The Interwoven Architecture of the Mu Transposase Couples DNA Synapsis to Catalysis
Identifieur interne : 002554 ( Istex/Corpus ); précédent : 002553; suivant : 002555The Interwoven Architecture of the Mu Transposase Couples DNA Synapsis to Catalysis
Auteurs : Hector Aldaz ; Eugene Schuster ; Tania A. BakerSource :
- Cell [ 0092-8674 ] ; 1996.
English descriptors
- Teeft :
- Acidic, Active monomer, Active protein, Active site, Active sites, Active subunit, Active subunits, Active tetramer, Active transposase, Agarose, Binding activity, Binding domain, Binding sites, Biol, Catalysis, Catalytic, Catalytic residues, Catalyze, Catalyze strand transfer, Chaconas, Chem, Cleavage, Cleavage sites, Cleavage step, Covalently, Craigie, Domain, Domain iiia, Domain iiib, Embo, Fragment, Genome, Harshey, Iiia, Inactive subunits, Inactive transposase, Integrase, Jayaram, Joinable, Joinable substrate, Mizuuchi, Monomer, Oligonucleotides, Partner substratea, Personal communication, Phage, Phage genome, Possible arrangements, Recombinase, Recombination, Recombination sites, Recombining dnas, Savilahti, Stcs, Strand, Strand transfer, Subunit, Subunit arrangement, Surette, Synapsis, Tetramer, Tetramer assembly, Tetramers, Total reaction, Total reaction mixture, Trans, Transposase, Transposase acts, Transposase subunits, Transposase tetramer, Transposition, Transposition reactions, Unjoinable, Unjoinable substrate, Unjoinable substrates, Unjoined, Unpublished data.
Abstract
Abstract: Mu transposition occurs exclusively using a pair of recombination sites found at the ends of the phage genome. To address the mechanistic basis of this specificity, we have determined both where the individual subunits of the tetrameric transposase bind on the DNA and where they catalyze DNA joining. We demonstrate that subunits do not catalyze recombination at the site adjacent to where they are bound, but rather on the opposite end of the phage genome. Furthermore, subunits bound to two different sites contribute to catalysis of one reaction step. This interwoven subunit arrangement suggests a molecular explanation for the precision with which recombination occurs using a pair of DNA signals and provides an example of the way in which the architecture of a protein–DNA complex can define the reaction products.
Url:
DOI: 10.1016/S0092-8674(00)81102-2
Links to Exploration step
ISTEX:365354F68B0CD54059610042AED543B886AEC623Le document en format XML
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Baker</name><affiliation><mods:affiliation>E-mail: tabaker@MIT.EDU</mods:affiliation></affiliation><affiliation><mods:affiliation>Department of Biology, Massachusetts Institute of Technology, 68–523, 77 Massachusetts Avenue, Cambridge MA 02139, USA</mods:affiliation></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">ISTEX</idno><idno type="RBID">ISTEX:365354F68B0CD54059610042AED543B886AEC623</idno><date when="1996" year="1996">1996</date><idno type="doi">10.1016/S0092-8674(00)81102-2</idno><idno type="url">https://api.istex.fr/ark:/67375/6H6-04P1GBFN-C/fulltext.pdf</idno><idno type="wicri:Area/Istex/Corpus">002554</idno><idno type="wicri:explorRef" wicri:stream="Istex" wicri:step="Corpus" wicri:corpus="ISTEX">002554</idno></publicationStmt><sourceDesc><biblStruct><analytic><title level="a" type="main" xml:lang="en">The Interwoven Architecture of the Mu Transposase Couples DNA Synapsis to Catalysis</title><author><name sortKey="Aldaz, Hector" sort="Aldaz, Hector" uniqKey="Aldaz H" first="Hector" last="Aldaz">Hector Aldaz</name><affiliation><mods:affiliation>Department of Biology, Massachusetts Institute of Technology, 68–523, 77 Massachusetts Avenue, Cambridge MA 02139, USA</mods:affiliation></affiliation></author><author><name sortKey="Schuster, Eugene" sort="Schuster, Eugene" uniqKey="Schuster E" first="Eugene" last="Schuster">Eugene Schuster</name><affiliation><mods:affiliation>Department of Biology, Massachusetts Institute of Technology, 68–523, 77 Massachusetts Avenue, Cambridge MA 02139, USA</mods:affiliation></affiliation></author><author><name sortKey="Baker, Tania A" sort="Baker, Tania A" uniqKey="Baker T" first="Tania A" last="Baker">Tania A. 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To address the mechanistic basis of this specificity, we have determined both where the individual subunits of the tetrameric transposase bind on the DNA and where they catalyze DNA joining. We demonstrate that subunits do not catalyze recombination at the site adjacent to where they are bound, but rather on the opposite end of the phage genome. Furthermore, subunits bound to two different sites contribute to catalysis of one reaction step. This interwoven subunit arrangement suggests a molecular explanation for the precision with which recombination occurs using a pair of DNA signals and provides an example of the way in which the architecture of a protein–DNA complex can define the reaction products.