THE J O U R N A L OF BIOLOGICAL CHEMISTRY 1 i . t 1984 by The American Society of Biological Chemists, Inc. Vol. 259, No. 7 , Issue of April 10, pp. 4576-4581. 1984

Printed In 1J.S.A.

Potent Catenation of Supercoiled and Gapped DNA Circles by Topoisomerase I in the Presence of a Hydrophilic Polymer*

(Received for publication, October 7, 1983)

Robert L. Low& Jon M. Kaguni, and Arthur Kornberg From the Department of Biochemistry, Stanford University School of Medicine, Stanford, California 94305

An exceptionally potent DNA catenation activity, identified in an extract from Escherichia coli, has been purified and partially characterized. Catenation re- sults from the sequential action of the following two polypeptides: B, 34 kDa and identical to exonuclease 111; and a, 101 kDa and identical to DNA topoisomerase I (w protein). An additional requirement is that a small proportion of the circles be nicked in order to provide the substrate for exonuclease I11 to generate gaps, es- timated to be about 100 nucleotides long. Following exonuclease I11 digestion, one molecule of topoisomer- ase I can interlock per minute at 30 "C about 20 super- coiled and gapped DNA circles into a massively cate- nated network. The reaction requires Mg2+ and a hy- drophilic polymer (polyvinyl alcohol or polyethylene glycol) at about 770, but neither ATP nor spermidine. The hydrophilic polymer appears to drive catenation by condensing the DNA; decatenation by topoisomer- ase I proceeds upon removal of the polymer.

In the first few minutes of replication of plasmids bearing the Escherichia coli replication origin (oriC) by a crude enzyme system (1, Z), the supercoiled circles became part of an exten- sively catenated network. Inasmuch as catenation was not attributable to any of the known topoisomerases (3-5), puri- fication of the responsible activity was undertaken. Catena- tion of a variety of supercoiled circles proved to depend on the following three components: (i) a minority of gapped circles, generated by exonuclease I11 action on nicked circles; (ii) topoisomerase I in its recognized use of single-stranded regions to knot or catenate DNA (6); and (iii) a hydrophilic polymer at a concentration that can condense DNA (7) and aggregate proteins (8) to drive the reaction. This previously unrecognized potent catenation activity of E. coli topoisomer- ase I may prove to have physiological significance and may apply to other type I topoisomerases as well.

MATERIALS AND METHODS

Bacterial Growth and Preparation of Fraction II-E. coli HMS83 (PoDIZ, polBI, thy, lys, lac, rha, str') (9) was grown in 100-liter cultures (IO), harvested, and used as a source for purification of the catenation activity. Extracts were prepared by a lysozyme, heat-lysis method and precipitated with ammonium su1fat.e (0.39 g/ml of lysate) to yield a concentrate (Fraction 11) as described (10). The E. coli exonuclease I11 mutant, BW9109 ( l l ) , was grown in a 3-liter culture and harvested

* This work was supported by grants from the National Institutes of Health and the National Science Foundation. The costs of publi- cation of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertise- nent" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

j Present address, Department of Pathology, Washington Univer- sity School of Medicine, St. Louis, MO 63110.

~ _ _ ~ _ _ ~ __ __- __

in midlog; a Fraction I1 was prepared in a similar way. Chemicals and Resins-Chemicals and resins were purchased from

sources as listed (12) unless otherwise indicated. PVA' (type 11), polyethylene glycol (20 kDa), and ethidium bromide were obtained from Sigma.

Nucleic Acids and Enzymes-ColEl, M13 and 4x174 RF DNAs were prepared by described methods (13). Nicked M13 RFII DNA was made by digesting the covalently closed RFI with pancreatic DNase until about 50% of the circles were converted to RFII. Follow- ing phenol extraction, the nicked circles (RFII) were purified by sucrose gradient centrifugation: 10 to 30% linear gradients in 50 mM Tris'HC1 (pH 8.01, 250 mM NaCI, 0.5 mM EDTA at 45,000 rpm for 3 h in an SW 50.1 rotor a t 4 "C. Plasmid pDPT275 DNA (14), either unlabeled or 3H-labeled (2.4 X lo3 cpm/pg), and M13oriC26 RF DNA (15) were purified as outlined (16).

The restriction enzyme Xhol was from New England Biolabs and used according to their recommendations. Pancreatic DNase I was from Boehringer-Mannheim. Purified E. coli DNA topoisomerase I and anti-topoisomerase I IgG were gifts from James Wang (Harvard University). Exonuclease 111 was a gift from Bernard Weiss (Johns Hopkins University).

