Proc. NatL Acad. Sci. USA Vol. 78, No. 2, pp. 843-847, February 1981 Biochemistry Catenation and knotting of duplex DNA by type 1 topoisomerases: A mechanistic parallel with type 2 topoisomerases (nickdng-closing enzyme/w protein/linking number/DNA condensation/circular DNA) PATRICK 0. BROWN* AND NICHOLAs R. COZZARELLI*t *Departnment of Biochemistry and tDepartment of Biophysics and Theoretical Biology, The University of Chicago, Chicago, Illinois 60637 Communicated by Bernard Roizman, November 13, 1980 ABSTRACT Escherichia coli a protein, a type 1 topoisomer- DNA molecules is nicked. The requirement for a nick was dem- ase, can catenate and knot duplex DNA circles. Previously, these onstrated by Y.-C. Tse and J. C. Wang, who independently activities were thought to be limited to type 2 topoisomerases. Catenation by a, requires a nick in one of the participating mol- discovered catenation by w (personal communication). Exten- ecules, but it is not necessary that both be nicked. Catenation does sive sequence homology between reacting molecules is not re- not depend on sequence homology and is greatly stimulated by quired. We therefore propose an alternative model for type 1 DNA-condensing agents such as spermidine. A eukaryotic type 1 topoisomerase reactions, analogous to our sign inversion model topoisomerase can also interlock duplex DNA circles. These ac- for gyrase (6), in that its characteristic feature is the deliberate tivities cannot easily be explained by the widely held topoisomer- passage of one segment of DNA through an enzyme-bridged ase model in which a reversible nick in DNA allows free rotation about the unbroken strand. We suggest instead passage of a DNA break in another. Our model differs from the swivel mechanism segment through a transient enzyme-bridged break in a single in that the enzyme holds fast to both sides of the nick to prevent DNA strand. This is analogous to the sign inversion mechanism free axial rotation, the change in Lk is intrinsically limited to of the type 2 topoisomerases, and thus expresses an essential one in each cycle of DNA breakage and reunion, enzyme-cat- mechanistic unity among topoisomerases. As expected for relax- alyzed movement of the DNA is not limited to axial rotation, ation by a single-strand passage, a, changes the linking number and a base-paired region is not required. of DNA in steps of 1. MATERIALS AND METHODS Enzymes that alter topological invariants of DNA molecules are called topoisomerases. Until recently, the detection of topo- Enzymes. w purified from E. coli by chromatography on isomerases has depended on their ability to relax negatively DEAE-Sephacel, Sephacryl S-200, and phosphocellulose was supercoiled DNA or, in the case of DNA gyrases, to introduce used for most of the experiments. Results were checked by negative supercoils (1, 2). These reactions change a topological using additional purified preparations, kindly provided by R. invariant called the linking number (Lk), the net number of E. Depew (Northeastern Ohio Universities College of Medi- times one strand of a closed circular DNA molecule is wound cine) and by J. C. Wang (Harvard University). Purified DNA- around the other. It is now clear that other topological invariants untwisting enzyme from rat liver nuclei was a gift from J. Chain- can be altered by topoisomerases. Phage T4 topoisomerase (3) poux (University of Washington). All four topoisomerase prep- and bacterial DNA gyrases (4, 5) can reversibly catenate and arations were >80% pure. knot DNA rings, and catenating enzymes have been isolated DNA. Native monomers of plasmids ColE1 and pAO3 and from several eukaryotic sources (2). These topological isomer- phage 4X174 (OX) replicative form (RF)I DNA were prepared izations are all readily explained by a mechanism we have called as described (4, 11). Nicked DNA was prepared by treatment sign inversion (6), in which a duplex DNA segment is passed with DNase I (Worthington) until 40% of the starting material through a transient break across both strands of another such remained intact, and then purified by sedimentation (4). segment (6, 7). The sign inversion mechanism always changes Reactions. Catenation reactions were at 37C and the mix- Lk in steps of 2, and topoisomerases with this property are des- tures contained, in addition to enzyme and DNA, 20 mM ignated type 2 topoisomerases (3). Tris'HCl (pH 7.6), 20 mM KCl, 6 mM MgCl2, 5 mM spermi- Type 1 topoisomerases instead introduce transient single- dine'HCl, 2 mM dithiothreitol, 30% (vol/vol) glycerol, and strand breaks and are shown in this report to change Lk in steps bovine serum albumin at 50 ug/ml. Reaction products were of 1. According to the swivel model for these topoisomerases, treated with proteinase K at 0.1 gg/ml for 30 min at 370C. the enzyme would nick one DNA strand, allow the two ends Crosslinking. DNA was crosslinked by irradiation with long- to rotate around the unbroken strand, then reseal the