</abstract><qualityIndicators><score>8.572</score><pdfWordCount>9646</pdfWordCount><pdfCharCount>56582</pdfCharCount><pdfVersion>1.3</pdfVersion><pdfPageCount>13</pdfPageCount><pdfPageSize>612 x 792 pts (letter)</pdfPageSize><refBibsNative>true</refBibsNative><abstractWordCount>131</abstractWordCount><abstractCharCount>832</abstractCharCount><keywordCount>0</keywordCount></qualityIndicators><title>The Interwoven Architecture of the Mu Transposase Couples DNA Synapsis to Catalysis</title><pmid><json:string>8612278</json:string></pmid><pii><json:string>S0092-8674(00)81102-2</json:string></pii><genre><json:string>research-article</json:string></genre><host><title>Cell</title><language><json:string>unknown</json:string></language><publicationDate>1996</publicationDate><issn><json:string>0092-8674</json:string></issn><pii><json:string>S0092-8674(00)X0086-4</json:string></pii><volume>85</volume><issue>2</issue><pages><first>257</first><last>269</last></pages><genre><json:string>journal</json:string></genre></host><namedEntities><unitex><date><json:string>1996</json:string></date><geogName></geogName><orgName><json:string>Biotech</json:string><json:string>Howard Hughes Medical Institute Massachusetts Institute of Technology</json:string><json:string>National Institutes of Health</json:string><json:string>National Science Foundation Young Investigator Award</json:string><json:string>Massachusetts Institute of Technology</json:string><json:string>United States Public Health Service grant GM499224</json:string></orgName><orgName_funder><json:string>United States Public Health Service grant GM499224</json:string></orgName_funder><orgName_provider></orgName_provider><persName><json:string>Eugene Schuster</json:string><json:string>Alan Engleman</json:string><json:string>Harri Savilahti</json:string><json:string>M. R. Boocock</json:string><json:string>Alan Grossman</json:string><json:string>Robert Sauer</json:string><json:string>David Roth</json:string><json:string>Dan Lee</json:string><json:string>E. Kremenstova</json:string><json:string>Tracy Smith</json:string><json:string>Glen Research</json:string><json:string>Kiyoshi Mizuuchi</json:string><json:string>M. J. McIlwraith</json:string><json:string>D. Pincus</json:string><json:string>Phoebe Rice</json:string><json:string>Tania A. Baker</json:string></persName><placeName><json:string>Discussion Mu Transposase Subunits Bridge</json:string><json:string>Eppendorf</json:string><json:string>MA</json:string></placeName><ref_url></ref_url><ref_bibl><json:string>Craigie and Mizuuchi, 1986</json:string><json:string>Kim and Landy, 1992</json:string><json:string>Bainton et al., 1991</json:string><json:string>Allison and Chaconas, 1992</json:string><json:string>Mizuuchi et al., 1995</json:string><json:string>Boocock et al., 1995</json:string><json:string>Lee et al., 1994</json:string><json:string>Baker et al., 1991</json:string><json:string>Yang and Steitz, 1995a</json:string><json:string>Surette et al., 1987</json:string><json:string>Wu and Chaconas, 1995</json:string><json:string>Benjamin and Kleckner, 1992</json:string><json:string>reviewed by Mizuuchi, 1992b</json:string><json:string>Chen et al., 1992, 1993</json:string><json:string>Duby et al., 1994</json:string><json:string>Craig, 1995</json:string><json:string>Yang and Jayaram, 1994</json:string><json:string>Leung et al., 1989</json:string><json:string>Stark et al., 1992</json:string><json:string>Arciszewska and Sherratt, 1995</json:string><json:string>Lavoie et al., 1991</json:string><json:string>reviewed by Mizuuchi, 1992a</json:string><json:string>Nakayama et al., 1987</json:string><json:string>Yang et al., 1995</json:string><json:string>Baker et al., 1993, 1994</json:string><json:string>Dyda et al., 1994</json:string><json:string>Chen et al., 1992</json:string><json:string>Kaufman and Rio, 1992</json:string><json:string>Zou et al., 1991b</json:string><json:string>Surette et al., 1991</json:string><json:string>Kim et al., 1995</json:string><json:string>Baker and Luo, 1994</json:string><json:string>Fayet et al., 1990</json:string><json:string>Mizuuchi et al., 1992</json:string><json:string>Doak et al., 1994</json:string><json:string>Zou et al., 1991a</json:string><json:string>Willis et al., 1993</json:string><json:string>Bujacz et al., 1995</json:string><json:string>reviewed by Grindley and Leschziner, 1995</json:string><json:string>Surette and Chaconas, 1992</json:string><json:string>Pan et al., 1993</json:string><json:string>Berg and Howe, 1989</json:string><json:string>Levchenko et al., 1995</json:string><json:string>Droge et al., 1990</json:string><json:string>Kulkosky et al.</json:string><json:string>Baker et al., 1993</json:string><json:string>Qian and Cox, 1995</json:string><json:string>Mizuuchi et al., 1991</json:string><json:string>Craigie and Mizuuchi, 1987</json:string><json:string>Schumm and Howe, 1981</json:string><json:string>Groenen et al., 1986</json:string><json:string>Kim and Harshey, 1995</json:string><json:string>Craigie et al., 1984</json:string><json:string>Chaconas et al. (1985)</json:string><json:string>Adzuma and Mizuuchi (1991)</json:string><json:string>Baker and Mizuuchi, 1992</json:string><json:string>Savilahti et al., 1995</json:string><json:string>Rice and Mizuuchi, 1995</json:string><json:string>van Luenen et al., 1994</json:string><json:string>Yang and Steitz, 1995b</json:string></ref_bibl><bibl></bibl></unitex></namedEntities><ark><json:string>ark:/67375/6H6-04P1GBFN-C</json:string></ark><categories><wos><json:string>1 - science</json:string><json:string>2 - cell biology</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 - developmental 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 - General Biochemistry, Genetics and 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></inist></categories><publicationDate>1996</publicationDate><copyrightDate>1996</copyrightDate><doi><json:string>10.1016/S0092-8674(00)81102-2</json:string></doi><id>365354F68B0CD54059610042AED543B886AEC623</id><score>1</score><fulltext><json:item><extension>pdf</extension><original>true</original><mimetype>application/pdf</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-04P1GBFN-C/fulltext.pdf</uri></json:item><json:item><extension>zip</extension><original>false</original><mimetype>application/zip</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-04P1GBFN-C/bundle.zip</uri></json:item><istex:fulltextTEI