Catenation Reactions-The 25-pl reactions contained 50 mM Hepes (pH 8.0), 40 mM KCI, 11 mM MgC12, 0.2 mg/ml of bovine serum albumin, 6.7% (w/v) PVA, 100 to 500 ng of DNA as indicated, and 10 to 80 units of enzyme. One unit is defined as the amount of enzyme that catalyzes catenation of 1 ng of DNA/min a t 30 "C. Except where indicated, reactions were incubated 8 min a t 30 "C. In general, activity was measured by trapping labeled catenated DNA on a Millipore filter. With this assay, t.he reactions contained 500 ng of ("HI pDPT275 DNA and were terminated by addition of 100 wl of Buffer A (1% SDS, 0.5 M NaCI, 20 mM EDTA, and 50 mM Tris.HC1 (pH 8.0)), maintained at 37 "C, and immediately filtered, dropwise, on nitrocellulose filters (Millipore, type HAWP, 45 pm). The filters were washed with 2 ml of Buffer A, dried, and counted in toluene scintil- lation fluid. Less than 2% of the input DNA was retained on the filter in the absence of catenation.

For agarose gel electrophoresis, the reactions contained labeled or unlabeled DNA, were terminated by addition of 1% SDS, 20 mM EDTA, 0.01% bromphenol blue, and run on an 0.8% agarose gel, usually overnight, a t 1 to 1.5 V/cm in 40 mM Tris acetate (pH 8.0). 2 mM EDTA. Gels were stained 40 min a t 23 "C with 0.5 pg/ml of ethidium bromide, destained for 15 min in water, and photographed with Polaroid film, type 57.

Exonuclease ZII Assays-The 20-pl reactions contained 100 r n M Tris. HCI (pH 8.0), 2 mM dithiothreitol, 5 mM MgC12,900 ng of nicked [3H]M13 RF DNA (5.7 X lo3 cpm/pg), and 0.5 to 5 ng of enzyme. After a 15-min incubation at 30 "C, the reactions were spotted drop- wise onto squares (2 X 2 cm) of DEAE-cellulose (DE81) paper. The squares were washed for 5 min a t room temperature in 500 ml of 0.3 M ammonium formate, rinsed briefly with ether, and counted in a liquid scintillation counter. With extensive digestion, the limit of near 50% hydrolysis was reached. One unit is defined as the amount of enzyme that hydrolyzes 1 nmol of nucleotide in 30 min at 30 "C (17).

Protein Assuy-Protein concentrations were measured by the Coo- massie brilliant blue method of Bradford (18) using bovine serum albumin as a standard.

_______- The abbreviations used are: PVA, polyvinyl alcohol; SDS, sodium

dodecyl sulfate; RF, circular double-stranded replicative form; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid.

4576

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Potent Catenation by Topoisomerase I and Exonuclease I I I 4577

A. I C D

FIG. 1. Catenat ion requirements . l ieact ions contained 225 ng of .Yll:3ori('26 DSX. 6 , 7 ' , [ \ v / v ) I'YA, 11 mM MgCI,, and 0.5 p g of' Fraction I I protein except as indicated. Other components, reaction conditions, and the agarose electrophoresis were as detailed under "Materials and Methods." A, titration of I'VA and Mi". H . titration of Fraction 11. C', digest analvsis of catenated DNA. Lane I, M13oriCY6 DNA (22.5 ng); lane 2, same DNA digested with 5 units ofthe restriction enzvme XhoI, 60 min, 30 "C: lane 3 , catenated Mlhri( '26 DNA (225 ng): lone '1, same DNA as in /one 9 heated IO min. 6.5 "C; lane 5, same as DNA in lane 4 hut in addition exposed to 0.1 mg/ml of' proteinase K. 20 min, 30 "C: /one 6. 225 ng of catenated M13oriC26 DNA sedimented onto a ISr; (w/v) glycerol shell, phenol purified. and digested with 5 units of Xhnl , 60 min, 90 "C. I ) . catenation with other than oriC DNA. Standard catenation reactions (right / m e of each pair) contained 0.5 pg of Fraction I I protein; components otherwise listed under "Materials and Methods:" and DNAs as follows: ColEl DNA (400 ng), 4X RF (375 ng). r11>I'TYi5 DNA (600 na) . and MI3 RF (300 na). IJntreated DNAs are shown in the /eft /ones, respectivelv. Electrophoresis was 11 h at 1.5 V/cm.

