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Downloaded from symposium.cshlp.org on December 20, 2012 - Published by Cold Spring Harbor Laboratory Press Escherichia coli Type-1 Topoisomerases: Identification, Mechanism, and Role in Recombination F. Dean, M.A. Krasnow, R. Otter, et al. Cold Spring Harb Symp Quant Biol 1983 47: 769-777 Access the most recent version at doi:10.1101/SQB.1983.047.01.088 References This article cites 45 articles, 19 of which can be accessed free at: http://symposium.cshlp.org/content/47/769.refs.html Article cited in: http://symposium.cshlp.org/content/47/769#related-urls Email alerting service Receive free email alerts when new articles cite this article - sign up in the box at the top right corner of the article or click here To subscribe to Cold Spring Harbor Symposia on Quantitative Biology go to: http://symposium.cshlp.org/subscriptions Copyright © 1983 Cold Spring Harbor Laboratory Press Downloaded from symposium.cshlp.org on December 20, 2012 - Published by Cold Spring Harbor Laboratory Press Escherichia coli Type-I Topoisomerases: Identification, Mechanism, and Role in Recombination F. DEAN,* M.A. KRASNOW,* R. OTTER,* M.M. MATZUK,? S.J. SPENGLER,t AND N.R. COZZARELLI? *Department of Biochemistry, University of Chicago, Chicago, Illinois 60637; ?Department of Molecular Biology, University of California, Berkeley, California 94720 Topoisomerases catalyze the concerted breakage and reunion of DNA strands. The energy of the broken phosphodiester bond is conserved in a covalent linkage of the enzyme to DNA, allowing the break to be resealed in the absence of an external energy source. Topoisomerases carefully control the breakage and reunion process, never leaving the DNA while it is broken. These enzymes carry out diverse reactions including supercoiling, catenation, knotting, recombination, and aspects of DNA replication. Type-2 topoisomerases make a transient double-stranded break and pass duplex DNA through the break. This changes the topological linking number of DNA (Lk) in steps of two, as explained by the sign-inversion mechanism (Brown and Cozzarelli 1979; Liu et al. 1980; Gellert 1981). Type-1 topoisomerases, on the other hand, break only one strand at a time and alter the Lk in steps of one (Champoux 1978; Wang and Liu 1979; Cozzarelli 1980b). In this paper we focus on the type-1 topoisomerases of Escherichia coli, enzymes that have recently received increased attention. Five topics are considered. First, in the analysis of the electrophoretic mobility of negatively supercoiled topoisomers, we discovered that adjacent DNA bands on a gel need not differ by one in linking number. Second, we identified a new E. coli topoisomerase, topoisomerase III. Third, the mechanism of catenation of duplex DNA rings by E. coli topoisomerase I has been elucidated. Fourth, we describe the involvement of a type- 1 topoisomerase, the resolvase of Tn3, in site-specific recombination. In catenation and recombination by type-I topoisomerases, duplex DNA is topologically rearranged via the breakage and rejoining of DNA a single strand at a time. Fifth, we consider what cellular processes might be particularly suited for the involvement of type-l, but not type-2, topoisomerases. composite acrylamide-agarose gel (Peacock and Dingman 1968). Nearly all of the topoisomers from fully relaxed to native are resolved well by a single gel, and thus a small change in topoisomer distribution can be discerned. Using this high-resolution system, we discovered that neighboring electrophoretic bands do not always differ in Lk by one. As the Lk of pAO3 DNA is decreased, topoisomer mobility initially increases, as expected. However, as the number of titratable negative supercoils increases beyond seven, there is a reduction in electrophoretic mobility (Fig. 1). The reduction is not a general consequence of an increase in superhelical density beyond a critical point, because as greater numbers of positive supercoils are introduced, electrophoretic mobility continues to rise (Fig. 1). We suggest instead that increased underwinding