uri="https://api.istex.fr/ark:/67375/6H6-04P1GBFN-C/fulltext.tei"><teiHeader><fileDesc><titleStmt><title level="a" type="main" xml:lang="en">The Interwoven Architecture of the Mu Transposase Couples DNA Synapsis to Catalysis</title></titleStmt><publicationStmt><authority>ISTEX</authority><publisher scheme="https://scientific-publisher.data.istex.fr">ELSEVIER</publisher><availability><licence><p>©1996 Cell Press</p></licence><p scheme="https://loaded-corpus.data.istex.fr/ark:/67375/XBH-HKKZVM7B-M">elsevier</p></availability><date>1996</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 type="content">Section title: Article</note><note type="content">Figure 1: Schematic of MuA Monomer and Assembly of Strand-Transfer Complex (A) Schematic of the domain structure of Mu transposase. The divisions are based on two protease-hypersensitive sites in the protein. Domain I contains two site-specific DNA binding regions: Domain IA recognizes a sequence required for assembly of transposase under physiological conditions (IAS binding). Domain IB recognizes the end-type MuA binding sites (end binding). Domain II contains the three catalytic residues; determination of the structure of domain II reveals that it is comprised of two subdomains. C-terminal subdomain probably has nonspecific DNA binding activity (NS-DNA binding). Domain IIIA has nonspecific DNA binding and a cryptic nuclease activity. Domain IIIB interacts with two proteins that control the activity of transposase: MuB and ClpX. The transposase derivatives used in this study are shown below the graphic (see text for references). (B) Tetramer formation can be promoted by substrates containing the two right-end binding sites of the Mu genome (donor DNA). A monomer binds to each of the two 22 bp binding sequences located on each substrate. The 3′ terminal A on one strand of each substrate becomes joined to the target DNA by the strand-transfer reaction to create the STC.</note><note type="content">Figure 2: R1* and R2* Substrates Cross-Link to Transposase with Similar Efficiencies (Left) The substrates used for protein–DNA cross-linking. The sequence shown differs slightly from the natural R1 and R2 sequence. The individual oligonucleotides were annealed as described in Experimental Procedures to create the substrates. To make the R1-modified substrate (R1*), oligonucleotides tb237, tb238, and tb239 were annealed with tb225; whereas to make the R2-modified substrate (R2*), oligonucleotides tb227 and tb237 were annealed with tb225. In the R1* and R2* substrates, tb237 was the only oligonucleotide labeled with 32P (shown by the asterisk). The location of IdU is represented by the Us in the sequence. The 3′ adenosine that participates directly in strand transfer is circled. This residue is absent from the unjoinable substrates. (Right) Cross-linking efficiencies were assessed by incubating each substrate with either wild-type transposase (WT), or the ΔDE+ deletion derivative, or a mixture of the two proteins. After UV irradiation, the products were analyzed by SDS–PAGE and autoradiography. A signal of approximately the same intensity was observed in each lane, revealing that similar amounts of labeled DNA have cross-linked to the protein or proteins in each reaction mixture.</note><note type="content">Figure 3: Active Transposase Is Preferentially Bound to the R1 Sites in the STCs (A) Schematic of the experimental design. The inactive subunits are represented by the stippled circles, the active subunits by the clear circles. The thick lines are the Mu end fragments, with the asterisk showing the position of the IdU and 32P. In the first stage, transposase tetramers form, and those with a properly positioned active subunit carry out strand transfer into the target DNA. Irradiation with UV light covalently joins the 32P-labeled short oligonucleotide to the transposase bound specifically to the modified site (asterisk indicates 32P transferred to the protein). The STCs are separated from the inactive tetramers. The ratio of inactive to active transposase labeled with 32P in the total reaction compared with the STCs is determined by SDS–PAGE and autoradiography. (B) and (C) Active and inactive transposase labeled by cross-linking with the R1* (B) and R2* (C) substrates. Forms of transposase present in the reaction mixtures are as marked above the lanes. As expected, very little STC was recovered in reactions that contained only the inactive (DE−) transposase (lanes 4). The numbers on the bottom of each gel allow a quantitative comparison to be made between the percentage of active subunits in the total reaction and that in the STCs.</note><note type="content">Figure 4: Possible Arrangements of Active Subunits Bound to the R1 Positions in Complexes that Carry Out Strand Transfer of One of the Two Mu End Fragments In the graphic, as well as in the following experiment, one of these two ends is only inefficiently joined by strand transfer, because the 3′ end of the donor DNA fragment is missing the terminal adenosine; this end is called UJ. Catalysis in cis refers to the mechanism in which the R1-bound subunit catalyzes the strand transfer of the same Mu end fragment as it is bound. Catalysis in trans refers to the mechanism in which the joining of one Mu end fragment is catalyzed by the R1-bound subunit on the partner fragment. The portion of the R1 monomer that is being assessed for activity is domain II (the DDE motif residues), while cross-linking is probably identifying the end-site binding domain (domain IB).