RESULTS

A Potent Catenation Activity in a Crude E. coli Enzyme Fraction: Rquirement for PVA and Mg""In st,udies o f t h e replication of oriC plasmids by an ammonium sulfate fraction from a n E . coli lysate (1, 2), the DNA was observed very early in the reaction to have been converted to a form that failed to enter an 0.8% agarose electrophoretic gel.2 This template conversion proceeded in the absence of ATP, other ribonucle- oside triphosphates, and deoxyribonucleoside triphosphates, and required MgCI, and about 7 % PVA (Fig. 1A). Similar levels of other hydrophilic polymers, such as polyethylene glycol, were also effective (data not shown). A remarkably small amount (0.25 pg of protein) of the crude ammonium sulfate fraction (Fraction 11) sufficed (Fig. 1R); this amount is only 0.1% of that needed for the oriC replication reaction (1, 2).

The converted DNA resembled a catenated network.:' Heat treatment (6.5 "C, 10 min), with or without a subsequent exposure to proteinase K (0.1 mg/ml, 20 min, 30 "C), failed to liberate the DNA from the gel origin (Fig. 1C). However, digestion of the phenol-purified DNA agregate with XhoI, a restriction enzyme which cleaves M130riC26 DNA at two sites (15), quantitatively converted the immobilized DNA to the two known linear restriction fragments (Fig. 1C). These results are suggestive of a catenated network (19).

C'atcnation 1)oc.s Not Require the oriC Sequence-Several other circular duplex DNAs tested, including ColE1, 6x174 RF, the R100 plasmid pDPT275, and MI3 RF, were as active as M13oriC26 as substrates for catenation (Fig. ID).

Catenation Proceeds Rapidly and Can Re Assayed by Reten-

' R. S. Fuller, .I. M. Kaguni. and A. Kornherg. unpublished ohser-

I' Klertron microscopic examination showed a massive network of vations.

looped DNA st rands.

tion on a Millipore Filter-Catenation was assayed by trapping ["HIDNA on a nitrocellulose filter washed with 1% SDS, 0.5 M NaCl, and 50 mM EDTA. (This wash prevents DNA binding proteins from trapping uncatenated DNA.) Assayed either by filter retention (Fig. 2 A ) or gel electrophoresis (Fig. 2B) , the time course of catenation appeared to be the same; with 500

A. 400

A ~ 300 C - P

E 200 a n

"- 100

.- m - z I

I

0 6 2 4 6 8

TIME (min)

0 2 3 4 6 8

TIME (min) FIG. 2. Time course of catenation reaction. As described under

"Materials and Methods," standard catenation reactions containing 500 ng of ['HH]pDPT275 DNA (2.4 X 10' cpm/pg) and 0.5 pg of Fraction I I were incuhated at 30 "C. terminated with lrE (w/v) SDS and 20 mM EDTA, and analvzed hv Millipore filter binding ( A ) or agarose gel electrophoresis ( E ) .

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Potent Catenation by Topoisomeraqe I and Exonuclease I I I

IgG. Furthermore, the tu:@ catenase was unaffected by inhib- itors of gyrase (10 pM novobiocin or 2 mM oxolinic acid). (vi) The characteristic pattern of DNA cleavage using !i'-:''P-

either topoisomerase I or protein n (20). E. I 1 1

400 - - labeled single-stranded DNA was virtually identical with

Catenation of Purified M13 RFI Is Enhanced by M I 3 RFII- - The catenation activity of DNA topoisomerase I depends on

spermidine, a trivalent cation, and requires that one circle in the reaction be nicked (21, 22). In the PVA-dependent reac-

and quantitatively with supercoiled DNAs that are minimally contaminated with nicked forms. It is noteworthy that puri-

- fied @ lacks endonuclease activity as measured with either M13oriC26 or M13 RFI supercoils, either in the presence or absence of PVA.4 When M13 RFI was further purified by sucrose gradient centrifugation, a dependency on nicked DNA became demonstrable. Catenation of purified M13 RFI (220 ng) was negligible after 6 min at 30 "C, but became complete