stabilizes a DNA structure such as a cruciform (Lilley 1980; Panayotatos and Wells 1981). Since extrusion of a DNA segment into a cruciform reduces the length of the DNA in the circle while I i [ I Positive 20 I I .,tl" j- 15 N e g o t i v e ~ /,, ,o5 /~',' f" o I 0 Assay of Topoisomerases and the Relationship of Linking Number to Electrophoretic Mobility I 25 t 2 I 4 I 6 ILko-Lkl I 8 I I0 I 12 Figure t. Electrophoretic mobility of plasmid pAO3 topoisomers. Samples of the 1683-bp pAO3 DNA (Oka et al. 1979) were prepared containing various amounts of positive (0) and negative (9 supercoils. After electrophoresis in a composite acrylamideagarose gel (Peacockand Dingman 1968), the DNA was visualized by ethidium bromide staining. The absolute value of the difference in the Lk between relaxed and topoisomer DNAs (ILko-Lkl) is plotted vs. electrophoretic mobility in arbitrary units, with a mobility of zero corresponding to the position of nicked DNA. Topoisomer assignments were confirmed using two-dimensional gel electrophoresis in which the second dimension was in the presence of the intercalating dye, chloroquine (Shure et al. 1977). Topoisomerases are usually assayed by the relaxation of supercoils in a plasmid, as measured by the production of topoisomers with reduced electrophoretic mobility (Crick et al. 1979). However, individual topoisomers of native plasmids in the size range usually employed (4-6 kb) are not well resolved by agarose gel electrophoresis, and only a large reduction in supertwist density can be detected. For a sensitive assay of relaxation, we have used the 1683-bp plasmid, pAO3, and a 769 Downloaded from symposium.cshlp.org on December 20, 2012 - Published by Cold Spring Harbor Laboratory Press 770 D E A N ET AL. Table 1. E. coli Topoisomerases Name I (o~) Structure II (gyrase) a2~2 II' cl2~2 topA gyrA, gyrB gyrA, gyrB? ~1 c~2 tnpR III Resolvase c~1 Gene ? Protomer molecular weight ( • 10-3) Type Relaxes positive supertwists 101 105; 95 105; 50 75 1 2 2 1 no no yes ? + + - + - 21 1 ? - - Inhibited by Nal Novo The a and Bdesignationsfor protomercompositiondo not indicategeneticrelatedness(shownin a separatecolumn). The structuresindicatedfor topoisomeraseHI and resolvaseare likely,but not as rigorouslydemonstratedas for the otherenzymes.OnlyE. coli strainsthatharborTn3, 3,6,or related transposonshave resolvase.Excludedfrom the table are phage-encodedtopoisomerases.Nal and Novoindicatenalidixicacidand novobiocin,respectively. the Lk remains constant, the negative superhelical density of the DNA in the circle drops. The DNA is then less compact and migrates more slowly than a topoisomer that is less underwound. A New E. coli Topoisomerase The properties of the three previously identified E. coli topoisomerases are summarized in Table 1. Topo- isomerase I (co protein) is a 101,000-dalton polypeptide encoded by topA (Wang and Liu 1979; Sternglanz et al. 1981; Trucksis and Depew 1981). It is a type-1 enzyme that relaxes negatively supercoiled DNA. Topoisomerase II (DNA gyrase) is a type-2 enzyme that introduces negative supercoils into DNA at the expense of ATP and slowly removes them in the absence of ATP (Cozzarelli 1980a). The subunit encoded by gyrA dictates the sensitivity of gyrase to nalidixic acid. The gyrB-coded subunit is responsible for ATP binding and is the target for novobiocin. Topoisomerase I I ' (Brown et al. 1979; Gellert et al. 1979) contains the gyrA subunit and is therefore sensitive to nalidixic acid. It also contains a subunit that is homologous to only part of the B subunit and cannot introduce negative supercoils. It is a type-2 topoisomerase that is unique among E. coli topoisomerases in its ability to relax both negatively and positively supercoiled DNAs (Brown et al. 1979). We have discovered a new topoisomerase, topoisomerase III. The enzyme is a type-1 topoisomerase that relaxes negatively supercoiled DNA. Electrophoresis of highly purified material in the presence of SDS reveals predominantly a pair of polypeptides, 77,000 and 75,000 daltons, that comprise 80% of the preparation. The ratio of the two