</note><note type="content">Figure 5: Active Transposase Is Preferentially Bound to the R1 Sites on the Unjoined Mu End Fragment (A) Substrates lacking a 3′ terminal A are not efficiently joined to the target DNA by strand transfer. Agarose gel analysis of the products of transposition reactions using the R1*UJ substrate show complexes that disappear upon adding SDS. STC marks the position of the STCs (which comigrate with the nicked form of the target DNA); the fuzzy band that migrates more rapidly than the STC represents the donor–DNA–transposase complexes. The substrates and types of transposase present in each reaction are as shown above the lanes; when the CJ or CUJ substrates were present, they were added at an approximately equal concentration as the labeled substrate. Lanes 1–6 and 7–12 are identical except that in 7–12, the samples were treated with SDS prior to loading the gel; this is also the case with lanes 13–16 (minus SDS) and 17–20 (plus SDS). When the R1*UJ substrate is labeled (left) the only samples with a detectable level of strand-transfer product (which migrates in nearly the same position as the STC in the plus SDS lanes) are the reactions containing the active transposase and no CJ substrate (lanes 7 and 8). In contrast, when a joinable substrate (R1*J) is labeled (right), a similar amount of radioactivity is recovered in the STC, and the strand-transfer products indicating the joinable end are in fact covalently joined to the target DNA. Cross-linking preceded electrophoresis. (B) The forms of transposase labeled by cross-linking with R1*UJ in the samples from the experiment shown in (A). On the left are the samples for the total reaction mixture; on the right are those from the purified STCs. The percent of the transposase in each lane that is the active type (ΔDE+) is shown below each lane. The slow migrating band visible in lane 2 of the STC gel is label trapped at the border of the stacking gel.</note><note type="content">Figure 6: Active Transposase Is Not Needed at the R1 Site on the Joined Mu End Fragment for Strand Transfer R1*J substrate was mixed with increasing concentrations of the CUJ substrate as noted above the lanes. Lanes 1–3 show the forms of transposase labeled by the R1*J substrate in the STCs that contain no unjoinable substrate. Lanes 4–6 show protein profile when the amount of R1*J is held constant while increasing the amount of CUJ being added. The amount of transposase present in these reactions was also increased to compensate for the increase in the total DNA concentration. Nonetheless, the total number of complexes containing labeled substrate DNA decreased with the addition of CUJ DNA; therefore, fewer complexes were recovered.</note><note type="content">Figure 7: Domain III Can Be Donated to the Strand Transfer Complex by the R2-Bound Subunits (A) Graphic illustrating two possible arrangements of inactive subunits that could yield a complex active for strand transfer. The stippled subunits have defective active–site residues in domain II but carry domain III (the DE− subunits in the experiment shown below). The clear subunits carry the active–site residues but lack domain III (574DE+ in the experiment shown below). If domain II and domain III are swapped by the two R1-bound subunits, the mixed tetramers should only be able to catalyze strand transfer of one of the Mu end fragments (left). In contrast, if the R1- and R2-bound subunits swap domain II and domain III, the resulting complex may be able to carry out two strand-transfer reactions, because both R1-bound subunits can be of the DE+ type (right). (B) Mixtures of the 574DE+ and DE− promote strand transfer of two Mu end fragments. Shown is the phosphoimager scan of an agarose gel of the reaction products generated in reactions containing the forms of transposase as marked above the lanes. The donor DNA was a 32P-labeled joinable substrate; the target was unlabeled ΦX174 DNA in the supercoiled circular form. The upper band comigrates with the nicked form of the circular target DNA and was therefore judged to be the product with one of the two ends joined. The lower product migrates near the position of the linearized target DNA, as expected for the product that has two Mu DNA fragments joined to the target. (This gel was run using 1× TBE, which results in better separation of these two products than the standard gel conditions). The percent of the strand-transfer products having two joined ends was determined and is shown below each lane. This experiment contained MuB protein. (C) The R2 position on the joined Mu end fragment is bound by subunits that carry domain III. Reactions were carried out for 2 hr instead of the usual 1 hr because of the weak assembly activity exhibited by 574DE+ and contained MuB protein and ATP. The transposase derivatives and substrates present in each reaction are shown above the lanes.