Two Classes of DNA End-joining Reactions Catalyzed by Vaccinia Topoisomerase I*

The ability of a eukaryotic DNA topoisomerase I to catalyze DNA rearrangements was examined in vitro using defined substrates and purified enzyme. Site-specific DNA strand cleavage by vaccinia topoisomer- ase 1 across from a nick generated double-strand breaks that could be religated to a heterologous blunt-ended duplex DNA regardless of the sequence of the acceptor molecule. Topoisomerase bound covalently at internal positions could religate the bound strand to an incoming acceptor provided that DNA molecule had sequence homology to the region 3' of the scissile bond. These end-joining reactions suggest two potential modes of topoisomerase-mediated recombination that differ in their requirements for DNA homology. Plasmid DNA used as acceptor in strand transfer reactions was linearized quantitatively by digestion with a single-cut restriction enzyme. The 5"phosphate termini of the linear DNAs were converted to 5"OH ends by treatment of the DNAs with calf intestinal phos-phatase. Covalent adduct formation between topoisomerase and ra- diolabeled DNA was measured by label transfer to protein as described (7, 8).

The ability of a eukaryotic DNA topoisomerase I to catalyze DNA rearrangements was examined in vitro using defined substrates and purified enzyme. Sitespecific DNA strand cleavage by vaccinia topoisomerase 1 across from a nick generated double-strand breaks that could be religated to a heterologous bluntended duplex DNA regardless of the sequence of the acceptor molecule. Topoisomerase bound covalently at internal positions could religate the bound strand to an incoming acceptor provided that DNA molecule had sequence homology to the region 3' of the scissile bond. These end-joining reactions suggest two potential modes of topoisomerase-mediated recombination that differ in their requirements for DNA homology.
Eukaryotic DNA topoisomerase I (topo I)' is thought to promote illegitimate recombination in animal cells by virtue of its ability to break and rejoin DNA strands (1, 2). Top0 I binds to duplex DNA and cleaves the phosphodiester backbone of one strand. Bond energy is conserved via the formation of a covalent adduct between the 3"phosphate of the incised strand and a tyrosyl residue of the enzyme. Religation of the covalently bound strand across the same bond originally cleaved restores the integrity of the DNA duplex (possibly accompanied by a change in linking number), whereas religation to a heterologous acceptor strand generates a recombinant molecule.
Vaccinia topoisomerase, a virus-encoded eukaryotic top0 I, can catalyze sequence-specific strand transfer during genetic recombination in vivo (3,4). The topo-mediated DNA rearrangements (i.e. prophage excision) are distinguished by the presence of an oligopyrimidine binding/cleavage motif for top0 I at both recombining half-sites. Vaccinia topoisomerase selectively binds and incises duplex DNAs in vitro at a consensus pentamer sequence 5'-(C/T)CCTT (5-7). Using short duplex DNA substrates containing a single enzyme binding site, I have demonstrated that vaccinia top0 I can efficiently mediate intermolecular strand transfer to a heterologous acceptor (8). Further examination of the ability of the vaccinia enzyme to join DNA ends now illuminates two possible modes of topo-dependent recombination that share half-site specificity but differ in their requirement for sequence homology.
* This work was supported by National Institutes of Health Grant GM 46330, American Cancer Society Grant JFRA-274, and a scholarship from the Pew Charitable Trusts. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

EXPERIMENTAL PROCEDURES
All experiments were performed using the heparin-agarose fraction of vaccinia DNA topoisomerase (9). Strand transfer reactions were constituted as described (8); the nature of the cleavable DNA substrate and the heterologous DNA acceptor was varied in each experiment. Preparation of 5"radiolabeled scissile strand and hybridization to unlabeled complementary oligonucleotides were performed as described (6)(7)(8). That the nicked duplex substrate actually contained three hybridized strands was confirmed by native gel electrophoretic analysis as described (7) under conditions where single strands, twostrand tailed molecules, and three-strand-containing duplexes were resolved. Plasmid DNA used as acceptor in strand transfer reactions was linearized quantitatively by digestion with a single-cut restriction enzyme. The 5"phosphate termini of the linear DNAs were converted to 5"OH ends by treatment of the DNAs with calf intestinal phosphatase. Covalent adduct formation between topoisomerase and radiolabeled DNA was measured by label transfer to protein as described (7, 8).