varies in separate prepara- tions. They are the only polypeptides coincident with activity when the enzyme is analyzed by velocity sedimentation. The identity of their partial proteolysis patterns (Cleveland et al. 1977) is good evidence that they are encoded by a single gene and differ only in processing. The sedimentation value of 5.0S is consistent with a molecular weight of about 65,000 (Martin and Ames 1961), and thus topoisomerase III very likely consists of a single polypeptide of about 75,000 daltons. Several characteristics distinguish topoisomerase III from topoisomerase I. First, antibodies to topoisomerase I (Trucksis and Depew 1981) do not inhibit topoisomerase III. Second, topoisomerase III is present in a topA deletion strain, DM800 (Sternglanz et al. 1981), that has no detectable topoisomerase I activity. DM800 is a convenient strain for the purification of topoisomerase HI because the two enzymes behave similarly in several purification steps. Third, the partial proteolysis patterns of the topoisomerases are entirely different. Fourth, the two enzymes have different sequence specificity. Incubation of topoisomerase I or III with DNA in the absence of Mg §247followed by addition of SDS, resulted in cleavage of the DNA and attachment of the enzyme to the resulting broken end (Liu and Wang 1979). The cleavage sites were mapped using singlestranded [5'-32p]DNA. Display of the cleavage products by polyacrylamide gel electrophoresis showed no common cut sites (Fig. 2). Thirty topoisomerase I cleavage sites on single-stranded DNA have been sequenced (Tse et al. 1980; F. Dean and N. Cozzarelli, unpubl.). All have a cytosine residue four nucleotides to the 5 ' side of the break (Fig. 3). The breaks produced by topoisomerase III lack cytosine in this position. Topoisomerase III is clearly distinguished from topoisomerases II and I I ' on the basis that it is type 1, insen- Figure 2. Single-strandedDNA cleavage specificity of topoisomerases I and III. The substrate was a 687-base fragment of single-stranded DNA prepared from q~X DNA. Reactions (10 #1) contained 30% glycerol, 20 nu4 Tris-HCl (pH 7.6), 20 mM KCI, 0.5 nua dithiothreitol, 50 #g/ml of serum albumin, [5 '-a2p]DNA, and 15 ng of topoisomerase I or 6 ng of topoisomerase III. After 25 min at 52~ SDS was added to 1%. Samples were analyzed by polyacrylamide (12.5%) gel electrophoresis (Maxam and Gilbert 1980). Downloaded from symposium.cshlp.org on December 20, 2012 - Published by Cold Spring Harbor Laboratory Press E. COLI TYPE-1 TOPOISOMERASES cut| site 5' -6 -5 -4-3 -2 -I ~, I 2 3 4 A 8 5 0 713 G 5 16 0 6 C 8 130 9 T 9 8 08 9 II 9 li 7 7 :> 5 6 6 9 2 I0 2 6 8 6 8 912 9 5 8 4 3j 3' [ nicked /o.A S = Figure 3. Topoisomerase I cleavage sequences for single-stranded DNA. The incidence of nucleotides around 30 topolsomerase I cleavage sites (Tse et al. 1980; F. Dean and N. Cozzarelli, unpubl.) are compiled. The common attribute is the presence of a cytosine residue four nucleotides to the 5' side of the cut site. Z ,! O( sitive to nalidixic acid and ATP, and much smaller. Topoisomerase III is therefore a new topoisomerase; its properties are summarized in Table 1. It has approximately one quarter of the supercoil relaxation activity of topoisomerase I and is the second most active E. coil topoisomerase. Mechanism of Type-I Topoisomerases The discovery of catenation and knotting of intact duplex DNA rings provided strong evidence for the sign-inversion model for type-2 topoisomerases because these reactions require the passage of duplex DNA segments through each other (Brown and Cozzarelli 1979; Liu et al. 1980). The simple connection of these reactions to type-2 topoisomerases was unsettled by the discovery that type-1 enzymes could also catenate and knot DNA (Tse and Wang 1980; Brown and Cozzarelli 1981). The resolution of this paradox comes from a consideration of DNA substrate specificity. Catenation requires a donor circle and a recipient circle that is transiently broken by the enzyme. For type-1 enzymes, the recipient must be nicked, whereas the donor can be supercoiled, nicked, or