</note><note type="content">Figure 8: Proposed Model of the Mu Transposase Subunits in the STC Domain IB of two subunits binds to the L1 and R1 end-type binding sites near the ends of the Mu genome. Domain II contains the conserved acidic amino acids (ΔDE+) that are part of the active site that catalyzes the strand-transfer reactions. Based on the current analysis, we propose that the subunit bound via domain IB to the left end (L1 site), contributes the active–site residues in domain II for strand transfer of the right end, while the R1-bound subunit promotes strand transfer on the left end. Domain III also serves a vital role in transposition and is donated by the other two subunits in the tetramer. One of these subunits is bound to the R2 site on the right end, whereas the analogous subunit on the left end does not appear to be tightly bound to the DNA; both these subunits are called R2-bound in the figure.</note><note type="content">Table 1: Recovery of Active Subunits at the R1 Position on the Unjoined Mu DNA End in Strand Transfer Complexes Is Not Due to the Influence of Domain III</note><note type="content">Table 2: Cross-Linking of the ΔDE+ Subunits to the R2 Sites: Comparison of the Total Reaction to the Purified Strand Transfer Complex</note></notesStmt><sourceDesc><biblStruct type="inbook"><analytic><title level="a" type="main" xml:lang="en">The Interwoven Architecture of the Mu Transposase Couples DNA Synapsis to Catalysis</title><author xml:id="author-0000"><persName><forename type="first">Hector</forename><surname>Aldaz</surname></persName><affiliation>Department of Biology, Massachusetts Institute of Technology, 68–523, 77 Massachusetts Avenue, Cambridge MA 02139, USA</affiliation></author><author xml:id="author-0001"><persName><forename type="first">Eugene</forename><surname>Schuster</surname></persName><affiliation>Department of Biology, Massachusetts Institute of Technology, 68–523, 77 Massachusetts Avenue, Cambridge MA 02139, USA</affiliation></author><author xml:id="author-0002"><persName><forename type="first">Tania A</forename><surname>Baker</surname></persName><email>tabaker@MIT.EDU</email><note type="correspondence"><p>Correspondence: Tania A. Baker, 617 253 3594 (phone), 617 252 1852 (fax)</p></note><affiliation>Department of Biology, Massachusetts Institute of Technology, 68–523, 77 Massachusetts Avenue, Cambridge MA 02139, USA</affiliation></author><idno type="istex">365354F68B0CD54059610042AED543B886AEC623</idno><idno type="ark">ark:/67375/6H6-04P1GBFN-C</idno><idno type="DOI">10.1016/S0092-8674(00)81102-2</idno><idno type="PII">S0092-8674(00)81102-2</idno></analytic><monogr><title level="j">Cell</title><title level="j" type="abbrev">CELL</title><idno type="pISSN">0092-8674</idno><idno type="PII">S0092-8674(00)X0086-4</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="1996"></date><biblScope unit="volume">85</biblScope><biblScope unit="issue">2</biblScope><biblScope unit="page" from="257">257</biblScope><biblScope unit="page" to="269">269</biblScope></imprint></monogr></biblStruct></sourceDesc></fileDesc><profileDesc><creation><date>1996</date></creation><langUsage><language ident="en">en</language></langUsage><abstract xml:lang="en"><p>Abstract: Mu transposition occurs exclusively using a pair of recombination sites found at the ends of the phage genome. To address the mechanistic basis of this specificity, we have determined both where the individual subunits of the tetrameric transposase bind on the DNA and where they catalyze DNA joining. We demonstrate that subunits do not catalyze recombination at the site adjacent to where they are bound, but rather on the opposite end of the phage genome. Furthermore, subunits bound to two different sites contribute to catalysis of one reaction step. This interwoven subunit arrangement suggests a molecular explanation for the precision with which recombination occurs using a pair of DNA signals and provides an example of the way in which the architecture of a protein–DNA complex can define the reaction products.</p></abstract></profileDesc><revisionDesc><change when="1996-03-08">Modified</change><change when="1996">Published</change></revisionDesc></teiHeader></istex:fulltextTEI><json:item><extension>txt</extension><original>false</original><mimetype>text/plain</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-04P1GBFN-C/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="gr4" NDATA="IMAGE" name="gr4"></istex:entity><istex:entity SYSTEM="gr5" NDATA="IMAGE" name="gr5"></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="gr8" NDATA="IMAGE" name="gr8"></istex:entity></istex:docType><istex:document><converted-article version="4.5.2" docsubtype="fla" xml:lang="en"><item-info><jid>CELL</jid><aid>146</aid><ce:pii>S0092-8674(00)81102-2</ce:pii><ce:doi>10.1016/S0092-8674(00)81102-2</ce:doi><ce:copyright type="other" year="1996">Cell Press</ce:copyright></item-info><head><ce:dochead><ce:textfn>Article</ce:textfn></ce:dochead><ce:title>The Interwoven Architecture of the Mu Transposase Couples DNA Synapsis to Catalysis</ce:title><ce:author-group><ce:author><ce:given-name>Hector</ce:given-name><ce:surname>Aldaz</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>1</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>Eugene</ce:given-name><ce:surname>Schuster</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>1</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>Tania A</ce:given-name><ce:surname>Baker</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>1</ce:sup></ce:cross-ref><ce:cross-ref refid="AFF2"><ce:sup>2</ce:sup></ce:cross-ref><ce:cross-ref refid="COR1">*</ce:cross-ref><ce:e-address>tabaker@MIT.EDU</ce:e-address></ce:author><ce:affiliation id="AFF1"><ce:label>1</ce:label><ce:textfn>Department of Biology, Massachusetts