- P 300 - -

200 - - tion with topoisomerase I and 8, catenation proceeds rapidly

0.0 0.02 0.04 0.06

PROTEIN ,9 lngl

FIG. 3 . Purified protein a and topoisomerase I relaxation activities and @ titration curves for catenation. A, relaxation assays (24 ~ 1 ) contained 20 mM Hepes (pH 8.0). 20 mM KCI, 5 mM MgCl2, 2 mM dithiothreitol, 5% (v/v) glycerol, 0.1 mg/ml of bovine serum albumin, 225 ng of Ml3oriC26 DNA, and purified protein n or DNA topoisomerase I as indicated. Reactions were incubated 40 min at 3O'C, terminated with 1% (w/v) SDS and 20 mM EDTA, and analyzed by electrophoresis in a 0.8% (w/v) agarose gel. R, catenation assays with protein /j (9 X 10' units/mg), monitored by the filter- binding assay, contained the standard assay components detailed under "Materials and Methods" supplemented with either 200 units of protein (r (0) or topoisomerase I (0) catenation activity.

ng of the ['H]pDPT275 DNA (a level which nearly saturates the catenation reaction), conversion is about 50% complete in 3 to 4 min at 30 "C.

Fractionation of the Catenation Activity Defines Two Essen- tial Proteins, N and &-Initial attempts to purify the catena- tion activity of Fraction I1 on phosphocellulose, DNA-cellu- lose, and heparin-agarose gave poor and variable recoveries. Filtration on Sephacryl S-200 confirmed the existence of two separable, complementing factors. One, called N, was eluted with the bulk of the protein near the void volume; the second, called 8 , was eluted just after the position of serum albumin. Supplementing one or the other of these fractions provided assays for purifying each of these factors.

Protein N Is Identical to D N A Topoisomerase I (w Protein)- The evidence is as follows. ( i ) With the filter-binding cate- nation assay saturated with @, (Y was further purified (on Sephacryl S-200, DEAE-Sephacel, and blue dextran Sepha- rose columns) about 1000-fold to yield a product of 101 kDa which appeared as the major band on SDS gel analysis (data not shown) and was indistinguishable from topoisomerase I by this analysis. ( i i ) This most purified fraction relaxed su- percoiled DNA in the absence of PVA and showed a specific activity for relaxation comparable to that obtained with pu- rified DNA topoisomerase I (Fig. 3A); about 0.6 ng of either protein fully relaxed 225 ng of supercoiled M13oriC26 DNA in 40 min a t 30 "C. ( i i i ) DNA topoisomerase I was as effective as n in complementing @ in the PVA-dependent catenation assay; specific activities were 8-10 X 10" units/mg. (iv) In catenation assays containing either a saturating level of N or topoisomerase I, titration curves for /3 were identical (Fig. 3 H ) . In the absence of @, these levels of N and topoisomerase I were by themselves inactive. (v) The N:@ catenation reactions monitored either by agarose gels or filter retention were sensitive to anti-topoisomerase I rabbit IgG; about 50-60% inhibition was observed when 0.3 ng of purified (Y was first incubated for 10 min a t 4 "C with 1 pg of anti-topoisomerase

when supplemented with 100 ng of purified MI3 RFII DNA (Fig. 4). As little as 30 ng of the RFII DNA was sufficient to enhance complete catenation of 225 ng of RFI (data not shown). Thus, one nicked M13 circle sustains catenation of about 10 covalently closed circles.

Purified Protein @ Is Identical to Exonuclease 111-The evidence is as follows. ( i ) With the filter-binding catenation assay saturated with topoisomerase I, protein @ was purified (by BioRex 70, hydroxylapatite, Bio-Gel P100, and valyl- Sepharose chromatography) about 1600-fold to near homo- geneity to yield on SDS-gel analysis a single major band of 34 kDa coincident with an external marker of purified exo- nuclease I11 (Fig. 5). Sedimentation and gel filtration studies of pufified 8 revealed an s~~~.,,. of 3.1 and a Stokes radius of 24.5 A, indicative of a monomeric native structure (data not shown); these values are in agreement with those determined for exonuclease I11 (25-27). ( i i ) Purified exonuclease I11 sub- stituted for purified 8 in the PVA-dependent catenation re- action; both purified fractions showed the same specific activ- ity for catenation, 7-9 X 10' units/mg. (iii) In exonuclease assays using nicked [:'HIM13 RF as substrate, @ exhibited exonuclease activity (2.1 X lo5 units/mg) comparable to that of purified exonuclease I11 (2.9 X 10" unit/mg). (iv) A fraction

RFII NONE 100 ng

min 0 2 3 4 6 0 2 3 4 6

FIG. 4. Catenation of supercoiled circles is enhanced by nicked duplex circles. Standard catenation reactions containing 240 units of purified CY, 280 units of purified /j, and 330 ng of M13 RFI DNA purified bv sucrose gradient centrifugation were incubated without or with 100 ng of M13 RFII DNA at 30 "C for the indicated times.