RESULTS AND DISCUSSION
Two models have been invoked to account for recombination by eukaryotic top0 I. According to the "recombinase" model (4), top0 I forms a covalent intermediate at two sites destined to recombine. The bound proteins are approximated, and DNA single strands are transferred reciprocally to form a Holliday intermediate, which is then resolved by a separate endonuclease. Nonreciprocal recombination is explained by invasion or uptake of a single strand containing a 5"hydroxyl terminus that serves as an acceptor for religation. Strand transfer via this pathway (shown in Fig. L 4 ) is predicted to be facilitated by (if not entirely dependent on) homology of the incoming acceptor strand to the noncovalently bound segment of the scissile strand. Nonreciprocal strand transfer has been demonstrated in vitro using model substrates and either purified vaccinia top0 I or human top0 I (8,lO). Indeed, transfer to a single-strand acceptor by the vaccinia enzyme required 4 bp of homology 3' of the CCCTT motif (8).
The "deletion" model (1) posits top0 I cleavage opposite preexisting nicks or gaps in the nonscissile strand. Such a reaction at two sites on the same DNA strand would generate double-strand breaks at recombining half-sites with liberation of a linear molecule containing one covalently bound top0 molecule at the 3' end. Intramolecular religation of the linear segment yields an extrachromosomal circle; the parental DNA, containing a gapped segment with one covalently activated terminus, can reseal across the gap (with deletion formation) or religate to an unrelated DNA (analogous to a A. Homologous strand transler B. Breakage and blunt-end llgatlon translocation event). Strand transfer via this pathway (illustrated in Fig. 1B) might be predicted to be independent of the sequence of the acceptor molecule.
The question of whether vaccinia top0 I can induce doublestrand breakage and heterologous end joining was examined using synthetic model DNA substrates. A 5'-labeled 24-mer scissile strand containing a single CCCTT motif was hybridized to two 12-mer oligonucleotides complementary to the labeled strand to create a duplex molecule with a strand discontinuity across from the scissile phosphodiester bond; a control duplex substrate contained a continuous nonscissile strand (Fig. 2). Purified top0 I formed a covalent adduct with either substrate with similar yield and enzyme concentration dependence (Fig. 2 A ) . Religation of the covalently bound 5'labeled 12-mer segment of the scissile strand to a heterologous 18-mer acceptor was manifest by the creation of novel 30-mer labeled strand that could be resolved from the original 24-mer substrate by denaturing gel electrophoresis (Fig. 2B). Note that labeled DNA covalently linked to top0 did not enter the gel and was therefore not seen in the autoradiogram. In the case of the continuous duplex substrate, strand transfer occurred only with an acceptor strand homologous to the original substrate (Fig. 2B, lune 6). (Strand transfer to a homologous single-strand acceptor is described in detail in a recent report from this laboratory (8).) Completely different specificity was observed for the substrate containing an opposing nick in the noncleaved strand. The nicked substrate acted as an efficient donor in strand transfer to a blunt-ended duplex acceptor whose sequence was entirely unrelated to that of the 3' segment of the original scissile strand (Fig. 2B, lune 9). Blunt-end ligation to duplex DNA was relatively efficient insofar as recombinant products were also observed a t 10-fold lower concentrations of acceptor ( i e . a t a 2:l ratio of acceptor to input donor molecules; data not shown). Individual single strands were entirely inert as acceptors (Fig. 2B, lunes 10 and  11 ), even when added in vast excess (>lOO-fold) over input substrate (not shown).
Cleavage by top0 I to yield a covalently activated blunt end could also occur on a 3"tailed duplex substrate, provided that