Institute of Technology, 68–523, 77 Massachusetts Avenue, Cambridge MA 02139, USA</ce:textfn></ce:affiliation><ce:affiliation id="AFF2"><ce:label>2</ce:label><ce:textfn>Howard Hughes Medical Institute, Massachusetts Institute of Technology, 68–523, 77 Massachusetts Avenue, Cambridge MA 02139, USA</ce:textfn></ce:affiliation><ce:correspondence id="COR1"><ce:label>*</ce:label><ce:text>Correspondence: Tania A. Baker, 617 253 3594 (phone), 617 252 1852 (fax)</ce:text></ce:correspondence></ce:author-group><ce:date-received day="19" month="1" year="1996"></ce:date-received><ce:date-revised day="8" month="3" year="1996"></ce:date-revised><ce:abstract><ce:section-title>Abstract</ce:section-title><ce:abstract-sec><ce:simple-para>Mu transposition occurs exclusively using a pair of recombination sites found at the ends of the phage genome. To address the mechanistic basis of this specificity, we have determined both where the individual subunits of the tetrameric transposase bind on the DNA and where they catalyze DNA joining. We demonstrate that subunits do not catalyze recombination at the site adjacent to where they are bound, but rather on the opposite end of the phage genome. Furthermore, subunits bound to two different sites contribute to catalysis of one reaction step. This interwoven subunit arrangement suggests a molecular explanation for the precision with which recombination occurs using a pair of DNA signals and provides an example of the way in which the architecture of a protein–DNA complex can define the reaction products.</ce:simple-para></ce:abstract-sec></ce:abstract></head></converted-article></istex:document></istex:metadataXml><mods version="3.6"><titleInfo lang="en"><title>The Interwoven Architecture of the Mu Transposase Couples DNA Synapsis to Catalysis</title></titleInfo><titleInfo type="alternative" lang="en" contentType="CDATA"><title>The Interwoven Architecture of the Mu Transposase Couples DNA Synapsis to Catalysis</title></titleInfo><name type="personal"><namePart type="given">Hector</namePart><namePart type="family">Aldaz</namePart><affiliation>Department of Biology, Massachusetts Institute of Technology, 68–523, 77 Massachusetts Avenue, Cambridge MA 02139, USA</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Eugene</namePart><namePart type="family">Schuster</namePart><affiliation>Department of Biology, Massachusetts Institute of Technology, 68–523, 77 Massachusetts Avenue, Cambridge MA 02139, USA</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Tania A</namePart><namePart type="family">Baker</namePart><affiliation>E-mail: tabaker@MIT.EDU</affiliation><affiliation>Department of Biology, Massachusetts Institute of Technology, 68–523, 77 Massachusetts Avenue, Cambridge MA 02139, USA</affiliation><description>Correspondence: Tania A. Baker, 617 253 3594 (phone), 617 252 1852 (fax)</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">1996</dateIssued><dateModified encoding="w3cdtf">1996-03-08</dateModified><copyrightDate encoding="w3cdtf">1996</copyrightDate></originInfo><language><languageTerm type="code" authority="iso639-2b">eng</languageTerm><languageTerm type="code" authority="rfc3066">en</languageTerm></language><abstract lang="en">Abstract: Mu transposition occurs exclusively using a pair of recombination sites found at the ends of the phage genome. To address the mechanistic basis of this specificity, we have determined both where the individual subunits of the tetrameric transposase bind on the DNA and where they catalyze DNA joining. We demonstrate that subunits do not catalyze recombination at the site adjacent to where they are bound, but rather on the opposite end of the phage genome. Furthermore, subunits bound to two different sites contribute to catalysis of one reaction step. This interwoven subunit arrangement suggests a molecular explanation for the precision with which recombination occurs using a pair of DNA signals and provides an example of the way in which the architecture of a protein–DNA complex can define the reaction products.</abstract><note type="content">Section title: Article</note><note type="content">Figure 1: Schematic of MuA Monomer and Assembly of Strand-Transfer Complex (A) Schematic of the domain structure of Mu transposase. The divisions are based on two protease-hypersensitive sites in the protein. Domain I contains two site-specific DNA binding regions: Domain IA recognizes a sequence required for assembly of transposase under physiological conditions (IAS binding). Domain IB recognizes the end-type MuA binding sites (end binding). Domain II contains the three catalytic residues; determination of the structure of domain II reveals that it is comprised of two subdomains. C-terminal subdomain probably has nonspecific DNA binding activity (NS-DNA binding). Domain IIIA has nonspecific DNA binding and a cryptic nuclease activity. Domain IIIB interacts with two proteins that control the activity of transposase: MuB and ClpX. The transposase derivatives used in this study are shown below the graphic (see text for references). (B) Tetramer formation can be promoted by substrates containing the two right-end binding sites of the Mu genome (donor DNA). A monomer binds to each of the two 22 bp binding sequences located on each substrate. The 3′ terminal A on one strand of each substrate becomes joined to the target DNA by the strand-transfer reaction to create the STC.