relaxed (Tse and Wang 1980; Brown and Cozzarelli 1981). Neither specificity is expected for topoisomerase I, which binds preferentially to negatively supercoiled DNA (Liu and Wang 1979). However, a simple scheme explains these requirements (see Fig. 4). We postulate that a type-1 topoisomerase introduces a transient single-stranded break opposite the 771 0 intact , 0 30 Total~, final , I0 Figure 5. Binding of topoisomerase I to DNA. ColEI DNA was treated with DNase I in the presence of ethidium bromide to introduce approximately one nick per molecule (Greenfield et al. 1975). Both the nicked DNA ( 9 and intact native ColE1 DNA (O) were linearized with EcoRI restriction enzyme and end-labeled using [7-32p]ATP and polynucleotide kinase. The 10-/zl reactions containing 30% glycerol, 20 mM Tris-HC1 (pH 7.6), 20 mM KC1, 0.5 mM dithiothreitol, 50 #g/ml of serum albumin, 6.8 fmoles of DNA, and topoisomerase I (~0) were incubated at 37~ for 25 min. Reactions were diluted 100-fold in binding buffer (Fennewald et al. 1981) and applied dropwise to a nitrocellulose filter (BA85; Schleicher and Schuell). The filters were washed with 1 ml of binding buffer, dried, and counted. nick in the recipient ring. The donor ring passes through the resultant double-strand interruption; resealing the nick forms a catenane. We demonstrate below that the postulated operation of a type-1 enzyme opposite a nick is correct. Topoisomerase I has a higher affinity for singly nicked plasmid DNA than for intact DNA (Fig. 5). Fivefold more enzyme is required to bind 1 fmole of intact DNA to a nitrocellulose filter than is required to bind 1 fmole of nicked molecules. Thus, a single nick in a 6.6-kb DNA creates an opportunity for a novel interaction with topoisomerase I. The binding to a nick cannot be a kinetic dead end because it directs cleavage to the region. An 1108-bp fragment of ~bX174 DNA was prepared with 32p at the 5' end of the viral ( + ) strand and a single nick in the 9 \ bindopposite nick Figure 4. Model for catenation by type-1 topoisomerases. The enzyme, depicted as a C-shaped structure, binds to a nick in a recipient DNA ring. The strand opposite the nick is cleaved, with the 5' end becoming covalently bound to the enzyme and the 3' end remaining bound noncovalently. A segment from a donor DNA ring passes through the break created by the enzyme. The transient nick in the recipient DNA circle is resealed, and the DNA is released from the enzyme. passDNA~ throughbreak ( ~ sealbreak Q ) ~ Downloaded from symposium.cshlp.org on December 20, 2012 - Published by Cold Spring Harbor Laboratory Press 772 D E A N ET AL. Hphl Ava11" HaeTr Figure 6. Uniquely nicked ~X DNA. ~X replicative form-I DNA linearized at the unique Avail site was mixed with a threefold molar excess of ~X viral ( + ) single-stranded circles. The DNA was denatured by dialysis against 95% formamide and 10 mM EDTA (pH 8.3) for 1 hr at 22"C and renatured by dialysis first against 50% formamide, 0.2 MTris-HCl (pH 8.0), and 20 rnM EDTA for 3 hr and then against 0.1 MNaCl.and 10 rnM EDTA (pH 8.0) for 2 hr (Clayton et al. 1970). The resulting nicked duplex DNA circles were purified by agarose gel electrophoresis. Purified nicked circles were digested with HphI restriction nuclease and endlabeled using h,-32P]ATP and polynucleotide kinase. The DNA was then cut with HaelI nuclease, and the 1108-bp fragment containing the Avail restriction enzyme site was isolated. complementary ( - ) strand 70 bases from the labeled end (Fig. 6). Cleavages made in the viral strand by topoisomerase I were displayed by gel electrophoresis (see Fig. 7). The exact position of the break was determined by comparison with the mobility of standards produced by chemical cleavage (Maxam and Gilbert 1980) and by restriction enzyme digestion. Only one cleavage was seen and it was three nucleotides from the nick. When the substrate was denatured prior to treatment with topoisomerase I, at least 20 cleavage fragments were produced, including the one seen with the nicked duplex fragment. Unnicked duplex DNA was not cleaved under