R. L. Low and A. Kornberg, unpuhlished ohservations.

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Potent Catenation by Topoisomerase I and Exonuclease I I I 4579

MARKERS

BSA

OVA

CA

FIG. 5. Gel electrophoresis of purified exonuclease 111 and protein 8. Protein @ (valyl-Sepharose fraction, 0.5 p g ) was dialyzed :1 h at 4 "C against 10 mM Hepes (pH K O ) , 2 mM dithiothreitol, 0.5 mM EDTA, and 5% (w/v) glycerol to remove excess salt. The dialyzed protein /j, exonuclease I11 (0.45 pg) , and marker proteins ( M A , bovine serum alhumin, 0.5 pg; OVA, ovalbumin, 0.25 pg; CA, carbonic an- hydrase, 0.5 pg) were heat-denatured under reducing conditions (2 min, 100 "C in 0.2% SDS, 0.2 M dithiothreitol) and electrophoresed in a 30-ml 10% acrylamide (1/40 concentration bisacrylamide) slab gel containing 0.1% SDS in a Tris glycine buffer system (23). The gel was stained with silver (24) and was purposefully overloaded to emphasize the absence of other silver-staining protein bands.

11, prepared from the exonuclease I11 deletion mutant RW9109, showed a ratio of /3 to topoisomerase I activity reduced about 10-fold compared to that from HMS83 (data not shown).

Pretreatment of the DNA with Exonuclease 111 Allows Rapid Catenation by DNA Topoisomerase Alone-Identification of protein I j as exonuclease 111 and the requirement for a nicked circle made it likely that a "gapped" circle was the preferred template. After 500 ng of [:'H]pDPT275 DNA was exposed to 0.09 ng of exonuclease I11 (an amount that causes filter retention of 140 ng of DNA in a 3-min catenation reaction containing 7% PVA and 0.25 ng of topoisomerase I) for 5 min at 30 "C in the absence of PVA and purified with phenol, exonuclease 111 was no longer needed in the catenation reac- tion (Fig. 6A). With this gapped template and 0.5 ng of topoisomerase I, about 50% of the ["H]pDPT275 DNA was catenated in only 1 min a t 30 "C. Addition of single strand binding protein in amounts sufficient to coat the DNA in the gapped regions showed no effect on either the rate or extent of the reaction (data not shown). In the absence of topoi- somerase I, no DNA was catenated ( 4 0 ng were retained on a filter). The initial, linear portion of the titration curve for topoisomerase I under standard catenation conditions (Fig. 6H) indicates that about 1 molecule of enzyme catalyzes catenation of at least 60 circles in 3 min a t 30 "C. As indicated by separate gel assays, this catenation reaction still requires 6 to 8% PVA (data not shown).

As monitored by gel electrophoresis, exonuclease 111 has been previously found to convert plasmid DNA into non- catenated, high molecular weight networks in the presence of spermidine.5 Presumably, such networks result from wide- spread annealing, among several circles, of homologous single- stranded segments generated by extensive degradation by

~~

' M. Pastorcic and N. Cozzarelli, personal communication.

0 1 2 3 4 5 0.0 0.2 0.4 0.6 0.8 1.0

TIME Imm) DNA Toponsamerase I lngl

FIG. 6. A gapped template promotes rapid, PVA-dependent catenation with topoisomerase I alone. The exonuclease reaction mixture to prepare a gapped template contained the following in 960 PI: 50 mM Hepes (pH R.O), 2 mM dithiothreitol, 5 mM MgCI,, 50 pg of' [:'H]pDPTL75 DNA (2.4 X 10:' cpm/pg), and 9 ng of exonuclease 111. Following a 5-min, 30 "C incubation, the reaction was extracted once with phenol and twice with ether, precipitated with 2 volumes of' ethanol in the presence of 0.3 M sodium acetate, and resuspended in 50 p1 of 10 mM Tris.HCI (pH 7.5). 0.5 mM EDTA. Recovery of DNA was 955 . A, time course. Catenation assays by filter binding were as described under "Materials and Methods" with 500 ng of gapped ["HIDNA and 0.5 ng of topoisomerase I, but no exonuclease 111. H . titration of topoisomerase I. Catenation reactions identical to those in A , except for varied levels of topoisomerase I, were incuhated :1 min at 30 "C.

