MOLECULAR AND CELLULAR BIOLOGY, Jan. 1983, p. 126-131 Vol. 3, No. 10270-7306/83/010126-06$02.00/0Copyright © 1983, American Society for Microbiology

In Vivo Catenation and Decatenation of DNAtJANET E. MERTZ* AND TIMOTHY J. MILLER:

McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, Wisconsin 53706

Received 8 June 1982/Accepted 1 October 1982

We have noted previously that when circular, but not linear, DNA or chromatinwas injected into Xenopus laevis oocytes, much of it went through an intermediateform in which it did not readily enter an agarose gel; after a few hours, itreappeared as monomer DNA that had acquired its full complement of nucleo-somes (T. J. Miller and J. E. Mertz, Mol. Cell. Biol. 2:1581-1593, 1982). Wedetermined, using electron microscopy and a variety of biochemical techniques,the structure of this aggregated material. Most of it was oligomeric and multimericcatenanes of the injected sample. In addition, injection of DNA that had beencatenated in vitro with DNA gyrase resulted in the conversion of most of it back tomonomer circles. These findings demonstrate directly that both catenation anddecatenation ofDNA occur in vivo under physiological conditions. Whether thesereactions play a crucial role in nucleosome formation, as well as in DNAreplication and recombination, remains to be determined.

In procaryotic cells, DNA supercoiling hasbeen clearly shown to play roles in DNA replica-tion, transcription, and genetic recombination(see reference 6 for review). In eucaryotes,DNA possesses a negative superhelical densitybecause it interacts with histone proteins to formnucleosomes (7). Linear DNA, being incapableof storing energy in the form of supertwists, failsto reconstitute into proper chromatin in vivo(13). These findings indicate that DNA super-coiling may also play an important role in prop-erly arranging chromosomal proteins on DNA.DNA topoisomerases are a class of enzymes

that regulate the superhelical density of DNA(see references 3, 4, and 6 for recent reviews).In addition to removing and, in some cases,introducing supercoils into DNA, these enzymesform and resolve catenated (interlocked) andknotted DNA rings. Decatenation of DNA maybe particularly important biologically to enablethe separation of chromosomes after replicationand genetic recombination.The ability to both catenate and decatenate

DNA in vitro has been shown to be associatedwith topoisomerases from a variety of sources(2, 8, 10, 11). The proposed mechanism bywhich topoisomerases catenate DNA rings pre-dicts that the reaction should be reversible.However, whereas the catenation reaction re-quires low salt concentration and a polyvalent

t This paper is dedicated to Norman Davidson, a manwhose numerous contributions to chemistry and molecularbiology made experiments of the type described here feasible,on the occasion of his 65th birthday.

t Present address: Norden Laboratory, Lincoln, NE 68521.

cation such as spermidine, the decatenation re-action occurs only at higher salt concentrationor in the absence of polyvalent cations.While performing experiments concerned

with the reconstitution into chromatin of circularsimian virus 40 (SV40) DNA injected into thenuclei of Xenopus laevis oocytes, we noted thatmuch of the DNA went through an intermediateform in which it did not readily enter a 1.4%agarose gel even though it had been deprotein-ized by treatment with both proteinase K andphenol (13, 14); after a few hours, it reappearedas monomer supercoiled DNA (see Fig. 5 ofreference 14 for an example). Electrophoresisindicated that some of this aggregated materialmigrated in the positions of dimeric and trimericlength SV40 DNA molecules (see Fig. 4 ofreference 13 for examples). This same phenome-non of aggregation followed by disaggregationwas also observed when circular pBR322,ColEl, or Drosophila melanogaster H2A his-tone DNA was injected (T. J. Miller and J. E.Mertz, unpublished data). On the other hand,unit-length linear SV40 DNA failed to form thisaggregated material when treated in an analo-gous manner (see Fig. 1 and 4 of reference 13 forexamples). Therefore, this aggregated materialwas unlikely to be either recombinants or atrivial artifact of the experimental protocol.