these conditions. Under more vigorous cleavage conditions, other breaks are made by topoisomerase I (Fig. 8), but all still cluster in the area of the nick. The nick does not have a rigid directing effect on cleavage, and thus the two breaks do not possess a unique stagger as they do with gyrase cleavage (Morrison and Cozzarelli 1979). In every case a cytosine residue is four nucleotides to the 5 ' side of the break. Since the same sequence specificity is observed with single-stranded DNA and nicked DNA, we conclude that the enzyme recognizes a similar conformation in each case. The strong binding affinity of topoisomerase I for negatively supercoiled DNA is likely also due to its preference for an unwound region. We have suggested a model for relaxation of negative supercoils by topoisomerase I (Brown and Cozzarelli 1981) that is consistent with the data on binding and catenation (see Fig. 9). After the unpairing o f the strands by enzyme binding, one strand can be passed through a transient break in the other. This will change the Lk in steps of one. The cleaved strand and the passing strand are not base-paired in this one-strand signinversion mechanism. This is necessary because catenation is unaffected by sequence complementarity. In fact, topoisomerase I has little specificity for the DNA segment that is passed through a break; it allows a single strand in the process of relaxation, but it can pass nicked, relaxed, or supercoiled duplex DNA in catenation. Figure 7. Specificity of DNA cleavage by topoisomerase I. A 5'-end-labeled DNA fragment with a site-specific nick (Fig. 6) was incubated in the presence ( + ) or absence ( - ) of enzyme (E) as described in Fig. 5. Cleavage was induced by the addition of SDS to 1% (Liu and Wang 1979). Sequencing ladders were created by chemical cleavages (Maxam and Gilbert 1980) at the residues indicated at the top of the first three lanes. The two bands in the - E lane are from restriction by Avail and Hphl enzymes and provide 65-bp and 70-bp length standards. The additional band created by topoisomerase I has the 3'-terminal sequence pApCpCpTpT. 5'-A- C-c- T- T!G-C-T- - T - G pG - A - A - C-G-A- G-C~-T-A_ C-G- A-T-T- A~- A _ T-5' Figure 8. Topoisomerase I cleavage sites near a specific nick in DNA. The substrate for cleavage by topoisomerase I is shown in Fig. 6. Thin arrows indicate the cleavage sites, and the heavy arrow indicates the preferred site. There is a cytosine residue four nucleotides to the 5' side of each cut. Downloaded from symposium.cshlp.org on December 20, 2012 - Published by Cold Spring Harbor Laboratory Press E. COLI TYPE-1 TOPOISOMERASES 773 k nick ) ~/ tnp R) ~~ ligote~~ Figure 9. A one-strand sign-inversion mechanism for type-1 topoisomerases. Topoisomerase I binds the DNA such that the double helix is unwoundand a node is formed by the crossingof the two singlestrands. The enzymetransientlynicks one strand, remains bound to each broken end, and passes the other strand through the break. Sealingthe break results in a change in linking fixed at +l. Role of a Type-1 Topoisomerase in Recombination A more complex case of controlled breakage and reunion occurs in recombination where the segments flanking a pair of double-strand breaks are rearranged before rejoining. There is evidence that a topoisomerase can be responsible for this central event in recombination. The bacteriophage X Int protein is necessary for the site-specific recombination that mediates the integration and excision of k DNA from the E. coli chromosome. Int is a true topoisomerase since it removes supercoils from circular plasmid DNA (Kikachi and Nash 1979). We have begun a study of the breakage and reunion reaction of a site-specific recombination involved in DNA transposition. This is part of our continuing study RSFI050 I , F ~ r e 10. Role of Tn3 genes in transposition. The transposition of Tm~(heavyarrow) from one replicon( ) m another ( ~,~ ) is shown, t~A and mpR are the only required Tn3 genes, tnpA is needed for formation of a cointegrate structure in which the two replicons are joined by Tm~ bridges, and