exonuclease 111. In the present studies, exonuclease I11 itself did immobilize some of the plasmid DNA at the top of an agarose gel but the levels of enzyme required were a t least twenty times higher than those used to catenate DNA in the presence of topoisomerase I. Thus, exposure of pDPT275 DNA (500 ng) to exonuclease I11 (0.09 to 4.5 ng) for 10 min a t 30 "C showed minimal trapping of the DNA at only the higher concentrations (Fig. 7). Even after a 30-min incubation a t 30 "C with 0.09 ng of exonuclease 111, the level used to catenate DNA completely in the presence of topoisomerase I, no significant amount of DNA was retained at the gel origin (data not shown).

Catenation Is Reversible in the Absence of PVA-Catenated ["H]pDPT275 DNA was prepared, collected by centrifugation, phenol purified, and assayed for decatenation using topoi- somerase I (Fig. 8). After 60 min, about 50% of the product appeared to be decatenated, as indicated on agarose gel elec- trophoresis by the appearance of nicked RFII and relaxed RFI DNA. These data confirm that the immobilized DNA is catenated; reversibility of the reaction implies that catenation is driven by PVA.

DISCUSSION

Supercoiled circular DNA becomes rapidly and completely catenated by E. coli topoisomerase I when (i) a low level of gapped circles, generated by exonuclease I11 action on nicked circles, provide single-stranded regions estimated to be about 100 nucleotides long, and when (ii) a high concentration of a hydrophilic polymer drives the reaction. Although this obser- vation is consistent with the known properties of these several agents, the catalytic efficiency of topoisomerase I in convert- ing circular DNA rapidly into a massive catenated network had neither been observed previously nor anticipated.

The catenation of circular DNA manifests the ability of a topoisomerase to pass one segment of DNA through another. The capacity of E. coli topoisomerase I to knot single-stranded DNA and to catenate duplex rings is well established (21, 22, 28). These reactions do not require ATP and, typical of a type I topoisomerase, proceed by concerted breakage and reunion of a single strand of DNA; with a duplex DNA circle, one of

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4580 Potent Catenation by Topoisol

TIME (rnin) "

I reri Origin Origin

R F l l R F l I

R FI RFI

1 2 3 4 5 6 7

FIG. 7 ( l v f t ) . Levels of exonuclease 111 sufficient for catena- tion do not trap DNA in uncatenated networks. Reactions (2.5 p l ) contained 50 mM Hepes (pH 8.0). 2 mM dithiothreitol, 11 mM YlgC12. 0.1 mg/ml of' hovine serum albumin, 40 mM KCI, 7% (w/v) Pi'A. 500 ng of' [:'H]pDPT275 DNA. and exonuclease 111 as indicated. Alter a IO-min. 30 "C incuhation, DNA was analyzed hy 0.8% agarose gel electrophoresis as described under "Materials and Methods."

FIG. 7 (right). PVA-dependent catenation is reversible. ('atenated [:'H]pDI'T275 DNA was prepared in a standard catenation reaction as detailed under "Materials and Methods," scaled up 20- fold. After a IO-min, 30 "C incuhation, the reaction mixture (0.5 ml) was layered over a 25-pl shelf of 50 mM Tris. HCI (pH 8.0). 0.5 mM EDTA. 15r; (w/v) glycerol, and centrifuged 10 min at room temper- ature in an Eppendorf centrifuge at l.5,000 X g. The shelf was collected. and the catenated DNA was extracted once with phenol and twice with ether. The uncatenated and catenated [:'H]pDPT275 DNA, :io0 ng each. are shown in lanes 1 and 2, respectively. Decate- nation reactions (2.5 pl) containing 50 mM Hepes (pH 8.0). 11 mM MgCI?. 4 0 mhl KCI, 0.1 mg/ml o f hovine serum albumin, 500 ng of catenated [:'H]pDPT275 DNA, and 50 ng of topoisomerase I were incu1)ated at 30 "C for the indicated times (lanes 3-7) and analyzed by 0.85 agarose gel electrophoresis as described under "Materials and Methods."

the strands must be nicked. Homologous strand pairing is not involved.

