></ce:title><ce:author-group><ce:author><ce:given-name>Kyusung</ce:given-name><ce:surname>Park</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>a</ce:sup></ce:cross-ref><ce:cross-ref refid="FN2"><ce:sup>2</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>Dhruba K</ce:given-name><ce:surname>Chattoraj</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>a</ce:sup></ce:cross-ref><ce:cross-ref refid="COR1">*</ce:cross-ref><ce:e-address>chattoraj@nih.gov</ce:e-address></ce:author><ce:affiliation id="AFF1"><ce:label>a</ce:label><ce:textfn>Laboratory of Biochemistry, Center for Cancer Research National Cancer Institute National Institutes of Health Bethesda, MD 20892-4255, USA</ce:textfn></ce:affiliation><ce:correspondence id="COR1"><ce:label>*</ce:label><ce:text>Corresponding author</ce:text></ce:correspondence><ce:footnote id="FN2"><ce:label>2</ce:label><ce:note-para>K. Park, Invitrogen Corporation, 1620 Faraday Ave., Carlsbad, CA 90028, USA.</ce:note-para></ce:footnote></ce:author-group><ce:date-received day="23" month="2" year="2001"></ce:date-received><ce:date-revised day="30" month="4" year="2001"></ce:date-revised><ce:date-accepted day="30" month="4" year="2001"></ce:date-accepted><ce:abstract><ce:section-title>Abstract</ce:section-title><ce:abstract-sec><ce:simple-para>The DnaA protein is essential for initiation of DNA replication in a wide variety of bacterial and plasmid replicons. The replication origin in these replicons invariably contains specific binding sites for the protein, called DnaA boxes. Plasmid P1 contains a set of DnaA boxes at each end of its origin but can function with either one of the sets. Here we report that the location of origin-opening, initiation site of replication forks and directionality of replication do not change whether the boxes are present at both or at one of the ends of the origin. Replication was bidirectional in all cases. These results imply that DnaA functions similarly from the two ends of the origin. However, origins with DnaA boxes proximal to the origin-opening location opened more efficiently and maintained plasmids at higher copy numbers. Origins with the distal set were inactive unless the adjacent P1 DNA sequences beyond the boxes were included. At either end, phasing of the boxes with respect to the remainder of the origin influenced the copy number. Thus, although the boxes can be at either end, their precise context is critical for efficient origin function.</ce:simple-para></ce:abstract-sec></ce:abstract><ce:keywords><ce:section-title>Keywords</ce:section-title><ce:keyword><ce:text>origin of replication</ce:text></ce:keyword><ce:keyword><ce:text>plasmid P1</ce:text></ce:keyword><ce:keyword><ce:text>DnaA box</ce:text></ce:keyword><ce:keyword><ce:text>origin-opening</ce:text></ce:keyword><ce:keyword><ce:text>direction of replication</ce:text></ce:keyword></ce:keywords><ce:keywords class="abr"><ce:section-title>Abbreviations</ce:section-title><ce:keyword><ce:text>RI</ce:text><ce:keyword><ce:text>replication intermediate</ce:text></ce:keyword></ce:keyword><ce:keyword><ce:text>EtBr</ce:text><ce:keyword><ce:text>ethidium bromide</ce:text></ce:keyword></ce:keyword><ce:keyword><ce:text>2D</ce:text><ce:keyword><ce:text>two-dimensional</ce:text></ce:keyword></ce:keyword></ce:keywords></head></converted-article></istex:document></istex:metadataXml><mods version="3.6"><titleInfo lang="en"><title>DnaA boxes in the P1 plasmid origin: the effect of their position on the directionality of replication and plasmid copy number</title></titleInfo><titleInfo type="alternative" lang="en" contentType="CDATA"><title>DnaA boxes in the P1 plasmid origin: the effect of their position on the directionality of replication and plasmid copy number</title></titleInfo><name type="personal"><namePart type="given">Kyusung</namePart><namePart type="family">Park</namePart><affiliation>Laboratory of Biochemistry, Center for Cancer Research National Cancer Institute National Institutes of Health Bethesda, MD 20892-4255, USA</affiliation><description>K. Park, Invitrogen Corporation, 1620 Faraday Ave., Carlsbad, CA 90028, USA.</description><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Dhruba K</namePart><namePart type="family">Chattoraj</namePart><affiliation>E-mail: chattoraj@nih.gov</affiliation><affiliation>Laboratory of Biochemistry, Center for Cancer Research National Cancer Institute National Institutes of Health Bethesda, MD 20892-4255, USA</affiliation><description>Corresponding author</description><role><roleTerm type="text">author</roleTerm></role></name><typeOfResource>text</typeOfResource><genre type="research-article" displayLabel="Full-length article" authority="ISTEX" authorityURI="https://content-type.data.istex.fr" valueURI="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</genre><originInfo><publisher>ELSEVIER</publisher><dateIssued