tnpR is necessary for resolution of the two replicons. The final products are catenated here, as in vitro, but they are found separated in vivo. (Fennewald et al. 1981) of the mechanism of transposition of the E. coli transposon, Tn3 (Heffron 1982). There are two steps in the transposition of Tn3 from one replicon to another (Fig. 10) (Shapiro 1979). The first is the complex event in which the transposon is duplicated and the replicons are fused via directly repeated transposon bridges. Cointegrate formation requires the product of a single Tn3 gene, tnpA, and unidentified host factors. The second step is a site-specific recombination between the transposons that regenerates the two replicons, each now containing Tn3. The second reaction has been shown for the related transposon, -y~i, to require only the product of the tnpR gene, resolvase (Reed 1981; Reed and Grindley 1981). The tnpA- and tnpR-gene products of Tn3 were cloned under the control of the ), P, promoter and cI repressor as illustrated in Figure 11. This allows the induction of ill tnpA pMC1426 tnpR ,L ?npA to ~ to pk8 N PL , , , [ tnpR I blo , bla ~. b/o Clo [ Figure 11. Cloning of mpA and tnpR genes of Tn3 under control of the bacteriophage k promoter, PL. Parental plasmidsare illustrated linearizedat the unique EcoRI restrictionenzymesite. Vector pX8 (Reed 1981) was digested with C/aI nucleasefor tnpA insertionor C/aI and PvuI restrictionenzymesfor mpR insertion. In the resultantplasmids, thin lines indicatethe vector sequencesand the boxed areas are the cloned sequences. (I-Z) tnpR; ( 1 ) mpA. Downloaded from symposium.cshlp.org on December 20, 2012 - Published by Cold Spring Harbor Laboratory Press 774 D E A N E T AL. o § ~ - § - + - § - r e s o l v a s e NmimJ -BamHI minutes 0 2 5 15 60240 lille, +BomHI Figure 13. Specificity of relaxation by resolvase. Relaxation by Tn3 resolvase was assayed using pBR322, which has no res site or derivatives containing one or two res sites. Arrows indicate the number and relative orientation of the sites. Reaction conditions were as described in Fig. 12. ~7 V ~4iBm ; Figure 12. Time course of DNA recombination and relaxation by resolvase, pRR51 (1.8 #g). a 5750-bp plasmid containing two directly repeated copies of the resolution site (Reed 1981), was incubated with 50 ng of purified Trd resolvase at 37~ in a 192-/zl reaction mixture containing 50 mM NaCI, 20 mM Tris-HCl (pH 7.45), 10 mM MgCI2, and 1 mM dithiothreitol. Samples (16 td) were removed at the times indicated and either quenched with 0.4% SDS to monitor relaxation (top) or first treated with BamHl nuclease to monitor recombination (bottom). The products were electrophoresed through 1% agarose gels. The two resolution sites in pRR51 are shown as open and closed arrows for ease of visualization, and the unique BamHI site is indicated by an open triangle. BamHI linearizes only the smaller of the rings of the product catenane because there are no BamHl sites in the larger daughter ring. large quantities of these proteins, and hundreds of milligrams of resolvase have been prepared (M.A. Krasnow et al., unpubl.). The Tn3 resolvase, like its 3,(5 counterpart (Reed 1981), effectively converts a plasmid containing two directly repeated sites for recombination, called res, into a catenane of the two product rings (Fig. 12). Scission at the unique B a m H I nuclease site disrupts the catenane but leaves one product ring circular. Since this ring is nearly fully supercoiled, resolvase maintains close control of the broken ends during the recombination. Resolvase was shown to be a topoisomerase by its removal of supercoils from the substrate (Fig. 12). It changes the L k in steps of one and is therefore a type-1 topoisomerase; a comparison with other E. coli topoisomerases is shown in Table 1. The time course for the relaxation and recombination by resolvase was the same (Fig. 12). A more important correspondence in the two processes is that like recombination, relaxation required res sites in the same orientation (Fig. 13). A plasmid with no sites, one site, or two sites in head-to-head