In the experiments reported here, we showthat: (i) most of this aggregated material isoligomeric and multimeric catenanes of the in-jected sample; and (ii) catenated DNA injectedinto Xenopus oocytes resolves back to monomercircles.

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IN VIVO CATENATION AND DECATENATION OF DNA 127

TABLE 1. Summary of oligomeric DNAs producedin Xenopus oocytesa

Structure No. observed

Simple catenanesDimer ....... .............. 32Trimer ........ ............ 17Tetramer ........ ........... 4Pentamer ................... 1Hexamer ........ ........... 1

OtherNoncatenated dimer ..... ...... 9Monomer catenated with

noncatenated dimer .... ...... 1

a The DNA present in fractions 7 through 13 of thesucrose gradient depicted in Fig. 1 was pooled andbanded to equilibrium (type 50 rotor; 35,000 rpm for 47h at 15°C) in a density gradient of CsCl (averagedensity, 1.700) containing 10 mM Tris-hydrochloride(pH 7.6)-i mM EDTA to reduce the level of contami-nation with endogenous oocyte nucleic acids. After-wards, the material in the fractions containing most ofthe radioactivity was pooled and mounted for electronmicroscopy in 40% formamide essentially as describedby Davis et al. (5). Randomly selected areas of plati-num-shadowed grids were examined and photo-graphed. The lengths of the molecules were deter-mined; in all cases, each DNA ring measured within10% of the length or twice the length of monomerSV40 DNA molecules present in the same photograph.Excluded from this table are the numerous monomericand approximately trimeric length DNA moleculesthat were also observed. Although some of the lattermay have been noncatenated trimers of SV40 DNA,most of them were probably Xenopus mitochondrialDNA that was present as a contaminant in the sample.

stage 6) oocytes were isolated from adult femaleXenopus laevis, incubated at 19°C in HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid)-modified Barth solution and injected intranuclearlywith the indicated samples as described previously(15, 16).

Purification of radiolabeled DNAs from injected oo-cytes. After incubation at 19°C in HEPES-modifiedBarth solution for the indicated times, the reactionswere terminated by homogenization of the oocytes ortheir nuclei directly in proteinase K buffer (50 mMTris-hydrochloride [pH 7.4], 10 mM EDTA, 1% sodi-um dodecyl sulfate, 750 p.g of proteinase K per ml, 50pul per oocyte) and incubation at 37°C for 2 h. Thehomogenized mixtures were extracted twice with 0.1M Tris-hydrochloride (pH 9.0)-saturated phenol:chloroform:isoamyl alcohol (25:25:1). The nucleic ac-ids present in the samples were then precipitated withethanol and suspended in 10 mM Tris-hydrochloride(pH 7.6)-i mM EDTA-10 mM NaCl before sedimenta-tion or electrophoresis.

RESULTSXenopus oocytes catenate circular DNA. To

characterize the aggregated material formed inoocytes, monomer circular 3H-labeled SV40

5 10 15 20 25FRACTION NUMBER

FIG. 1. Sucrose gradient analysis of SV40 DNAafter incubation in Xenopus oocytes for 30 min. Fortyoocytes were each injected intranuclearly with 5 ng ofmonomeric 3H-labeled SV40 DNA at a concentrationof 250 ,ug/ml in injection medium. After incubation inHEPES-modified Barth solution for approximately 30min at 19°C, the nuclei were manually dissected out ofthe oocytes as described previously (15) and homoge-nized in 2 ml of proteinase K buffer. The DNA waspurified as described in the text, with greater than 95%of the input radioactivity being recovered. It was thensedimented (SW41 rotor; 39,500 rpm for 5 h at 4°C) in a5 to 20% (wt/vol) gradient of sucrose containing 10 mMTris-hydrochloride (pH 7.6)-i mM EDTA-0.10 MNaCl layered on top of a 1.0-ml cushion of 60%sucrose. Fractions (0.40 ml) were collected from thebottom of the gradient. The amount of 3H-labeledSV40 DNA present in one-tenth of each fraction wasdetermined by scintillation spectroscopy. The approx-imate positions of monomer-length supercoiled (I) andnicked circular (LI) SV40 DNAs were determined fromuninjected 3H-labeled SV40 DNA sedimented concur-rently in a parallel gradient.

