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• W2039751418 abstract "RecQ-like DNA helicases pair with cognate topoisomerase III enzymes to function in the maintenance of genomic integrity in many organisms. These proteins play roles in stabilizing stalled replication forks, the S phase checkpoint response, and suppressing genetic crossovers, and their inactivation results in hyperrecombination, gross chromosomal rearrangements, chromosome segregation defects, and human disease. Biochemical activities associated with these enzymes include the ability to resolve double Holliday junctions, a process thought to lead to the suppression of crossover formation. Using Escherichia coli RecQ and topoisomerase III, we demonstrate a second activity for this pair of enzymes that could account for their role in maintaining genomic stability: resolution of converging replication forks. This resolution reaction is specific for the RecQ-topoisomerase III pair and is mediated by interaction of both of these enzymes with the single-stranded DNA-binding protein SSB. RecQ-like DNA helicases pair with cognate topoisomerase III enzymes to function in the maintenance of genomic integrity in many organisms. These proteins play roles in stabilizing stalled replication forks, the S phase checkpoint response, and suppressing genetic crossovers, and their inactivation results in hyperrecombination, gross chromosomal rearrangements, chromosome segregation defects, and human disease. Biochemical activities associated with these enzymes include the ability to resolve double Holliday junctions, a process thought to lead to the suppression of crossover formation. Using Escherichia coli RecQ and topoisomerase III, we demonstrate a second activity for this pair of enzymes that could account for their role in maintaining genomic stability: resolution of converging replication forks. This resolution reaction is specific for the RecQ-topoisomerase III pair and is mediated by interaction of both of these enzymes with the single-stranded DNA-binding protein SSB. The RecQ family of 3′ → 5′ DNA helicases plays an important role in the maintenance of genomic integrity. Inactivation of many of the human homologs, such as BLM, WRN, and RECQ4, results in hyperrecombination, genome rearrangements, and propensity for cancer (Bennett and Keck, 2004Bennett R.J. Keck J.L. Structure and function of RecQ DNA helicases.Crit. Rev. Biochem. Mol. Biol. 2004; 39: 79-97Crossref PubMed Scopus (83) Google Scholar, Cobb and Bjergbaek, 2006Cobb J.A. Bjergbaek L. RecQ helicases: lessons from model organisms.Nucleic Acids Res. 2006; 34: 4106-4114Crossref PubMed Scopus (49) Google Scholar, Hickson, 2003Hickson I.D. RecQ helicases: caretakers of the genome.Nat. Rev. Cancer. 2003; 3: 169-178Crossref PubMed Scopus (561) Google Scholar, Mankouri and Hickson, 2004Mankouri H.W. Hickson I.D. Understanding the roles of RecQ helicases in the maintenance of genome integrity and suppression of tumorigenesis.Biochem. Soc. Trans. 2004; 32: 957-958Crossref PubMed Scopus (23) Google Scholar, Sharma et al., 2006Sharma S. Doherty K.M. Brosh Jr., R.M. Mechanisms of RecQ helicases in pathways of DNA metabolism and maintenance of genomic stability.Biochem. J. 2006; 398: 319-337Crossref PubMed Scopus (196) Google Scholar, Wu, 2007Wu L. Role of the BLM helicase in replication fork management.DNA Repair (Amst.). 2007; 6: 936-944Crossref PubMed Scopus (47) Google Scholar, Wu and Hickson, 2006Wu L. Hickson I.D. DNA helicases required for homologous recombination and repair of damaged replication forks.Annu. Rev. Genet. 2006; 40: 279-306Crossref PubMed Scopus (141) Google Scholar). The genome-stabilizing activity of RecQ helicases is often linked to the activity of topoisomerase III, a type IA enzyme that breaks and reseals single strands of DNA. This relationship was first established when mutations in the yeast gene Sgs1 were isolated as suppressors of the slow growth and hyperrecombination phenotype of loss-of-function mutations in Top3, the gene encoding yeast Top3 (Gangloff et al., 1994Gangloff S. McDonald J.P. Bendixen C. Arthur L. Rothstein R. The yeast type I topoisomerase Top3 interacts with Sgs1, a DNA helicase homolog: a potential eukaryotic reverse gyrase.Mol. Cell. Biol. 1994; 14: 8391-8398Crossref PubMed Scopus (606) Google Scholar). Sequence