Proc. Natl. Acad. Sci. USA Vol. 90, pp. 8571-8575, September 1993 Biochemistry Decatenation activity of topoisomerase IV during oriC and pBR322 DNA replication in vitro (chromosome deateaton/topolsomerase) HONG PENG AND KENNETH J. MARIANS Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, and Graduate Program in Molecular Biology, Cormell University Graduate School of Medical Sciences, New York, NY 10021 Communicated by Jerard Hurwitz, June 15, 1993 ABSTRACT Topoisomerase IV (Topo IV), encoded by DNA accumulated at the nonpermissive temperature only in paiC and parE, is required for partition of the daughter parC and parE strains, not in gyrA, gyrB, or parF strains. chromosomes in Escherichwa coil. This enzyme is kely respon- In this report, we have examined the action of Topo IV in sible for decatenating the lnked daughter chromosomes after plasmid replication systems reconstituted in vitro with puri- replication. In this report, we have examid the action ofTopo fied proteins. We show that Topo IV is required for the IV in both pBR322 and oriC DNA replication reconstituted in topological resolution ofthe daughter DNA molecules in both vitro with purified proteins. Gyrase fails to decatenate the unidirectionally and bidirectionally replicating systems, that linked daughter molecules under any condition in the oriC this decatenation step occurs prior to the completion ofDNA system and at physiological salt concentrations in the pBR322 replication, and that Topo IV-catalyzed decatenation of system, whereas Topo IV stimulates generation of monomer multiply linked DNA dimers is less sensitive to salt than that product DNA by 7- to 10-fold. Topo IV-catalyzed decatenation catalyzed by DNA gyrase. of isolated multiply linked DNA dimers was relatively insen- sitive to salt; it proceeded at 14% of the mamal rate even in the presence of 800 mM potassium glutamate. In contrast, MATERUILS AND METHODS decatenation in vitro by gyrase was inhibited completely under Replication Proteins and DNAs. ParC and ParE were puri- these conditions. Pulse-chase analysis indicated that Topo fied as will be described elsewhere (25). Topo IV was IV-catalyzed resolution of linked daughter DNA molecues reconstituted by mixing equimolar amounts ofParC and ParE occurred prior to completion of DNA replication, such that and incubating at 0°C for 30 min. The preparation of DnaA multiply linked daughter molecules did not arise. These results and HU was described by Parada and Marians (13); all other suggest that duriug DNA replication, gyrase acts primarily to replication proteins were as described by Minden and Mar- relieve accumulated poitive supercoiling and Topo IV acts to ians (14) and Wu et al. (15). Supercoiled pBROTB plasmid, segregte the daughter chromosomes. harbored in E. coli K38tus was prepared according to Mar- ians et al. (16). The construction of pBROTB will be de- Replication of circular chromosomes leaves the daughter scribed elsewhere (H. Hiasa and K.J.M.). chromosomes linked topologically in the form of catenanes DNA Replication. A standard oriC replication reaction (1). In bacteria, successful cell division requires that the mixture (12.5 ;d) contained 40 mM Hepes-KOH (pH 8.0), 10 daughter chromosomes be disengaged topologically (decat- mM Mg(OAc)2, 10 mM dithiothreitol, bovine serum albumin enation), presumably by the action of a topoisomerase, and at 100 ,ug/ml, 2 mM ATP, 0.4 mM GTP, 0.4 mM CTP, 0.4 mM that the daughter nucleoids be separated topographically UTP, 40 ,M [a-32PJdATP (3000-10,000 cpm/pmol), 40 pM (partition), one migrating to each of the daughter cells (2). dGTP, 40 ,uM dCTP, 40 pM TTP, 4mM phosphocreatine, 250 Six mutations (parA-F) have been identified that show a ng ofcreatine kinase, 50 uM NAD, DNA template (35 fmol), defect in these processes (2). parA and parD were allelic to 200 ng of DnaA, 150 ng of DnaB, 60 ng of DnaC, 160 ng of gyrB and gyrA, respectively (3-5), whereas parB was allelic DnaG, 1.0 unit of DNA polymerase III*, 25 ng ofDnaN, 250 to dnaG (6). The identification ofthe genes encoding the two ng of SSB, 10 ng of HU, and 50 ng of DNA gyrase. Where subunits of DNA gyrase as par loci was consonant with the indicated, 0.2 ng ofRNase H, 13 ng ofDNA ligase, and 24 ng observation that in mutant strains that were temperature- ofDNA polymerase I were included. Reaction mixtures were sensitive in gyrB, the nucleoid appeared dumbbell-shaped assembled on ice, and the reaction was started by adding and twice the normal size (7), suggesting that topological DnaA. Incubation was at 30°C for 15 min. A standard pBR322 resolution of the daughter chromosomes had not occurred. replication reaction mixture (12.5 pl) contained the