orientation was not relaxed at all. Three conclusions can be drawn from this experiment: First, the topoisomerase activity must be intrinsic to the breakage and reunion that marks recombination, because both have the same unique substrate requirements. Relaxation of D N A by Int did not show any sequence preference (Kikuchi and Nash 1979), and therefore the role of its topoisomerase activity in recombination was inferential. Second, resolvase must have at least two active sites, both of which have to be occupied for either to break and rejoin DNA. Third, the two res sites must be brought together in a fixed orientation so that the direct and inverted configuration can be distinguished. A mechanism for this distinction was suggested by an analysis of the interlinking complexity of the catenated product circles. Using ribbon models for DNA, it can be shown that intramolecular recombination can convert the supercoils of the substrate into intertwining of the product-catenated rings, if the supercoils are plec- Downloaded from symposium.cshlp.org on December 20, 2012 - Published by Cold Spring Harbor Laboratory Press TYPE-1 T O P O I S O M E R A S E S E. COLI SYNAPSI~" 4 RECOMBINATION )' '9 tJ Figure 14. Intramolecular recombination of a supercoiled substrate can lead passively to catenation of the product rings. The two resolution sites are shown as open and closed arrows. Synapsis is the parallel alignment of the resolution sites. Upon recombination, the interwound supercoils trapped between the recombination sites can be directly converted to interlinkings of the daughters. If n is the number of supercoils and M e is the twist lost during breakage and reunion, then (n--Me)~2 interlockings of product catenanes can result. tonemic rather than toroidal (Fig. 14) (Mizuuchi et al. 1980). This results because the DNA segments that are to be separated by recombination are interwound in the substrate. This passive model for interlinking predicts that the catenated products will be largely relaxed and highly intertwined. Neither is observed. The supercoil density of the resolved products is nearly that of the substrate, and the catenanes appear simple and homogeneous, intertwined only once. Since it is a reasonable assumption that the substrate supercoils are plectonemic (Camerini-Otero and Felsenfeld 1978), we suggest that resolvase removes the intertwining prior to recombination and actively introduces a single product interlock. This can be accomplished by a tracking mechanism for resolution (Fig. 15). One subunit of a dimeric enzyme initially binds nonspecifically to the DNA. The enzyme then slides bidirectionally along the DNA to a r e s site where it remains bound; the second subunit then repeats the search for a second r e s site. When both sites are bound, recombination and/or relax- NON-)'I SPECIFIC BINDING WALK TO SITE NON- ) BINDING / TO), 2ndSITE WALK 775 ation results. The search for the second r e s site will disentangle the supercoils such that substrate supercoiling will not affect the number of intertwinings of the product rings. Interlinking would instead be dictated by the mechanism of the enzyme, and the products need not be catenated at all. The tracking between the r e s sites also explains how the enzyme distinguishes the orientation of the r e s sites and thereby operates only when they are in the same orientation. If instead the second subunit released DNA and rebound in either orientation, then it would not distinguish the relative orientation of r e s sites. Reactions of Type-I Topoisomerases Type-2 topoisomerases catalyze the passage of duplex DNA segments through each other and can therefore reversibly catenate and knot intact duplex DNA rings. Because type-1 enzymes make only transient singlestranded breaks, they can (with the exception of sitespecific recombination enzymes) only knot and catenate rings that have a preexisting break. They seem, however, particularly suited to a number of reactions (Fig. 16). At the termination of replication of circular DNA, there are topological problems (Gefter 1975) that can be solved