analysis revealed that Sgs1 was a member of the RecQ family. Sgs1 was also isolated by two-hybrid analysis as a protein that interacted with both toposiomerases II and III (Watt et al., 1995Watt P.M. Louis E.J. Borts R.H. Hickson I.D. Sgs1: a eukaryotic homolog of E. coli RecQ that interacts with topoisomerase II in vivo and is required for faithful chromosome segregation.Cell. 1995; 81: 253-260Abstract Full Text PDF PubMed Scopus (372) Google Scholar). In these original reports, the authors recognized that the marriage of a DNA helicase with a type I topoisomerase could produce a unique DNA-unlinking activity where the helicase would unwind a segment of duplex DNA and the topoisomerase would act on the single-stranded (ss) DNA, perhaps akin to the reverse DNA gyrase that had recently been reported to be composed of helicase and type I topoisomerase domains in a single polypeptide chain (Confalonieri et al., 1993Confalonieri F. Elie C. Nadal M. de La Tour C. Forterre P. Duguet M. Reverse gyrase: a helicase-like domain and a type I topoisomerase in the same polypeptide.Proc. Natl. Acad. Sci. USA. 1993; 90: 4753-4757Crossref PubMed Scopus (132) Google Scholar). A more likely role for the Sgs1-Top3 activities, however, was considered to be at the point where replication forks converge. Positive supercoiling accumulates ahead of a progressing replication fork, requiring the action of topoisomerases to remove the positive supercoils to allow continued progress. Converging replication forks present a particular topological problem in circular DNAs that are replicated bidirectionally and linear chromosomes where free rotation of the parental DNA is constrained by attachment to cellular structures. Under these circumstances, progress of the forks decreases the parental duplex DNA available for the binding of type II topoisomerases that act to remove the positive supercoils (Figure 1). A point is reached wherein either the topoisomerase can no longer bind or so much positive supercoiling has built up that it presents a barrier to further replication. In their analysis of the replication of SV40 DNA, Sundin and Varshavsky, 1980Sundin O. Varshavsky A. Terminal stages of SV40 DNA replication proceed via multiply intertwined catenated dimers.Cell. 1980; 21: 103-114Abstract Full Text PDF PubMed Scopus (226) Google Scholar, Sundin and Varshavsky, 1981Sundin O. Varshavsky A. Arrest of segregation leads to accumulation of highly intertwined catenated dimers: dissection of the final stages of SV40 DNA replication.Cell. 1981; 25: 659-669Abstract Full Text PDF PubMed Scopus (232) Google Scholar concluded that the major pathway of resolution of these late θ-type replication intermediates (Figure 1, [ii]) was that the remaining region of parental DNA was unwound by continued synthesis of only the nascent leading strands at the forks, converting the remaining parental duplex turns directly into catenanes between the sister molecules, the lagging-strand gaps were then filled in, the nascent strands sealed, and the catenated sister molecules then unlinked by a type II topoisomerase (Figure 1, pathway A, [ii] → [iii] → [iv] → [v]). In this pathway, although it was possible that a type I topoisomerase could support replication fork progression, only a type II topoisomerase could segregate the linked sister molecules. Studies on SV40 DNA replication in vitro in crude extracts confirmed the existence of this pathway (Yang et al., 1987Yang L. Wold M.S. Li J.J. Kelly T.J. Liu L.F. Roles of DNA topoisomerases in simian virus 40 DNA replication in vitro.Proc. Natl. Acad. Sci. USA. 1987; 84: 950-954Crossref PubMed Scopus (235) Google Scholar). Our observation that a type I topoisomerase could decatenate replicating pBR322 DNA (Minden and Marians, 1986Minden J.S. Marians K.J. Escherichia coli topoisomerase I can segregate replicating pBR322 daughter DNA molecules in vitro.J. Biol. Chem. 1986; 261: 11906-11917Abstract Full Text PDF PubMed Google Scholar) led us to propose a second pathway of resolution of the late replication intermediate (LRI; Figure 1, [ii]). Here the topoisomerase would bind to a single-stranded region on the lagging-strand template and unlink the parental DNA as replication fork progression continued, resulting in sister molecules that were completely resolved topologically coincident