encoding="w3cdtf">2001</dateIssued><dateModified encoding="w3cdtf">2001-04-30</dateModified><copyrightDate encoding="w3cdtf">2001</copyrightDate></originInfo><language><languageTerm type="code" authority="iso639-2b">eng</languageTerm><languageTerm type="code" authority="rfc3066">en</languageTerm></language><abstract lang="en">Abstract: The DnaA protein is essential for initiation of DNA replication in a wide variety of bacterial and plasmid replicons. The replication origin in these replicons invariably contains specific binding sites for the protein, called DnaA boxes. Plasmid P1 contains a set of DnaA boxes at each end of its origin but can function with either one of the sets. Here we report that the location of origin-opening, initiation site of replication forks and directionality of replication do not change whether the boxes are present at both or at one of the ends of the origin. Replication was bidirectional in all cases. These results imply that DnaA functions similarly from the two ends of the origin. However, origins with DnaA boxes proximal to the origin-opening location opened more efficiently and maintained plasmids at higher copy numbers. Origins with the distal set were inactive unless the adjacent P1 DNA sequences beyond the boxes were included. At either end, phasing of the boxes with respect to the remainder of the origin influenced the copy number. Thus, although the boxes can be at either end, their precise context is critical for efficient origin function.</abstract><note type="footnote">Edited by M. Gottesman</note><note type="content">Section title: Regular article</note><note type="content">Figure 1: 2D gel analysis of replication intermediates (RIs) of miniP1 plasmids, pSP102 (a) and pKP176 (b). pKP176 is identical to pSP102 except for the orientation of the PstI fragment containing the cat gene. The top line shows relevant features of pSP102: DnaA box ( ), GATC (G) and iteron (→)of the origin; and repA and cat genes. Both the genes are transcribed in the left to right direction. The schematic in the top left corner shows the expected gel running patterns of RIs with bubble, simple-Y and double-Y forms. The gray circle and the gray arc represent linear molecules of 4.3 kb (length of a plasmid monomer) and longer lengths, respectively. The gels show the RIs after linearization with PvuI (P), BamHI (B), HindIII(H) and StyI (S). The RIs expected from symmetric (first three rows) and asymmetric (last row) bidirectional replication, initiating near the PvuI site and after linearization with different enzymes, are also shown schematically for pSP102. Asymmetric bidirectional replication is supported here, since the gel patterns differed after digestion with HindIII and StyI, whose sites are symmetrically disposed with respect to the PvuI site. The asymmetry is attributed to cat transcriptions since inverting the gene in pKP176 makes the HindIII pattern appear as StyI and vice versa.</note><note type="content">Figure 2: 2D gel analysis of plasmids containing the replication terminator, ter. The ter binding protein, Tus, was supplied by induction from pBAD30tus. pTH102 ((a) and (b)) is used here as a control plasmid to monitor effective Tus induction. The schematics are as for Figure 1 except that they also show the insertion site and the blocking orientation of ter (the gray arrows). RIs of pTH102 were digested with AvaI, and those of miniP1 plasmids either with PvuI (pKP157 and pKP158) ((c) and (d)) or with BamHI (pKP155 and pKP156) ((e) and (f)). Note that simple-Y forms are seen only when the RIs were linearized close to ter in the blocking orientation.</note><note type="content">Figure 3: 2D gel analysis of RIs of miniP1 with varied location of DnaA boxes. The RIs were isolated from DH5Δlac(λrepA) and linearized at the NruI site (Figure 1). The gel patterns are similar to each other and resemble that of the HindIII digested pSP102 (Figure 1) in that they contain predominantly double-Y form molecules. The triangular smear under the double-Y line may be indicative of branch migration and/or near fully replicated intermediates with variable branch points due to asymmetric progression of the two forks.