MATERIALS AND METHODSDNA and enzymes. Supercoiled (form I) 3H-labeled

SV40 DNA (>991% monomers; specific activity, ca. 1.5x 105 cpm/,Lg) was isolated from virus-infected mon-

key cells and purified as described previously (14).Although initially containing less than 5% nicked cir-cular molecules (form II), the preparation of DNAused in the experiments described here containedapproximately 60% nicked molecules because of radia-tion damage and nonspecific endonucleolytic cleav-ages that occurred during prolonged storage at 4°C.Catenated 3H-labeled ColEl DNA (specific activity,

ca. 3 x 104 cpm/,lg) was the generous gift of N.Cozzarelli. It was prepared as described previously (9)by incubation at a concentration of 2 pmol/ml withEscherichia coli DNA gyrase in the presence of 5 mMspermidine. The oligomeric molecules were separatedfrom the monomeric ones by sedimentation in a su-

crose gradient.Restriction endonucleases were obtained from com-

mercial sources. They were used as recommended bythe manufacturers.

Handling and injection of oocytes. Full-grown (early

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128 MERTZ AND MILLER ML EL IL

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FIG. 2. Electron micrographs of oocyte-produced catenanes of SV40 DNA. The experimental protocol wasas described in the footnote to Table 1. All of the micrographs in this composite are at the same magnification. a,Monomer SV40 DNA molecules; b, a catenated dimer and a supercoiled monomer; c and d, dimers consisting ofone supercoiled and one relaxed DNA molecule; e through h, trimer, tetramer, pentamer, and hexamer,respectively.

DNA was injected intranuclearly and reisolated30 min later. The DNA was treated with protein-ase K and sodium dodecyl sulfate and extractedwith phenol. Afterwards, it was sedimented in asucrose gradient. As shown in Fig. 1, 13% of theinjected radiolabeled DNA was distributedamong fractions 1 through 12 of the gradient. Onthe other hand, only 1% of the radioactivity wasfound in the corresponding fractions of a gradi-ent run in parallel with an uninjected sample ofthe same DNA preparation (data not shown).Therefore, incubation in Xenopus oocytes re-sults in some of the SV40 DNA undergoingchanges that cause it to sediment at a variety ofrates greater than that characteristic of eithersupercoiled or relaxed circular monomer SV4ODNA.The radiolabeled SV40 DNA in fractions 1

through 6 and 7 through 13 of the sucrosegradient were pooled separately, concentratedby precipitation with ethanol, and purified fur-ther by banding to equilibrium in CsCl density

gradients (see footnote to Table 1 for protocol).All of the radioactivity banded at the densitycharacteristic of pure SV4O DNA (data notshown). We therefore concluded that this fast-sedimenting material contains little or no Xeno-pus RNA or amplified ribosomal DNA, since thelatter two both have significantly higher densi-ties than SV40 DNA does.At this stage, the only contaminants remaining

in the viral DNA preparations should have beenXenopus chromosomal and mitochondrialDNAs. Since we injected 5 ng of SV40 DNA intoa tetraploid cell that contained only 12 pg ofchromosomal DNA, the first of these contami-nants could be largely ignored. By reisolatingthe injected DNA from nuclei rather than fromwhole oocytes we succeeded in eliminatingmost, but not quite all, of the mitochondrialDNA. Therefore, when the purified DNA fromfractions 7 through 13 of the sucrose gradientwas examined by electron microscopy, manymolecules approximately three times the length