with the completion of replication (Figure 1, pathway B, [ii] → [vi] → [vii] → [v]). Our findings that Escherichia coli topoisomerase III (Topo III) required binding to single-stranded DNA for activity (DiGate and Marians, 1988DiGate R.J. Marians K.J. Identification of a potent decatenating enzyme from Escherichia coli.J. Biol. Chem. 1988; 263: 13366-13373Abstract Full Text PDF PubMed Google Scholar), could segregate replicating pBR322 and oriC sister plasmid DNA molecules in vitro (Hiasa et al., 1994Hiasa H. DiGate R.J. Marians K.J. Decatenating activity of Escherichia coli DNA gyrase and topoisomerases I and III during oriC and pBR322 DNA replication in vitro.J. Biol. Chem. 1994; 269: 2093-2099Abstract Full Text PDF PubMed Google Scholar), and could replace topoisomerase IV as the cellular decatenase when overproduced (Nurse et al., 2003Nurse P. Levine C. Hassing H. Marians K.J. Topoisomerase III can serve as the cellular decatenase in Escherichia coli.J. Biol. Chem. 2003; 278: 8653-8660Crossref PubMed Scopus (61) Google Scholar) were consistent with this pathway. A popular review by Wang, 1991Wang J.C. DNA topoisomerases: why so many?.J. Biol. Chem. 1991; 266: 6659-6662Abstract Full Text PDF PubMed Google Scholar shortly after the discovery of yeast Top3 (Wallis et al., 1989Wallis J.W. Chrebet G. Brodsky G. Rolfe M. Rothstein R. A hyper-recombination mutation in S. cerevisiae identifies a novel eukaryotic topoisomerase.Cell. 1989; 58: 409-419Abstract Full Text PDF PubMed Scopus (440) Google Scholar) discussed the participation of type I and type II topoisomerases in these pathways of chromosome segregation. In considering how Sgs1 and Top3 might act on such LRIs, Rothstein and Gangloff, 1995Rothstein R. Gangloff S. Hyper-recombination and Bloom's syndrome: microbes again provide clues about cancer.Genome Res. 1995; 5: 421-426Crossref PubMed Scopus (36) Google Scholar modified pathway B, suggesting that resolution could be accomplished by Sgs1-catalyzed unwinding of the remaining parental duplex DNA between the converging replication forks in concert with Top3-catalyzed unlinking of the single strands. Although an attractive model as a role for the RecQ-like helicase-topoisomerase III pairs, the action of these enzymes in resolving converging replication forks has received little support. Most observed defects in chromosome segregation in yeast and Schizosaccharomyces pombe strains mutated in top3 and sgs1 (rqh1 in S. pombe) have been attributed to unresolved recombinant structures linking the sisters that arise from homologous recombination at stalled replication forks. The top3 phenotypes can be suppressed by mutation in homologous recombination genes (Oakley et al., 2002Oakley T.J. Goodwin A. Chakraverty R.K. Hickson I.D. Inactivation of homologous recombination suppresses defects in topoisomerase III-deficient mutants.DNA Repair (Amst.). 2002; 1: 463-482Crossref PubMed Scopus (44) Google Scholar, Shor et al., 2002Shor E. Gangloff S. Wagner M. Weinstein J. Price G. Rothstein R. Mutations in homologous recombination genes rescue top3 slow growth in Saccharomyces cerevisiae.Genetics. 2002; 162: 647-662Crossref PubMed Google Scholar), and Sgs1-Top3 has been shown to suppress genetic crossing over (Ira et al., 2003Ira G. Malkova A. Liberi G. Foiani M. Haber J.E. Srs2 and Sgs1-Top3 suppress crossovers during double-strand break repair in yeast.Cell. 2003; 115: 401-411Abstract Full Text Full Text PDF PubMed Scopus (459) Google Scholar). These observations focused attention on the likelihood that the Sgs1-Top3 pair acted to resolve double Holliday junctions (DHJ[s]) in a manner that prevents crossing over. This model has received considerable support, including the biochemical demonstration that such structures can be resolved in vitro by the human (Wu and Hickson, 2003Wu L. Hickson I.D. The Bloom's syndrome helicase suppresses crossing over during homologous recombination.Nature. 