A B
the duplex region encompassed the CCCTT motif (Fig. 3A).
Such cleaved molecules could function as donors for religation to a nonspecific blunt-ended duplex acceptor, but not to single strands (Fig. 3B). The ability of 3"tailed DNAs to be cleaved depended on the length of the single-strand tail; duplex molecules with 6-nucleotide tails were inactive as cleavage substrates, whereas DNAs with 12-mer tails were utilized by top0 I. 5"Tailed molecules in which CCCTT was single-stranded were inert for covalent adduct formation (Fig. 3A).
The specificity of vaccinia topoisomerase for blunt end joining was examined further using linear plasmid DNA as acceptor. Incubation of radiolabeled tailed donor DNA with top0 I resulted in the formation of a protein-DNA complex that was resolved from free DNA during native agarose gel electrophoresis (Fig. 4, lune 2). This complex was eliminated completely by digestion of the reaction products with proteinase K (not shown). Addition of unlabeled 5'-hydroxyl-termi-  nated blunt-ended linear pUC18 DNA resulted in transfer of the topo-bound DNA strand to the linear DNA fragment (Fig.  4, lane 3 ) . The 2.7-kb labeled reaction product was insensitive to proteolysis (not shown). Blunt-ended plasmid DNA containing a 5'-phosphoryl terminus was inert as an acceptor (Fig. 4, lane 4 ) , consistent with covalent transfer of the topobound strand directly to the termini of the plasmid. Linear plasmid DNA containing 5'-OH overhangs four bases in length (generated by restriction with EcoRI or HindIII) were ineffective as acceptors (Fig. 4, lanes 5 and 6). Thus, doublestrand breaks generated by top0 I can only recombine with blunt-ended partners. Linear plasmid DNA with 5'-OH overhangs could be ligated to topo-bound donor DNAs via the homology-dependent strand transfer pathway. This was demonstrated using duplex 12-mer substrates containing identical 8-bp sequences 5' of the scissile bond, but different 4-bp elements 3' of the CCCTT sequence (Fig. 5). The 3' sequences AGCT or AATT corresponded to the 4-base overhangs generated by restriction endonucleases HindIII and EcoRI. The level of covalent adduct formation as a function of enzyme concentration was the same for both 12-mers (Fig. 5A). Both substrates formed topo-DNA complexes that were detected by agarose gel electrophoresis (Fig. 5, panels B and C, lane 2 ) . Addition of unlabeled 5'-hydroxyl-terminated EcoRI-cut pUC18 DNA resulted in transfer of the topo-bound AATT-containing DNA strand to the linear DNA fragment (Fig. 5B, lane 3 ) . Stickyend ligation was quite efficient insofar as 83% of the radiolabeled scissile strand was transferred from the topo-DNA complex to the pUC18 acceptor (as measured by scintillation counting of sections of the dried agarose gel containing the linear pUC plasmid and the topo-DNA complex). The transferred strand was distributed equally to both termini of the linear acceptor. This was determined by isolating the labeled linear plasmid followed by secondary cleavage with ScaI to yield two fragments (1.7 and 1.0 kb; not shown). The ratio of radioactivity in the two ScaI fragments was 1.02. 5"Phosphate-terminated EcoRI-cut plasmid was inert as an acceptor (Fig. 5B, lane 5 ) . Transfer of the AATT-containing DNA to 5'-hydroxyl-terminated HindIII-cut plasmid (containing an AGCT overhang) was 120-fold less efficient than to the homologous sticky end (Fig. 5B, lane 4 ) . Similarly, the AGCTcontaining DNA was efficiently and specifically ligated to 5'hydroxyl-terminated HindIII-cut pUC18 (Fig. 5C, lane 4 ) , but not to 5'-hydroxyl-terminated EcoRI-cut plasmid, which was 76-fold less effective as an acceptor (Fig. 5C, lane 3 ) .

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These experiments provide biochemical evidence for two types of end-joining reactions catalyzed by a eukaryotic top0 I. Site-specific strand cleavage across from a nick generates double-strand breaks that can be religated to a heterologous duplex DNA regardless of the sequence of the acceptor molecule. Simple synthetic substrates or long natural linear DNAs can serve as acceptors, provided they have duplex 5'hydroxyl termini. This type of end-joining reaction is consistent with a role for top0 I in generating chromosomal translocations. It also supports the deletion model for top0 Imediated illegitimate recombination (1). Top0 I bound covalently a t internal positions within duplex DNA can religate the bound DNA strand to an incoming acceptor provided that molecule has sequence homology to the region 3' of the scissile bond. A role for this homologous strand transfer pathway in top0 I-catalyzed recombination is suggested by the finding that a majority of in uiuo recombination events involve parental half-sites with several bases of sequence identity 3' of the  1 and 6). Acceptor DNAs were included as follows: 5'-OH EcoRI-cut pUC18 (lanes 3 and 6 ) , 5"OH HindIII-cut pUC18 ( l a n e 4 ) , 5'-P04 EcoRI-cut pUCl8 (lane 5 ) . A control reaction received no exogenous acceptor (lane 2 ) . C, reaction mixtures included 400 fmol of the AGCT-containing DNA (closed circle). Control reactions contained no enzyme (lanes 1 and 6 ) . Acceptor DNAs were included as follows: 5'-OH EcoRI-cut pUC18 ( h e 3), 5'-OH HindIII-cut pUC18 (lanes 4 and 6 ) , 5'-P04 HindIII-cut pUC18 ( l a n e 5 ) . A control reaction received no exogenous acceptor (lane 2).