</note><note type="content">Figure 2: R1* and R2* Substrates Cross-Link to Transposase with Similar Efficiencies (Left) The substrates used for protein–DNA cross-linking. The sequence shown differs slightly from the natural R1 and R2 sequence. The individual oligonucleotides were annealed as described in Experimental Procedures to create the substrates. To make the R1-modified substrate (R1*), oligonucleotides tb237, tb238, and tb239 were annealed with tb225; whereas to make the R2-modified substrate (R2*), oligonucleotides tb227 and tb237 were annealed with tb225. In the R1* and R2* substrates, tb237 was the only oligonucleotide labeled with 32P (shown by the asterisk). The location of IdU is represented by the Us in the sequence. The 3′ adenosine that participates directly in strand transfer is circled. This residue is absent from the unjoinable substrates. (Right) Cross-linking efficiencies were assessed by incubating each substrate with either wild-type transposase (WT), or the ΔDE+ deletion derivative, or a mixture of the two proteins. After UV irradiation, the products were analyzed by SDS–PAGE and autoradiography. A signal of approximately the same intensity was observed in each lane, revealing that similar amounts of labeled DNA have cross-linked to the protein or proteins in each reaction mixture.</note><note type="content">Figure 3: Active Transposase Is Preferentially Bound to the R1 Sites in the STCs (A) Schematic of the experimental design. The inactive subunits are represented by the stippled circles, the active subunits by the clear circles. The thick lines are the Mu end fragments, with the asterisk showing the position of the IdU and 32P. In the first stage, transposase tetramers form, and those with a properly positioned active subunit carry out strand transfer into the target DNA. Irradiation with UV light covalently joins the 32P-labeled short oligonucleotide to the transposase bound specifically to the modified site (asterisk indicates 32P transferred to the protein). The STCs are separated from the inactive tetramers. The ratio of inactive to active transposase labeled with 32P in the total reaction compared with the STCs is determined by SDS–PAGE and autoradiography. (B) and (C) Active and inactive transposase labeled by cross-linking with the R1* (B) and R2* (C) substrates. Forms of transposase present in the reaction mixtures are as marked above the lanes. As expected, very little STC was recovered in reactions that contained only the inactive (DE−) transposase (lanes 4). The numbers on the bottom of each gel allow a quantitative comparison to be made between the percentage of active subunits in the total reaction and that in the STCs.</note><note type="content">Figure 4: Possible Arrangements of Active Subunits Bound to the R1 Positions in Complexes that Carry Out Strand Transfer of One of the Two Mu End Fragments In the graphic, as well as in the following experiment, one of these two ends is only inefficiently joined by strand transfer, because the 3′ end of the donor DNA fragment is missing the terminal adenosine; this end is called UJ. Catalysis in cis refers to the mechanism in which the R1-bound subunit catalyzes the strand transfer of the same Mu end fragment as it is bound. Catalysis in trans refers to the mechanism in which the joining of one Mu end fragment is catalyzed by the R1-bound subunit on the partner fragment. The portion of the R1 monomer that is being assessed for activity is domain II (the DDE motif residues), while cross-linking is probably identifying the end-site binding domain (domain IB).</note><note type="content">Figure 5: Active Transposase Is Preferentially Bound to the R1 Sites on the Unjoined Mu End Fragment (A) Substrates lacking a 3′ terminal A are not efficiently joined to the target DNA by strand transfer. Agarose gel analysis of the products of transposition reactions using the R1*UJ substrate show complexes that disappear upon adding SDS. STC marks the position of the STCs (which comigrate with the nicked form of the target DNA); the fuzzy band that migrates more rapidly than the STC represents the donor–DNA–transposase complexes. The substrates and types of transposase present in each reaction are as shown above the lanes; when the CJ or CUJ substrates were present, they were added at an approximately equal concentration as the labeled substrate. Lanes 1–6 and 7–12 are identical except that in 7–12, the samples were treated with SDS prior to loading the gel; this is also the case with lanes 13–16 (minus SDS) and 17–20 (plus SDS). When the R1*UJ substrate is labeled (left) the only samples with a detectable level of strand-transfer product (which migrates in nearly the same position as the STC in the plus SDS lanes) are the reactions containing the active transposase and no CJ substrate (lanes 7 and 8). In contrast, when a joinable substrate (R1*J) is labeled (right), a similar amount of radioactivity is recovered in the STC, and the strand-transfer products indicating the joinable end are in fact covalently joined to the target DNA. Cross-linking preceded electrophoresis. (B) The forms of transposase labeled by cross-linking with R1*UJ in the samples from the experiment shown in (A). On the left are the samples for the total reaction mixture; on the right are those from the purified STCs. The percent of the transposase in each lane that is the active type (ΔDE+) is shown below each lane. The slow migrating band visible in lane 2 of the STC gel is label trapped at the border of the stacking gel.