IJnlike earlier observations of topoisomerase I catenation activity, in which 15:l and 60:l ratios of enzyme to DNA circles were needed (21, 22) and incubation times extended to 30 and 60 min, one molecule of enzyme in this study can catenate 20 circles/min. A strong dependence on salt and a polyvalent cation ( e . g spermidine) to promote DNA aggre- gation (29) is not evident in the present study presumably because of the contribution made by the hydrophilic polymer. The effect of gapped circles had not been evaluated before. Exposure of specific nucleotide sequences preferred for single strand passage'' is presumably responsible for the action of the gapped circles in this study and may have significant effects on topoisomerase I catenation activity under other conditions as well. For catenation of nicked circles (in the presence of spermidine), the need for the much higher levels of topoisomerase I is presumably to exploit the small gapped regions that result from transient fraying at the nicks.

A comparison of the relative effectiveness of topoisomerase I in its relaxation and catenation activities can only be ap- proximate. As shown in Fig. 3, 0.25 ng (2 fmol) of topoi- somerase I relaxed about 50% of the circles (225 ng, 34 fmol)

I' F. Dean and N. Cozzarelli, personal communication.

nerase I and Exonuclease 111

in 40 min. Assuming 30 negative twists/circle and a linear rate for the 40-min interval, a topoisomerase molecule carried out about 7 nicking-closing (relaxation) events/min as com- pared to 20 catenations (see above).

When catenation reactions include excess exonuclease 111 to generate large gaps, some DNA aggregates form independ- ently of catenation (Fig. 7). This synaptase reaction. identified first with gapped DNA in the presence of spermidine,5 appears t o resemble the synapsis of homologous single-stranded circles with duplex circles mediated by recA protein and ATP (30, 31); addition of topoisomerase I catalyzes intertwining of these annealed strands to generate stable hemi-catenaries (32). In the present study, the predominant product appears to be a network of truly catenated duplex circles for several reasons. (i) The small gaps sufficient for rapid and extensive catenation do not provide significant immobilization of the DNA on agarose gels or Millipore filters in the absence of topoisomerase I. (ii) The gapped circles participate in a cata- lytic fashion, i.e. one gapped circle can accept nearly ten supercoiled forms; while, in hemi-catenation, a singly gapped circle can only trap at most one neighbor. ( i i i ) Most of the catenated network can be decatenated by topoisomerase I in the absence of PVA. T h e possibility that much larger gaps might favor a hemi-catenation reaction in the presence of PVA remains a possibility.

Gyrase reconstituted from its A and B subunits also cata- lyzes catenation in the presence of polyvinyl alcohol and in the absence of spermidine. However, this reaction requires ATP and levels of enzyme at least equivalent to the number of DNA circles. The specific activity of gyrase in such reac- tions, 2-4 X 10' units/mg (as measured by the filter-binding assay used for topoisomerase I), is only about one-hundredth tha t of the specific activity of topoisomerase I in the present study.

In view of the occurrence of single-stranded DNA regions (at replication forks, in DNA repair and recombination, and in viral replicative forms) and the likelihood that macromo- lecular crowding in the cell substitutes for a hydrophilic polymer, this catenation activity of E. coli topoisomerase I may be manifested in oioo and in other prokaryotic and eukaryotic cells where t-ype I topoisomerases are abundant.

Acknowledgments-We thank Dr. J . Wang for advice, Drs. N. Cozzarelli and F. Dean for their suggestions, particularly that exo- nuclease 111 might be a key component in the catenation reaction, and L. Bertsch for assistance in preparing this manuscript. We are indebted to F. Dean and N. R. Cozzarelli for performing DNA analyses.

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1. Fuller, R. S., Kaguni, J . M., and Kornherg, A. (1981) Proc. Natl.

2. Kaguni, J. M., Fuller, R. S., and Kornherg, A. (1982) Nature

3. Cozzarelli, N. R. (1980) Science (Wash. 11. C.) 207, 953-960 4. Wang, J. C. (1981) Enzymes 14, 331-344 6 . Gellert, M. (1981) Enzvmes 14, 345-366 6. Depew, R. E., Liu, L. F., and Wang, d. C. (1978) J . Biol. Chem.

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R L Low, J M Kaguni and A Kornbergthe presence of a hydrophilic polymer.

Potent catenation of supercoiled and gapped DNA circles by topoisomerase I in

1984, 259:4576-4581.J. Biol. Chem. 

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