2003; 426: 870-874Crossref PubMed Scopus (827) Google Scholar) and Drosophila (Plank et al., 2006Plank J.L. Wu J. Hsieh T.S. Topoisomerase IIIα and Bloom's helicase can resolve a mobile double Holliday junction substrate through convergent branch migration.Proc. Natl. Acad. Sci. USA. 2006; 103: 11118-11123Crossref PubMed Scopus (117) Google Scholar) BLM-Topo IIIα pairs. Here we demonstrate the ability of the E. coli RecQ-Topo III pair to resolve stalled, converging replication forks in vitro in an LRI structure produced by the replication of oriC plasmid DNA. The resolution reaction displays functional specificity for the RecQ-Topo III pair and functional cooperation between the two proteins that is mediated by interaction with the single-stranded DNA-binding protein (SSB). To generate an LRI structure with two converging, stalled replication forks, we made use of the activity of Tus to block replication fork progression when bound to its DNA-binding sequence Ter (Hill and Marians, 1990Hill T.M. Marians K.J. Escherichia coli Tus protein acts to arrest the progression of DNA replication forks in vitro.Proc. Natl. Acad. Sci. USA. 1990; 87: 2481-2485Crossref PubMed Scopus (85) Google Scholar, Lee et al., 1989Lee E.H. Kornberg A. Hidaka M. Kobayashi T. Horiuchi T. Escherichia coli replication termination protein impedes the action of helicases.Proc. Natl. Acad. Sci. USA. 1989; 86: 9104-9108Crossref PubMed Scopus (112) Google Scholar). The Tus-Ter system acts to form a replication termination zone on the E. coli chromosome (Kuempel et al., 1989Kuempel P.L. Pelletier A.J. Hill T.M. Tus and the terminators: the arrest of replication in prokaryotes.Cell. 1989; 59: 581-583Abstract Full Text PDF PubMed Scopus (41) Google Scholar). We engineered a bidirectionally replicating oriC plasmid template carrying two TerB sites oriented to block the oncoming replication forks (Figure 2A, [i]). Replication of the template in vitro with purified proteins yields form II DNA as the major product in the absence of Tus and, as we have demonstrated previously (Hiasa and Marians, 1994Hiasa H. Marians K.J. Tus prevents overreplication of oriC plasmid DNA.J. Biol. Chem. 1994; 269: 26959-26968Abstract Full Text PDF PubMed Google Scholar), an LRI in the presence of Tus (Figure 2A, [ii]). Because the LRI is essentially twice the size of the input template DNA, it migrates slowly during neutral agarose gel electrophoresis (Figure 2B). Our previous studies (Hill and Marians, 1990Hill T.M. Marians K.J. Escherichia coli Tus protein acts to arrest the progression of DNA replication forks in vitro.Proc. Natl. Acad. Sci. USA. 1990; 87: 2481-2485Crossref PubMed Scopus (85) Google Scholar) on the replication fork-blocking activity of Tus demonstrated that at the stalled fork, the 3′ OH of the nascent leading strand was ahead of the 5′ end of the last Okazaki fragment (creating a variably sized gap in the nascent lagging strand) and that the leading strand progressed until it physically collided with Tus bound to the DNA. In the LRI substrate used in this report, the 3′ OH ends of the two nascent leading strands are separated by 130 bp of unreplicated parental duplex DNA (Figure 2A, [iv]). Resolution of the LRI by RecQ and Topo III yields gapped, form II sister DNA molecules (Figure 2A, [iii]). The LRI was converted to form II DNA in the presence of RecQ, Topo III, and SSB (Figure 2C). All three proteins were required for the resolution reaction. SSB was included in these reactions because the protein will be bound to any ssDNA at a replication fork in the cell. As we show below, SSB in fact appears to mediate functional cooperation between Topo III and RecQ in the resolution reaction. As expected, the combination of Topo III and SSB could not resolve the LRI, nor could the combination of RecQ and SSB. In the latter case, the parental duplex DNA in the LRI was unwound; however, because there was no topoisomerase present to unlink the parental strands, each duplex turn of the unreplicated parental DNA in the LRI that is unwound gives rise to an ss catenane between the sister molecules. In the data shown in this paper, a ladder of bands is generated (these ladders can be seen more clearly in figures below), each rung differing by steps of one in the number of linkages between the sister DNA molecules. (Restriction enzyme analysis of the LRI and the ss catenanes is given in Figure S1; see the Supplemental Data available with this article online. A ladder of bands is generated as opposed to one band on the 13th rung of the ladder because of the manner in which replication terminates during preparation of the LRI; see the legend to Figure 2.) This observation suggested that the resolution reaction need