</note><note type="content">Figure 6: Active Transposase Is Not Needed at the R1 Site on the Joined Mu End Fragment for Strand Transfer R1*J substrate was mixed with increasing concentrations of the CUJ substrate as noted above the lanes. Lanes 1–3 show the forms of transposase labeled by the R1*J substrate in the STCs that contain no unjoinable substrate. Lanes 4–6 show protein profile when the amount of R1*J is held constant while increasing the amount of CUJ being added. The amount of transposase present in these reactions was also increased to compensate for the increase in the total DNA concentration. Nonetheless, the total number of complexes containing labeled substrate DNA decreased with the addition of CUJ DNA; therefore, fewer complexes were recovered.</note><note type="content">Figure 7: Domain III Can Be Donated to the Strand Transfer Complex by the R2-Bound Subunits (A) Graphic illustrating two possible arrangements of inactive subunits that could yield a complex active for strand transfer. The stippled subunits have defective active–site residues in domain II but carry domain III (the DE− subunits in the experiment shown below). The clear subunits carry the active–site residues but lack domain III (574DE+ in the experiment shown below). If domain II and domain III are swapped by the two R1-bound subunits, the mixed tetramers should only be able to catalyze strand transfer of one of the Mu end fragments (left). In contrast, if the R1- and R2-bound subunits swap domain II and domain III, the resulting complex may be able to carry out two strand-transfer reactions, because both R1-bound subunits can be of the DE+ type (right). (B) Mixtures of the 574DE+ and DE− promote strand transfer of two Mu end fragments. Shown is the phosphoimager scan of an agarose gel of the reaction products generated in reactions containing the forms of transposase as marked above the lanes. The donor DNA was a 32P-labeled joinable substrate; the target was unlabeled ΦX174 DNA in the supercoiled circular form. The upper band comigrates with the nicked form of the circular target DNA and was therefore judged to be the product with one of the two ends joined. The lower product migrates near the position of the linearized target DNA, as expected for the product that has two Mu DNA fragments joined to the target. (This gel was run using 1× TBE, which results in better separation of these two products than the standard gel conditions). The percent of the strand-transfer products having two joined ends was determined and is shown below each lane. This experiment contained MuB protein. (C) The R2 position on the joined Mu end fragment is bound by subunits that carry domain III. Reactions were carried out for 2 hr instead of the usual 1 hr because of the weak assembly activity exhibited by 574DE+ and contained MuB protein and ATP. The transposase derivatives and substrates present in each reaction are shown above the lanes.</note><note type="content">Figure 8: Proposed Model of the Mu Transposase Subunits in the STC Domain IB of two subunits binds to the L1 and R1 end-type binding sites near the ends of the Mu genome. Domain II contains the conserved acidic amino acids (ΔDE+) that are part of the active site that catalyzes the strand-transfer reactions. Based on the current analysis, we propose that the subunit bound via domain IB to the left end (L1 site), contributes the active–site residues in domain II for strand transfer of the right end, while the R1-bound subunit promotes strand transfer on the left end. Domain III also serves a vital role in transposition and is donated by the other two subunits in the tetramer. One of these subunits is bound to the R2 site on the right end, whereas the analogous subunit on the left end does not appear to be tightly bound to the DNA; both these subunits are called R2-bound in the figure.</note><note type="content">Table 1: Recovery of Active Subunits at the R1 Position on the Unjoined Mu DNA End in Strand Transfer Complexes Is Not Due to the Influence of Domain III</note><note type="content">Table 2: Cross-Linking of the ΔDE+ Subunits to the R2 Sites: Comparison of the Total Reaction to the Purified Strand Transfer Complex</note><relatedItem type="host"><titleInfo><title>Cell</title></titleInfo><titleInfo type="abbreviated"><title>CELL</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">1996</dateIssued></originInfo><identifier type="ISSN">0092-8674</identifier><identifier type="PII">S0092-8674(00)X0086-4</identifier><part><date>1996</date><detail type="volume"><number>85</number><caption>vol.</caption></detail><detail type="issue"><number>2</number><caption>no.</caption></detail><extent unit="issue-pages"><start>137</start><end>290</end></extent><extent unit="pages"><start>257</start><end>269</end></extent></part></relatedItem><identifier type="istex">365354F68B0CD54059610042AED543B886AEC623</identifier><identifier type="ark">ark:/67375/6H6-04P1GBFN-C</identifier><identifier type="DOI">10.1016/S0092-8674(00)81102-2</identifier><identifier type="PII">S0092-8674(00)81102-2</identifier><accessCondition type="use and reproduction" contentType="copyright">©1996 Cell 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>Cell Press, ©1996</recordOrigin></recordInfo></mods><json:item><extension>json</extension><original>false</original><mimetype>application/json</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-04P1GBFN-C/record.json</uri></json:item></metadata><serie></serie></istex></record>Pour manipuler ce document sous Unix (Dilib)
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