nick. wavelength ultraviolet light (UV Products model B-1OA) in a Supercoil relaxation and. even knotting (8) and renaturation of 10-,ul volume containing 0.4 ug of DNA, 25 ng of 4,5',8-tri- single-stranded circles (9, 10) by type 1 enzymes have been methylpsoralen (trioxsalen, Calbiochem), 10 mM Tris-HCl (pH thought to occur by this swivel mechanism. It is hard to conceive 7.8), and 0.5 mM EDTA. To monitor crosslinking, products of of a plausible scheme whereby a swivel could catenate duplex a parallel reaction using Cfo I restriction endonuclease frag- DNA circles. But we report here our surprising discovery that ments of OX RFI DNA were denatured and then renatured in the two archetypal type 1 topoisomerases-Escherichia coli w the presence of a 20-fold molar excess of 4X174 viral single- protein and rat liver nicking-closing enzyme-efficiently cate- stranded DNA. Uncrosslinked fragments failed to self-renature nate duplex rings, provided that at least one of the reacting and were resolved- from crosslinked fragments by polyacryla- mide gel electrophoresis and quantitated by scanning the ethid- The publication costs ofthis article were defrayed in part by page charge ium bromide-stained gel. payment. This article must therefore be hereby marked "advertise- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Abbreviations: OX, phage 4X174; RF, replicative form. 843 Downloaded by guest on September 25, 2021 844 Biochemistry: Brown and Cozzarelli Proc. Nad Acad. Sci. USA 78 (1981) RESULTS ca Catenates Duplex DNA Circles. Interlocking or fusion of DNA rings can readily be assayed by the reduction of their elec- trophoretic mobility in agarose gels. A characteristic ladder of slower-moving forms is produced or, if the products are suf- ficiently large, the DNA remains at the origin (4). By using this itA assay, catenation by gyrase and topoisomerase II' (2) were easily detected in crude extracts of E. coli. Two additional activities were discovered when the extract was fractionated by DEAE-Sephacel chromatography. Surpris- ingly, one of these coincided with a supercoil-relaxation activity FIG. 2. Catenation of nonhomologous DNA. A 200-MAl catenation that had properties characteristic of w. The protein, purified reaction mixture containing 1 ,ug each of nicked pA03 and nicked OX on the basis of the relaxation activity, was a 10,000-dalton poly- RF DNA and 2 ,±g of co was incubated for 3.hr and spread for electron peptide that comigrated on NaDodSO4polyacrylamide gels microscopy. The catenane shown contains four pAO3 and two OX RF with authentic A. Fig. 1A shows catenation of nicked #X174 molecules. (x51,000.) RFI DNA by the purified protein at molar ratios to DNA circles ranging from 0.3 (lane g) to 150 (lane b). The proof that the tions of cations for catenation were nearly identical to their crit- multimers are catenanes is presented below. ical concentrations for induction of DNA aggregation under The ratio of catenation to relaxation activity was constant similar ionic conditions (12), suggesting that the driving force throughout purification. Two other preparations purified by for catenation was provided primarily by polycation-promoted using different procedures by R. E. Depew and by J. C. Wang DNA aggregation. had similar specific activities. For all three preparations, 10-20 Under standard conditions for DNA aggregation, supercoil times more enzyme was needed to catenate than to relax half relaxation was inhibited so that the ratio of relaxation events of the DNA substrate, under the respective optima. As ex- (unit changes in Lk) to net catenation events was about 10. pected for w (1), catenation was inhibited by single-stranded Catenation Does Not Depend on Extensive Sequence Ho- DNA and required Mg(II) (Fig. 1B, lanes band c). We conclude mology. co and other type 1 topoisomerases have always been that the catenation activity is intrinsic to ct. assumed to act at base-paired regions (1, 8, 13). To determine The optimal conditions for catenation included low ionic if extensive sequence homology was required for catenation, strength and the presence of a polycationic DNA-condensing we used unrelated DNA molecules that could be distinguished agent, such as spermidine (Fig. 1B, lane a), spermine (lane e), by electron microscopy. With a mixture of pAO3 (1683 base or cobaltic hexammine (lane f) (12). The threshold concentra- pairs) (11) and #X DNA (5386 base pairs), heterologous products predominated (Fig. 2). (Although most of the products of the spermidine-promoted reactions were networks containing >100 A B circles, electron micrographs of more easily interpreted small a bc d e f g a b c d e f oligomers are presented.) Homologous circles are not prefer- Top of gel-- e-Top of gel entially linked. In a reaction with supercoiled ColEl and an equal mixture, by weight, of nicked ColE1 and nicked OX RFI DNA, the ratio of nicked ColEl-closed ColEl to nicked OX-closed ColEl dimers was very close to 1.