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In Vitro Concatemer Formation Catalyzed by Vaccinia Virus DNA Polymerase

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In Vitro Concatemer Formation Catalyzed by Vaccinia Virus DNA Polymerase Virology 278, 562–569 (2000) doi:10.1006/viro.2000.0686, available online at http://www.idealibrary.com on View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector In Vitro Concatemer Formation Catalyzed by Vaccinia Virus DNA Polymerase David O. Willer, Xiao-Dan Yao, Melissa J. Mann,1 and David H. Evans2 Department of Molecular Biology and Genetics, The University of Guelph, Guelph, Ontario N1G 2W1, Canada Received July 18, 2000; returned to author for revision August 18, 2000; accepted October 6, 2000; published online November 22, 2000 During poxvirus infection, both viral genomes and transfected DNAs are converted into high-molecular-weight concate- mers by the replicative machinery. However, aside from the fact that concatemer formation coincides with viral replication, the mechanism and protein(s) catalyzing the reaction are unknown. Here we show that vaccinia virus DNA polymerase can catalyze single-stranded annealing reactions in vitro, converting linear duplex substrates into linear or circular concatemers, in a manner directed by sequences located at the DNA ends. The reaction required Ն12 bp of shared sequence and was stimulated by vaccinia single-stranded DNA-binding protein (gpI3L). Varying the structures at the cleaved ends of the molecules had no effect on efficiency. These duplex-joining reactions are dependent on nucleolytic processing of the molecules by the 3Ј-to-5Ј proofreading exonuclease, as judged by the fact that only a 5Ј-32P-end label is retained in the joint molecules and the reaction is inhibited by dNTPs. The resulting concatemers are joined only through noncovalent bonds, but can be processed into stable molecules in E. coli, if the homologies permit formation of circular molecules. This reaction provides a starting point for investigating the mechanism of viral concatemer formation and can be used to clone PCR-amplified DNA. © 2000 Academic Press Key Words: concatemer; DNA polymerase; I3L; poxvirus; SSB; recombination; replication; single-stranded annealing; vaccinia virus. INTRODUCTION infected cells, being converted by the replicative machin- ery into high-molecular-weight concatemers encoding The Poxviridae comprise a family of large double- embedded recombinants (Evans et al., 1988). It is un- stranded DNA viruses capable of replicating in the cyto- known whether these replication and recombination re- plasm of infected cells (Moss, 1996). The prototype, vac- actions will eventually prove to be functionally separable, cinia virus, encodes ϳ260 proteins within a 190-kbp but all of the genetic, kinetic, and biochemical evidence genome (Johnson et al., 1993), a genome that is thought to date suggests that the two processes are somehow to encode most, if not all, of the gene products required inextricably linked (Colinas et al., 1990; Evans et al., 1988; for viral replication, repair, and recombination (reviewed Fisher et al., 1991; Merchlinsky, 1989). in Traktman, 1990). A characteristic feature of poxvirus The linkage between replication and recombination replication reactions is that, during the course of viral has hampered genetic dissection of this process be- DNA replication, early gene products convert virus DNA Ϫ cause, although DNA mutations typically block recom- into high-molecular-weight concatemers. These mole- bination (Merchlinsky, 1989), replication is also essential cules are initially composed of dimeric viral genomes for the production and analysis of recombinants. Conse- linked in a head-to-head or tail-to-tail manner, which are quently, it has been difficult to draw mechanistic conclu- thought to be produced by a rolling hairpin mechanism sions from such experiments as those showing that viral (Moyer and Graves, 1981). However, more complex con- DNA polymerase mutations create recombination defi- catemers can be detected later in infection, containing ciencies (Colinas et al., 1990; Merchlinsky, 1989; Willer et genomes that are also linked in a head-to-tail configura- al., 1999a). However, superficially similar reactions to tion (DeLange, 1989). Such molecules cannot be formed those catalyzed by poxvirus enzymes are also observed by a purely replicative process (e.g., rolling hairpins or in phage-infected E. coli, and such studies can at least rolling circles) and their existence suggests that recom- provide general insights into possible mechanisms. bination within the terminal inverted repeats must occur Lambda phage uses a combination of rolling circle rep- during the later stages of viral genome replication. Trans- lication and red-mediated recombination reactions to fected molecules undergo similar processing in virus- produce