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IN VIVO CATENATION AND DECATENATION OF DNA 129

FIG. 3. Electron micrograph of approximately one-third of a DNA network. The complex DNA structureshown here was obtained from fractions 1 through 6 of the sucrose gradient depicted in Fig. 1 and was purifiedand prepared for electron microscopy as described in the footnote to Table 1. The large spherical particlescontaminating the sample were picked up accidentally during purification of the DNA and may representglobules of mineral oil.

of monomer SV40 DNA were seen. Besidesthese and monomer SV40 DNA, we also ob-served a variety of other structures (see Fig. 2for examples and Table 1 for summary). Threethings of note about these latter DNA moleculeswere: (i) they sometimes consisted of both su-percoiled and relaxed DNA rings (Fig. 2c and d);(ii) most of the DNA rings within one structurewere linked to a central "key" molecule; and(iii) most clearly appeared to cross another ringtwice rather than once. These findings are allindicative of catenanes.The DNA from fractions 1 through 6 of the

sucrose gradient was also examined by electronmicroscopy. In this latter case, huge DNA net-works were observed (Fig. 3). They containedcentrally connecting fibers and were similar inappearance to the interlocked DNA networksseen when circular DNAs are catenated in vitro

with preparations of purified topoisomerases(see reference 10 for example).To exclude further the possibility that these

molecules were intermediates in DNA replica-tion or recombination rather than catenanes,each of these DNA preparations was incubatedwith BamHI, a restriction endonuclease thatcleaves SV40 DNA at one site. The resultantmolecules were then electrophoresed in a 1.4%agarose gel, along with appropriate size mark-ers. Fluorographic analysis of the gel indicatedthat essentially all of the radioactivity migratedin the position of unit-length linear SV40 DNA(data not shown). We therefore conclude thatmost of the aggregated radiolabeled materialproduced when the 3H-labeled SV40 DNA wasinjected into oocytes consisted of catenatedoligomers and multimers of circular SV40 DNAmolecules.

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130 MERTZ AND MILLER

Xenopus oocytes decatenate interlocked DNArings. The catenanes that form when circularDNA is injected into oocytes disappear withprolonged incubation. However, since 20 to 40%of the injected molecules are degraded becauseof failure to hit the nucleus (13, 17), we could notdirectly exclude the possibility that the cate-nanes had also been degraded rather than re-solved back to monomer circles.To solve this problem, 3H-labeled ColEl

DNA was catenated in vitro with purified DNAgyrase. The resulting oligomeric catenanes werepurified and then injected into the nuclei ofXenopus oocytes; 21 h later, the reaction wasterminated, and the radiolabeled DNA was puri-fied and analyzed by electrophoresis in a 0.8%agarose gel (Fig. 4). The results indicate that,whereas <5% of the input DNA was monomer-ic in length, >95% of the product was. Further-more, since 60 to 70% of the radioactivity wasrecovered, a majority of the catenated materialwas converted to supercoiled monomers. (Theslight retardation in the mobilities of the super-coiled and linear monomeric DNAs present inlane 2 of the gel shown in Fig. 4 was probablythe result of this sample containing a largeamount of nonradiolabeled Xenopus oocyte nu-cleic acid.) We therefore conclude that Xenopusoocytes can decatenate, as well as catenate,circular DNA molecules.

DISCUSSIONThe experiments reported here demonstrate

directly that both catenation and decatenation ofDNA occur in vivo under physiological condi-tions. Therefore, the finding by others that thesereactions can be performed in vitro is unlikely tobe a biochemical artifact. However, the reactionconditions used to generate catenanes in vitrowere always different from those used to resolvethem back to monomer rings. Recently, Kras-now and Cozzarelli (9) have shown that the rolein vitro of polyvalent cations such as spermidinemay be solely to enable aggregation of thesubstrate DNA, thereby facilitating the juxtapo-sition of DNA segments involved in the reac-tion. Since DNA normally exists in a compactedstate in association with histone proteins, elec-trostatic repulsion between negatively chargedDNA molecules would not be expected to hinderthe catenation and decatenation reactions invivo. In support of this hypothesis is our obser-vation that SV40 minichromosomes (i.e., SV40DNA isolated from virus-infected monkey cellsin association with histone proteins) also formcatenanes when injected into Xenopus oocytes(see Fig. 8 of reference 14).One peculiar observation is the finding that

this aggregated material is also produced whencircular, but not linear, DNA is injected into the