not be concerted, that is, RecQ could unwind the parental duplex first followed by the action of Topo III to unlink the catenated DNA, as opposed to a requirement for a complex of RecQ and Topo III acting together to unwind and unlink in unison. That the reaction is not concerted and can be separated into unwinding and unlinking steps is shown in figures below. Resolution of DHJs by the hBLM-hTopo IIIα pair also does not require a physical interaction between the two proteins; however, it does require the HRDC (helicase and RNaseD C-terminal) domain of RecQ (Wu et al., 2005Wu L. Chan K.L. Ralf C. Bernstein D.A. Garcia P.L. Bohr V.A. Vindigni A. Janscak P. Keck J.L. Hickson I.D. The HRDC domain of BLM is required for the dissolution of double Holliday junctions.EMBO J. 2005; 24: 2679-2687Crossref PubMed Scopus (130) Google Scholar). The HRDC domain, found in many members of the RecQ helicase family (ScSgs1, EcRecQ, and hBLM included), can bind DNA, but the mode of DNA binding seems to differ among different members of the family. In the case of hBLM, a variant protein deleted for the HRDC was able to unwind forked DNA substrates but was defective in either binding or resolving DHJs, suggesting that the domain was responsible for specific recognition of the substrate. We tested whether this domain was required for resolution of the LRI. RecQΔHRDC (consisting of the N-terminal 523 amino acids of RecQ) supported LRI resolution nearly as efficiently as intact RecQ (Figure 3), with less than a 2-fold difference in specific activity between the two proteins. Because RecQ unwinding of the LRI is initiated at a forked structure, this observation is consistent with the demonstration by Wu et al., 2005Wu L. Chan K.L. Ralf C. Bernstein D.A. Garcia P.L. Bohr V.A. Vindigni A. Janscak P. Keck J.L. Hickson I.D. The HRDC domain of BLM is required for the dissolution of double Holliday junctions.EMBO J. 2005; 24: 2679-2687Crossref PubMed Scopus (130) Google Scholar that removal of the HRDC domain from EcRecQ inactivated the ability of the enzyme to unwind a DHJ but not a forked structure. However, whether RecQ is recognizing either a structural feature of the LRI or something else is unclear. Binding and unwinding by RecQ of forked substrates with a complete nascent leading strand (i.e., with the 3′ OH of the leading strand at the fork branchpoint), such as in the LRI, are poor. In the absence of SSB, preferred RecQ substrates have just the opposite of the fork structure in the LRI: a complete nascent lagging strand (i.e., with the 5′ end of the lagging strand at the fork branchpoint) and no nascent leading strand (Hishida et al., 2004Hishida T. Han Y.W. Shibata T. Kubota Y. Ishino Y. Iwasaki H. Shinagawa H. Role of the Escherichia coli RecQ DNA helicase in SOS signaling and genome stabilization at stalled replication forks.Genes Dev. 2004; 18: 1886-1897Crossref PubMed Scopus (104) Google Scholar). These observations suggest that RecQ binding to a specific structural element during resolution of the LRI in the presence of SSB is not likely to be a major component of recognition. In yeast (Gangloff et al., 1994Gangloff S. McDonald J.P. Bendixen C. Arthur L. Rothstein R. The yeast type I topoisomerase Top3 interacts with Sgs1, a DNA helicase homolog: a potential eukaryotic reverse gyrase.Mol. Cell. Biol. 1994; 14: 8391-8398Crossref PubMed Scopus (606) Google Scholar) and S. pombe (Goodwin et al., 1999Goodwin A. Wang S.W. Toda T. Norbury C. Hickson I.D. Topoisomerase III is essential for accurate nuclear division in Schizosaccharomyces pombe.Nucleic Acids Res. 1999; 27: 4050-4058Crossref PubMed Scopus (97) Google Scholar), Sgs1 (Rqh1 in S. pombe) and Top3 interact genetically, and the human (Johnson et al., 2000Johnson F.B. Lombard D.B. Neff N.F. Mastrangelo M.A. Dewolf W. Ellis N.A. Marciniak R.A. Yin Y. Jaenisch R. Guarente L. Association of the Bloom syndrome protein with topoisomerase IIIα in somatic and meiotic cells.Cancer Res. 2000; 60: 1162-1167PubMed Google Scholar, Wu et al., 2000Wu L. Davies S.L. North P.S. Goulaouic H. Riou J.F. Turley H. Gatter K.C. Hickson I.D. The Bloom's syndrome gene product interacts with topoisomerase III.J. Biol. Chem. 2000; 275: 9636-9644Crossref PubMed Scopus (275) Google Scholar), Drosophila (Plank et al., 2006Plank J.L. Wu J. Hsieh T.S. Topoisomerase