</note><note type="content">Figure 4: (a) Probing of miniP1 origin-opening in vivo by reactivity to KMnO4. The host was dnaC(ts), and contained a miniP1 plasmid (pDKC412-418) and a second compatible plasmid (pALA177) that supplied RepA. The KMnO4-modified bases in the origin region were visualized by primer extension. No particular region of hypersensitivity to KMnO4 was apparent at 30°C with the exception of the A+T region of the origin containing the GATC sequences (marked as G) (lanes 1, 3, 5, 7, 9 and 11). At 42°C, the sensitivity increased in the region (bracket) containing the GATC sequence proximal to the DnaA boxes on the left ( ) and decreased in the remainder of the lane (lanes 2, 4, 6, 8, 10 and 12). The pattern was essentially identical when the only DnaA boxes present were next to the iterons (lane 12). (b) The efficiency of origin-opening and copy numbers of the plasmids used in (a). The schematics show the region of P1 DNA (white bar) present in the plasmids. Additionally, the plasmids had the Ω-Cm cassette in all cases (not shown). The efficiency of origin-opening was estimated as described in Materials and Methods. The copy numbers are averages of at least three independent measurements and relative to pSP102 copy number of eight.56</note><note type="content">Figure 5: (a) Origin-opening in miniP1 with consensus DnaA boxes. Experiments were done as for Figure 4(a). No particular region of hypersensitivity to KMnO4 is apparent at the permissive temperature (lanes 1, 3, 5, 7, 9 and 11). Upon increase in temperature, the opening signal, when present, is at the same position as seen in Figure 4(a) (lanes 2, 8, 10 and 12). Opening was undetectable in origins with a single consensus DnaA box (lanes 4 and 6). Increasing the number of boxes to two affected opening in a position sensitive manner. When the boxes flanked the origin (lane 8) or when both the boxes were on the left (lanes 10) opening was enhanced. In contrast, a plasmid with two boxes at the downstream position could be constructed only when the boxes were also present on the left; even then the opening was marginal (lanes 12). (b) Efficiency of origin-opening and the copy number of miniP1 plasmids with consensus DnaA boxes (shaded gray). The plasmids had the Ω-Cm cassette as in Figure 4, all oriented identically.</note><note type="content">Figure 6: Copy number of pUC19-P1ori chimeric plasmids containing a consensus DnaA box and a 9 or 5 bp spacer. The schematics are as for Figure 4(b). The 9 or 5 bp spacer is indicated with an open rectangle at the site of insertion. The ori fragments were present between the HindIII and EcoRI sites of the vector, pUC19. The copy number was measured in a polAts host at the non-permissive temperature, 42°C. RepA was provided in trans from an integrated λ phage.</note><note type="content">Figure 7: Efficiency of origin-opening and copy number of pUC19-P1ori chimeric plasmids containing natural DnaA boxes. A P1ori is cloned in the same manner as for Figure 6 except for pKP133 and pKP145 where the 225 bp HindIII fragment containing P1ori was inverted with respect to plasmids, pALA631 and pKP131, respectively. Origin-opening and copy number were determined as for Figures 4 and 6, respectively.</note><subject><genre>article-category</genre><topic>Regular article</topic></subject><subject lang="en"><genre>Keywords</genre><topic>origin of replication</topic><topic>plasmid P1</topic><topic>DnaA box</topic><topic>origin-opening</topic><topic>direction of replication</topic></subject><subject lang="en"><genre>Abbreviations</genre><topic>RI : replication intermediate</topic><topic>EtBr : ethidium bromide</topic><topic>2D : two-dimensional</topic></subject><relatedItem type="host"><titleInfo><title>Journal of Molecular Biology</title></titleInfo><titleInfo type="abbreviated"><title>YJMBI</title></titleInfo><genre type="journal" authority="ISTEX" authorityURI="https://publication-type.data.istex.fr" valueURI="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</genre><originInfo><publisher>ELSEVIER</publisher><dateIssued encoding="w3cdtf">2001</dateIssued></originInfo><identifier type="ISSN">0022-2836</identifier><identifier type="PII">S0022-2836(00)X0232-X</identifier><part><date>2001</date><detail type="volume"><number>310</number><caption>vol.</caption></detail><detail type="issue"><number>1</number><caption>no.</caption></detail><extent unit="issue-pages"><start>1</start><end>280</end></extent><extent unit="pages"><start>69</start><end>81</end></extent></part></relatedItem><identifier type="istex">911BED3F96E7A055BF967158D03F96F4DE6CEC34</identifier><identifier type="ark">ark:/67375/6H6-2ZCNFMDK-W</identifier><identifier type="DOI">10.1006/jmbi.2001.4741</identifier><identifier type="PII">S0022-2836(01)94741-2</identifier><accessCondition type="use and reproduction" contentType="copyright">©2001 Academic Press</accessCondition><recordInfo><recordContentSource authority="ISTEX" authorityURI="https://loaded-corpus.data.istex.fr" valueURI="https://loaded-corpus.data.istex.fr/ark:/67375/XBH-HKKZVM7B-M">elsevier</recordContentSource><recordOrigin>Academic Press, ©2001</recordOrigin></recordInfo></mods><json:item><extension>json</extension><original>false</original><mimetype>application/json</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-2ZCNFMDK-W/record.json</uri></json:item></metadata></istex></record>Pour manipuler ce document sous Unix (Dilib)
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