same Schmid (8) and Kato et al. (9) demonstrated that parE was nucleotide and buffer components as the oriC reaction mix- linked closely toparC, and Kato et al. (10) demonstrated that tures and 0.24 ,ug of RNA polymerase, 24 ng of DNA both purified Escherichia coli ParC and ParE were required polymerase I, 0.2 ng of RNase H, 125 ng of SSB, 110 ng of to reconstitute topoisomerase IV (Topo IV) activity. parC DnaB, 28 ng of DnaC, 40 ng of DnaG, 14 ng of DnaT, 15 ng and parE showed considerable homology to gyrA and gyrB, of PriA, 4 ng of PriB, 4 ng of PriC, 1.0 unit of DNA respectively (9, 11). A sixth par locus, parF, could be found polymerase HII*, 25 ng of DnaN, 50 ng of DNA gyrase, and just downstream ofparC (8). Luttinger et al. (11) suggested DNA template (35 fmol). Reaction mixtures were assembled that ParF, which has a hydrophobic amino-terminal region, on ice, and the reaction was started by adding RNA poly- might serve to anchor Topo IV to the membrane. merase. Incubation was at 30°C for 20 min. Replication The role of Topo IV in decatenating replication interme- reactions were terminated by the addition of EDTA to 25 diates has been supported by the studies ofAdams et al. (12) mM. A portion ofthe reaction mixtures was precipitated with that showed that replication catenanes of pBR322 plasmid trichloroacetic acid andfiltered through glassfiberfilters, and total DNA synthesis was measured by liquid scintillation The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: Topo III and IV, topoisomerases III and IV; LRI, late in accordance with 18 U.S.C. §1734 solely to indicate this fact. replicative intermediate; SC Di, supercoiled DNA dimers. 8571 Downloaded by guest on September 30, 2021 8572 Biochemistry: Peng and Marians Proc. Natl. Acad. Sci. USA 90 (1993) spectrometry. Another portion of the reaction mixtures was A mixed with one-fifth volume of a dye mixture containing 50 ligase + mM EDTA, 12.5% glycerol, 2% sarkosyl, 0.05% xylene Topo IV 0 0.8 4 20 100 0 0.8 4 20 100 cyanol, and 0.05% bromophenol blue and analyzed by elec- (fmol) trophoresis through vertical 0.8% (or 1.0%) agarose (SeaKem ME; FMC) gels (14 x 10 x 0.3 cm) at 2 V/cm for 16 h in an - LRI electrophoresis buffer of50 mM Tris HCl (pH 7.9 at 23°C), 40 mM NaOAc, and 1 mM EDTA. Gels were dried under vacuum onto Whatman 3M paper and autoradiographed with dimer~ 1rSC DI Amersham Hyperfilm-MP. _III 11 Decatenation of Multiply Linked DNA Dimers. Multiply _ linked DNA dimers were purified as described by Marians (17) by sucrose gradient centrifugation from oriC replication reactions (in the absence of Topo IV) increased in size by 120-fold. Decatenation reaction mixtures (5 ,ul) containing 50 mM Hepes-KOH (pH 8.0), 6 mM MgCl2, 5 mM dithiothrei- 1 2 3 4 5 6 7 8 9 10 tol, bovine serum albumin at 100 pig/ml, DNA dimers (4 fmol as monomer), and 40 fmol ofTopo IV were incubated at 30°C 80 for 2.5 min. The reactions were stopped by the addition of EDTA to 20 mM. Gel loading dye was then added, and the reactions were analyzed by electrophoresis through vertical t5$ 60 0.8% agarose gels at 2 V/cm for 18 h. The gels were dried and autoradiographed. EL 20 ._ RESULTS - C 4) C 0 Topo IV-Catalyzed Segregation of Replicating Daughter DNA Molecules. The template used for these studies was 2E 2 0 pBROTB, a 6-kb pBR322 plasmid that carries oriC [including 1- the A+T-rich region to the left of the 13-mers (18)] and two TerB sequences (19) with a 1-kb spacer between them. Thus, 0 20 40 60 80 100 pBROTB can be replicated via either a pBR322-type or TopoiVi (fmol) the oriC-type initiation mechanism, depending on replication FIG. 1. Topo IV-catalyzed decatenation of replicating daughter proteins provided. DNA molecules during oriC DNA replication. (A) Standard oriC Topoisomerase function is required, during the replication DNA replication reaction mixtures containing RNase H, DNA of a circular DNA, for removal of the positive supercoils polymerase I, and the indicated amounts of Topo IV were incubated generated by the advancing replication fork and for the in the presence (lanes 6-10) or absence (lanes 1-5) of DNA ligase, topological resolution ofthe linked daughter molecules. Prior processed, and analyzed as described in Materials and Methods. to the discovery of Topo IV (9, 10), of the E. coli topoi- Shown is an autoradiogram of the DNA products analyzed by somerases, only gyrase was found to be capable ofremoving electrophoresis through a 0.8% agarose gel. (B) The autoradiogram positive supercoils (by converting them directly to negative of the gel was scanned with a Millipore Bioimage densitometer, and of the fraction of the total activity present as monomer product (form ones) (20).