the recombinant-containing concatemers, which are consumed by phage-packaging reactions. [If DNA concatemers are extracted from poxvirus-infected 1 Present address: The Robarts Research Institute, Siebens-Drake cells and contain cos-sites, the DNA can also be pack- Building, Room 107, 1400 Western Road, London, Ontario N6G 2V4, Canada. aged into recombinant phage particles (Parks and Evans, 2 To whom correspondence and reprint requests should be ad- 1991).] Red-mediated phage recombination reactions are dressed. Fax: (519) 837-2075. E-mail: [email protected]. not biochemically complicated. They require only an ex- 0042-6822/00 $35.00 Copyright © 2000 by Academic Press 562 All rights of reproduction in any form reserved. VACCINIA VIRUS DNA POLYMERASE 563 onuclease to expose complementary DNA sequences and process joint molecules (␭ exonuclease), and a single-stranded DNA-binding protein (␤-protein) to cata- lyze strand annealing (Karakousis et al., 1998; Li et al., 1998; Stahl et al., 1997). Such “single-stranded annealing” reactions are intrinsically nonconservative and occur in many species, including mammalian and yeast cells. In yeast cells, where competition between recombination pathways can be closely monitored, single-stranded an- nealing is the primary route by which double-stranded breaks are repaired within repeated sequences (re- viewed in Paques and Haber, 1999). This creates “pop- out” recombinants in yeast, a well-established process wherein recombination between two directly repeated sequences deletes a total of one repeat plus any inter- vening sequences. This type of recombination event is FIG. 1. DNA annealing catalyzed by vaccinia DNA polymerase. also well characterized in the poxviral literature (Ball, pDW101 was cut with EcoRI or XhoI to produce 2.96-kbp molecules 1987). sharing 33 bp of overlapping sequence homology (A). Annealing reac- These observations raise the question of whether any tions (20 ␮L) were then prepared containing 0.35 ␮g of each DNA of the enzymes known to be encoded by vaccinia virus substrate plus 0.1 ␮g of vaccinia virus polymerase. After incubation for might similarly catalyze single-stranded annealing and the indicated times, the reaction products were fractionated using agarose gel electrophoresis and visualized by ethidium bromide stain- concatemer-forming reactions. Thus far only two virus- ing (B). Alternatively, reactions were prepared containing 0.35 ␮gof encoded early gene products exhibit the requisite activ- each DNA substrate plus the indicated quantities of DNA polymerase ities. Experiments with temperature-sensitive viruses and incubated for 20 min at 37°C (C). A 6-kbp reaction product is have shown that the primary DNA exonuclease activity formed in (B) and (C). detectable in vaccinia-infected cell extracts is the 3Ј- to-5Ј exonuclease of vaccinia DNA polymerase (Chall- berg and Englund, 1979; Willer et al., 1999a) and, despite polymerase converted 45% of the input DNA into dimeric reports of other virion nucleases (Rosemond-Hornbeak products over a 10-min period, with a concomitant loss of and Moss, 1974), this remains to date the only known monomeric molecules (Fig. 1B). The yield of dimer de- virus-encoded DNA exonuclease. In contrast vaccinia clined slightly thereafter, stabilizing with about 35% of the encodes several DNA-binding proteins, although most molecules converted to product. The reaction kinetics are late gene products that probably serve structural suggested that there might be a short delay prior to the functions. The principal replicative high-affinity single- appearance of dimers, which was not caused by a delay stranded DNA-binding protein (SSB) is thought to be the in reaching thermal equilibrium. The reaction yield was I3L gene product (Rochester and Traktman, 1998; Tseng also dependent on protein concentration (Fig. 1C). et al., 1999). In this communication we show that vaccinia Duplex annealing selectively pairs homologous ends DNA polymerase and gpI3L are the only proteins needed to catalyze concatemer formation in vitro and thus pro- This pairing reaction required two different DNA sub- vide a possible biochemical route to dissecting a pro- strates that shared some small amount of overlapping cess that has long resisted genetic investigation. The end homology. Controls showed that the reaction cannot simplicity of the reaction suggests that it might also find produce joint molecules when the only available sub- some use as an alternative cloning method. strates were a pool of identical duplexes (Fig. 2, lanes 1–6). To determine which ends of the two substrates RESULTS were being joined, we further digested EcoRI- and XhoI- cut molecules with ScaI, producing DNA fragments that Vaccinia DNA polymerase catalyzes concatemer can be differentiated by size (Fig. 2, inset). Vaccinia DNA formation
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