IIIα and Bloom's helicase can resolve a mobile double Holliday junction substrate through convergent branch migration.Proc. Natl. Acad. Sci. USA. 2006; 103: 11118-11123Crossref PubMed Scopus (117) Google Scholar), yeast (Bennett et al., 2000Bennett R.J. Noirot-Gros M.F. Wang J.C. Interaction between yeast Sgs1 helicase and DNA topoisomerase III.J. Biol. Chem. 2000; 275: 26898-26905Abstract Full Text Full Text PDF PubMed Google Scholar), and S. pombe (Laursen et al., 2003Laursen L.V. Ampatzidou E. Andersen A.H. Murray J.M. Role for the fission yeast RecQ helicase in DNA repair in G2.Mol. Cell. Biol. 2003; 23: 3692-3705Crossref PubMed Scopus (61) Google Scholar) RecQ-like helicase and topoisomerase pairs interact physically. The hTopo IIIα interaction domain of hBLM is essential for suppression of sister chromatid exchanges in vivo (Hu et al., 2001Hu P. Beresten S.F. van Brabant A.J. Ye T.Z. Pandolfi P.P. Johnson F.B. Guarente L. Ellis N.A. Evidence for BLM and topoisomerase IIIα interaction in genomic stability.Hum. Mol. Genet. 2001; 10: 1287-1298Crossref PubMed Google Scholar), even though it is not required for biochemical resolution of DHJs (Wu et al., 2005Wu L. Chan K.L. Ralf C. Bernstein D.A. Garcia P.L. Bohr V.A. Vindigni A. Janscak P. Keck J.L. Hickson I.D. The HRDC domain of BLM is required for the dissolution of double Holliday junctions.EMBO J. 2005; 24: 2679-2687Crossref PubMed Scopus (130) Google Scholar). On the other hand, DmBLM does not interact physically with DmTopo IIIβ and the latter enzyme cannot substitute for DmTopo IIIα in a DHJ resolution reaction (Plank et al., 2006Plank J.L. Wu J. Hsieh T.S. Topoisomerase IIIα and Bloom's helicase can resolve a mobile double Holliday junction substrate through convergent branch migration.Proc. Natl. Acad. Sci. USA. 2006; 103: 11118-11123Crossref PubMed Scopus (117) Google Scholar). These observations suggest that interaction between RecQ-like helicase-topoisomerase III pairs is required for some manifestation of their activity, be it biochemical or specific localization in the cell. However, neither a physical nor genetic interaction has been demonstrated between EcRecQ and EcTopo III. To assess whether there was functional specificity to the RecQ-Topo III pair, we investigated the ability of other E. coli topoisomerases and DNA helicases to support the LRI resolution reaction. E. coli possesses four topoisomerases, two type IA, Topo I and Topo III, and two type II, DNA gyrase and Topo IV (Champoux, 2001Champoux J.J. DNA topoisomerases: structure, function, and mechanism.Annu. Rev. Biochem. 2001; 70: 369-413Crossref PubMed Scopus (2041) Google Scholar). Topo III was clearly very efficient in supporting the resolution reaction, with half-maximal activity obtaining at about 1 nM concentration (Figures 4A and 4B). As expected, Topo IV could not support the resolution reaction at all (Figure 4C). Topo IV cleaves and reseals both strands of the duplex at the same time; it will not act on ssDNA and thus should not be able to unlink the ss catenanes generated by RecQ unwinding of the LRI. Topo I, on the other hand, is a type I enzyme and, in theory, could unlink the ss catenanes. Topo I activity in the resolution reaction was very weak. At high concentrations of Topo I, reduction of the number of linkages between the two sister DNA molecules could be observed (a change in the distribution of the linkages is evident), but the enzyme appeared unable, even at a concentration of 100 nM, of completely decatenating the substrate (Figure 4C). The relative inactivity of Topo I compared to Topo III in the resolution reaction may relate to differences in preferred DNA binding sites or to the relative ability of the two enzymes to interact with DNA in the presence of SSB. Topo I does prefer to bind to regions where single-stranded and double-stranded DNA abut (Kirkegaard and Wang, 1985Kirkegaard K. Wang J.C. Bacterial DNA topoisomerase I can relax positively supercoiled DNA containing a single-stranded loop.J. Mol. Biol. 1985; 185: 625-637Crossref PubMed Scopus (125) Google Scholar), whereas Topo III does not show this preference and will bind to any region of ssDNA. E. coli possesses at least one dozen DNA helicases. In considering which ones to assay, we decided to test those that have been demonstrated to possess activity on