by conversion of the incomplete rings to catenanes (Sundin and Varshavsky 1980), and these catenanes could be separated by type-1 enzymes (Fig. 16a). The replication of duplex DNA in which the two strands are covalently joined by a terminal hairpin, such as vaccinia DNA (Geshelin and Berns 1974; Baroudy et al. 1982), could be via single-strand catenanes in which the hairpins are intertwined (Fig. 16b). Separation requires a topoisomerase that operates on single strands. Recombination generally proceeds through a Holliday-structure intermediate (Dressier and Potter 1982) in which duplex DNA segments are joined covalently through two of the four strands (Fig. 16c). Both the formation and resolution of such structures could be by a type-1 topoisomerase such as resolvase. Another process im- ) ( RECOMBINE) Figure 15. Tracking model for resolvase-mediated recombination. A resolvase dimer (stippled area) binds DNA nonspecifically and translocates randomly along the DNA until reaching ares site (arrow) where it binds tightly and specifically. The second subunit then binds nonspecificallyto adjacent DNA, trapping a loop of DNA. One-dimensionaldiffusion of the second subunit with respect to the DNA causes the loop to expand and contract; resolvase serves as a loose annulus. This segregates the supercoils and prevents net interwinding of the DNA segments between the sites. When the second subunit reaches the second res site in the proper orientation, it is tightly bound, and the enzyme changes conformation, activating breakage and reunion. Strand exchange is directed by resolvase with a specific geometry, resulting in singly interlinked recombinants. Downloaded from symposium.cshlp.org on December 20, 2012 - Published by Cold Spring Harbor Laboratory Press 776 DEAN ET AL. (a) Decatenate interrupted circles (b) Decatenate single-strand loops -- 9 -- 9 (c) Recombination via Holliday structure (3 (d) Strand transfer 9169 (e) Rolling circle nick replicate join Figure 16. Reactionsof type-1 topoisomerases. The 3' and 5' ends of DNA are indicatedby arrowheads and closed circles, respectively. Parallel lines indicatebase-pairedstrands. (a) Interruptedduplexrings such as those in SV40 DNA replicationare decatenated; (b) terminal hairpins as may be formed in vacciniaDNA replicationare resolved. (c) Hollidaystructure is formed at a homologousregion betweentwo circular DNA moleculesand subsequentlyconvertedto a dimeric recombinationproduct; (d) a single strand of DNA is transferred from a gapped duplex ring to an intact duplex ring to form a D loop; (e) replicationof q~Xand fd viral DNAs. A topoisomerase, cistron-A, or gene-2 product (1) nicks the replicative form I, but closure is delayed until a single-strandedcircular product is synthesized. plicated in recombination is the transfer of a singlestranded segment from one duplex to another (Radding 1978) (Fig. 16d). Single-strand passage by a type-1 enzyme would solve the topological problems of such an intermolecular helix. This is an interpretation of the facilitation of recA-protein-mediated strand transfer by E. coli topoisomerase I (Cunningham et al. 1981). This winding problem also occurs in the annealing of complementary single-stranded circles and this reaction can be carried out with type-1 enzymes (Champoux 1977; Kirkegaard and Wang 1978). The ~X cistron-A protein and the related gene-2 protein of filamentous phages (Kornberg 1980) are examples of still another topoisomerase-mediated process (Fig. 16e). Here, the singlestrand breakage and reunion reactions are temporarily interrupted by synthesis of a single-stranded DNA circle. These enzymes relax supercoils and are therefore topoisomerases (Geider and Meyer 1979; Langeveld et al. 1980). Thus, type-1 topoisomerases promote a variety of DNA manipulations that allow facile participation of single strands of DNA in cellular processes. ACKNOWLEDGMENTS This work was supported by National Institutes of Health research grants GM-21397 and GM-22729 from the National Institute of General Medical Sciences. 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