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FIG. 4. Decatenation of DNA in Xenopus oocytes.A 20-nl portion of catenated 3H-labeled ColEl DNA,prepared as described in the text, was injected intranu-clearly into each of 40 oocytes at a DNA concentrationof approximately 50 jig/ml. After incubation of 19°Cfor 21 h, the oocytes were homogenized in proteinaseK buffer. The DNA in the sample was purified asdescribed in the text and electrophoresed at 33 V for16 h in a 0.8% agarose gel submerged in 4x TA buffer(0.16 M Tris-acetate [pH 8.3], 0.08 M sodium acetate,8 mM EDTA). Afterward, the gel was impregnatedwith En3Hance (New England Nuclear Corp.) andfluorographed at -70°C with Kodak XAR-5 film. Theamount of DNA in each band was determined bydensitometry as described previously (13). 1 and IIindicate the positions of supercoiled and relaxed circu-lar DNA molecules, respectively. Lane 1, approxi-mately 0.8 ,ul of the catenated ColEl DNA sampleused in the injections; lane 2, the catenated ColElDNA after incubation in oocytes; lane 3, uninjectedmarkers of ColEl DNA.

cytoplasm of oocytes (see Fig. 1 of reference13). Mattoccia et al. (12) have reported thatcomplex DNA is only formed when nuclear andcytoplasmic extracts of Xenopus oocytes areincluded together in in vitro reactions. There-fore, a topoisomerase activity or a componentneeded for its functioning may exist in thecytoplasm of Xenopus oocytes.

Examination by electron microscopy of thefast-sedimenting material indicated that some ofit was noncatenated dimers (Table 1). Theorigin of this material is unknown. Likely expla-nations are: (i) these molecules preexisted in theDNA preparation used in the experiment; and(ii) they were produced by recombination duringthe incubation in the oocytes. Consistent with

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IN VIVO CATENATION AND DECATENATION OF DNA 131

the latter hypothesis is the finding that thesestructures are also seen when SV40 DNA isincubated in a cell-free extract of Xenopus oo-cytes (1).

Lastly, the most interesting question raised bythese studies is whether catenation and decaten-ation of DNA play a crucial role in nucleosomeformation as well as in DNA replication andrecombination. Findings consistent with an affir-mative answer to this question include: (i) topo-isomerases are needed to relieve the supertwiststhat are generated when DNA wraps aroundcores of histone proteins (7); (ii) a correlationexists between the appearance in oocytes ofinjected DNA molecules that are fully reconsti-tuted into chromatin and the disappearance ofDNA catenanes (14); and (iii) linear DNA nei-ther catenates nor forms chromatin containingregularly spaced nucleosomes in vivo (13).However, based upon our present knowledge

about topoisomerases, an equally reasonablehypothesis is that these enzymes play a purelypassive role, with their primary function being toresolve catenanes that form during DNA replica-tion, recombination, and packaging. Looked atfrom this latter point of view, the observedcatenation and decatenation of circular DNAinjected into Xenopus oocytes may be a trivialconsequence of the experimental conditionsused. The high DNA concentration at which thesample was injected initially favored the catena-tion reaction; however, as the DNA moleculesgradually diffused with time throughout the nu-cleus, decatenation became predominant. Fur-ther experiments will be needed to distinguishbetween these two hypotheses.

ACKNOWLEDGMENTS

We are indebted to Nicolas Cozzarelli for discussions andfor supplying us with in vitro catenated ColEl DNA. We alsothank Catharine Conley for technical assistance and RichardBurgess, Bill Sugden, and Howard Temin for helpful com-ments on the manuscript.