stalled fork structures similar to the one that is present in the LRI. Thus, we compared the activity of UvrD, Rep, RuvAB, RecG, and PriA to that of RecQ (Figure 5). Interestingly, of these half-dozen DNA helicases that are active at replication forks, only RecQ could support the resolution reaction. Some apparent processing of the LRI could be observed at high concentrations of UvrD. However, coincidently, an ∼2.0 kb DNA species is also observed. This size corresponds to the distance that the counterclockwise moving fork travels on the pBROTB I 535-80 template DNA from oriC to the Ter site, indicating that rather than binding to the leading-strand template and unwinding the unreplicated parental DNA directly, as RecQ does, UvrD was binding the lagging-strand template and unwinding all of the nascent DNA before coming around to the unreplicated parental DNA. (The 3.0 kb fragment from the clockwise-moving fork was not observed, suggesting some specificity to UvrD binding.) The data presented in Figure 4, Figure 5 support the concept of functional cooperation between RecQ and Topo III: of the two type I E. coli topoisomerases that could possibly support the resolution reaction, only Topo III was capable of doing so. Similarly, of a number of E. coli DNA helicases known to be able to modify the structure of stalled replication forks, only RecQ supported the resolution reaction. To provide additional support for this argument, we sought evidence of direct enzymatic synergy between the two proteins. As shown in Figure 2, in the presence of SSB, RecQ will unwind the LRI, irrespective of whether Topo III is present. To assess the effect of Topo III on RecQ unwinding, we compared the extent of unwinding by RecQ alone and in the presence of either Topo I or Topo III (Figure 6). The total amount of LRI unwound (the sum of ss catenated DNA and form II present as products of the reaction) by RecQ was unaffected by the presence of Topo I. In contrast, at low concentrations of RecQ, the presence of Topo III stimulated unwinding by greater than 4-fold, strengthening our argument that the RecQ-Topo III pair manifests a functional specificity in the LRI resolution reaction. The functional specificity of the RecQ-Topo III pair demonstrated above seems inconsistent with the lack of a physical or genetic interaction between RecQ and Topo III and the likelihood that structure-specific binding of the substrate by RecQ was not a significant factor in substrate recognition. However, both RecQ and Topo III were shown to interact with SSB in a proteome-wide interaction screen (Butland et al., 2005Butland G. Peregrin-Alvarez J.M. Li J. Yang W. Yang X. Canadien V. Starostine A. Richards D. Beattie B. Krogan N. et al.Interaction network containing conserved and essential protein complexes in Escherichia coli.Nature. 2005; 433: 531-537Crossref PubMed Scopus (908) Google Scholar) and SSB was captured by RecQ in a TAP tag affinity-purification analysis (Shereda et al., 2007Shereda R.D. Bernstein D.A. Keck J.L. A central role for SSB in Escherichia coli RecQ DNA helicase function.J. Biol. Chem. 2007; 282: 19247-19258Crossref PubMed Scopus (110) Google Scholar). We therefore investigated the role of SSB in mediating the LRI resolution reaction. Several proteins that are involved in the maintenance of genomic integrity are known to interact with the extreme C terminus of SSB. Therefore, the ability of two variant SSBs, one lacking the last eight C-terminal amino acid residues (SSBΔC8; Shereda et al., 2007Shereda R.D. Bernstein D.A. Keck J.L. A central role for SSB in Escherichia coli RecQ DNA helicase function.J. Biol. Chem. 2007; 282: 19247-19258Crossref PubMed Scopus (110) Google Scholar) and the other being SSB113 (SSBP176S, originally isolated as lexC113; Glassberg et al., 1979Glassberg J. Meyer R.R. Kornberg A. Mutant single-strand binding protein of Escherichia coli: genetic and physiological characterization.J. Bact" @default.
• W2039751418 created "2016-06-24" @default.
• W2039751418 creator A5003565422 @default.
• W2039751418 creator A5068066930 @default.
• W2039751418 date "2008-06-01" @default.
• W2039751418 modified "2023-09-29" @default.
• W2039751418 title "Resolution of Converging Replication Forks by RecQ and Topoisomerase III" @default.
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