This work was supported by Public Health Service researchgrants CA-07175 and CA-22443 from the National CancerInstitute. T.J.M. was supported by fellowship PF-1678 fromthe American Cancer Society.

LITERATURE CITED1. Attardi, D. G., G. Martini, E. Mattoccia, and G. P. Toc-

chini-Valentini. 1976. Effect of Xenopuis laevis oocyteextract on supercoiled simian virus 40 DNA: formation ofcomplex DNA. Proc. Natl. Acad. Sci. U.S.A. 73:554-558.

2. Baldi, M. I., P. Benedetti, E. Mattoccia, and G. P. Toc-chini-Valentini. 1980. In vitro catenation and decatenationof DNA and a novel eucaryotic ATP-dependent topoiso-merase. Cell 20:461-467.

3. Cozzarelli, N. R. 1980. DNA gyrase and the supercoilingof DNA. Science 207:953-960.

4. Cozzarelli, N. R. 1980. DNA topoisomerases. Cell 22:327-328.

5. Davis, R. W., M. Simon, and N. Davidson. 1971. Electronmicroscope heteroduplex methods for mapping regions ofbase sequence homology in nucleic acids. Methods Enzy-mol. 21:413-428.

6. Gellert, M. 1981. DNA topoisomerases. Annu. Rev. Bio-chem. 50:879-910.

7. Germond, J. E., B. Hirt, P. Oudet, M. Gross-Bellard, P.Chambon. 1975. Folding of the DNA helix in chromatin-like structures from simian virus 40. Proc. Natl. Acad.Sci. U.S.A. 72:1843-1847.

8. Hsieh, T.-S., and D. Brutlag. 1980. ATP-dependent DNAtopoisomerase from D. melanogaster reversibly catenatesduplex DNA rings. Cell 21:115-125.

9. Krasnow, M. A., and N. R. Cozzarelli. 1982. Catenation ofDNA rings by topoisomerases: mechanism of control byspermidine. J. Biol. Chem. 257:2687-2693.

10. Kreuzer, K. N., and N. R. Cozzarelli. 1980. Formationand resolution of DNA catenanes by DNA gyrase. Cell20:245-254.

11. Liu, L. F., C.-C. Liu, and B. M. Alberts. 1980. Type IIDNA topoisomerases: enzymes that can unknot a topo-logically knotted DNA molecule via a reversible double-strand break. Cell 19:697-707.

12. Mattoccia, E., D. Gandini Attardi, and G. P. Tocchini-Valentini. 1976. DNA-relaxing activity and endonucleaseactivity in Xenopus laevis oocytes. Proc. Natl. Acad. Sci.U.S.A. 73:4551-4554.

13. Mertz, J. E. 1982. Linear DNA does not form chromatincontaining regularly spaced nucleosomes. Mol. Cell. Biol.2:1608-1618.

14. Miller, T. J., and J. E. Mertz. 1982. Template structuralrequirements for transcription in vivo by RNA polymer-ase II. Mol. Cell. Biol. 2:1595-1607.

15. Miller, T. J., D. L. Stephens, and J. E. Mertz. 1982.Kinetics of accumulation and processing of simian virus40 RNA in Xenopus laevis oocytes injected with simianvirus 40 DNA. Mol. Cell. Biol. 2:1581-1594.

16. Stephens, D. L., T. J. Miller, L. Silver, D. Zipser, andJ. E. Mertz. 1981. Easy-to-use equipment for the accuratemicroinjection of nanoliter volumes into the nuclei ofamphibian oocytes. Anal. Biochem. 114:299-309.

17. Wyllie, A. H., R. A. Laskey, J. Finch, and J. B. Gurdon.1978. Selective DNA conservation and chromatin assem-bly after injection of SV40 DNA into Xenoputs oocytes.Dev. Biol. 64:178-188.

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