World

Journal

of Microbiology

and Biotechnology

9, 583486

Genetic recombination of homologous plasmids catalysed by cell-free extracts topo-isomerase mutant strains of Saccharomyces cerevisiae

of

A.M. MacLeod, G.D. Ferroni* and P. Unrau Cell-free extracts of the yeast Saccbaromyces cerevisiae can be used to catalyse the recombination of bacterial plasmids in vitro. Recombination between homologous plasmids containing different mutations in the gene encoding tetracycline resistance is detectable by the appearance of tetracycline-resistance following transformation of the recombinant plasmid DNA into Escherichia coli DH5. This in vitro recombination system was used to determine the involvement of eukaryotic topo-isomerases in genetic recombination. Cell-free extracts prepared from a temperature-sensitive topo-isomerase II mutant (top&~) of S. cerevisiae yielded tetracycline-resistant recombinants, when the recombination assays were performed at both a non-restrictive temperature (WC) and the restrictive temperature (3 F’C). Th is result was obtained whether or not ATP was present in the recombination buffer. Extracts from a non-conditional topo-isomerase I mutant (topl-I) of S. cerevisiae yielded tetracyclineresistant recombinants, as did a temperature-sensitive double mutant (top&l/fop1-8) at the restrictive temperature. The results of this study indicate that neither topo-isomerase I nor topo-isomerase II was involved in the recombinational activity examined. Key words: Cell-free

extracts, plasmids, recombination,

Succharomyces cerevisiae,

Topo-isomerases, the enzymes that mediate the supercoiling of DNA, were first described by Wang (1971) and Gellert et al. (1976); subsequently, they were implicated in cellular processes such as DNA replication, gene regulation, and recombination in bacteria (Drlica 1984). Topo-isomerase I relaxes negatively-supercoiled DNA and does not have a requirement for ATP. In prokaryotes, topo-isomerase II introduces negative supercoiling in the presence of ATP (Liu 1983) and has relaxation activity in the absence of ATP. In eukaryotes, topo-isomerase II relaxes negatively-supercoiled DNA only and this relaxation reaction requires ATP. Prokaryotic topo-isomerases have been shown to play a role in recombination, and this is consistent with their ability to catalyse changes in helical density and to introduce transient strand breaks in DNA. The negative-supercoiling A.M. MacLeod and G.D. Ferroni are with the Department of Biology, Laurentian University, Sudbury, Ontario, P3E 2C6, Canada; fax (705) 675 4859. P. Unrau is with the Atomic Energy of Canada Ltd, Chalk River Nuclear Laboratories, Chalk River, Ontario, KOJ lJ0, Canada. ‘Corresponding author @ 7993 Rapid

Communications

of Oxford

topo-isomerase

mutants.

activity of DNA gyrase is important for phage lambda integrative recombination in vitro, as the provision of a supercoiled substrate in the absence of gyrase was sufficient to promote recombination (Gellert et al. 1976). Also, recombination hot spots have been associated with potential Z-forming DNA sequences (Skowronski et al. 1984), the least twisted isomeric form, which suggests the importance of the supercoiling reaction mediated by gyrase in the recombination process. Evidence for eukaryotic topo-isomerase I involvement in recombination is lacking. Krniec ef al. (1983) showed that circular duplex DNA molecules could be topologically linked to other circular single-stranded or circular duplex DNA molecules by Usfilago Reel protein and topoisomerase I, indicating a possible indirect role for the enzyme in recombination. Furthermore, sites preferentially acted upon by topo-isomerase I have been associated with cross-over points in non-homologous (illegitimate) recombination (Bullock et al. 1985). The fact that eukaryotic topo-isomerase II does not supercoil DNA (Liu 1983) leaves

Ltd World Journal

of Microbiology

and Bmtechnology, Vol 9, 1993

583

A.M. Macleod,

G.D. Ferroni and P. Unrau

doubt as to its involvement in recombination, unless the involvement is based on the subunit exchange model proposed for gyrase by Gellert (1981). Ikeda (1986) reported that phage T4 DNA topo-isomerase II, which is similar to the eukaryotic enzyme in that it does not supercoil DNA in the presence or absence of ATP, mediates illegitimate recombination in vitro. This indicates that the eukaryotic enzyme could be involved in recombination by the same subunit exchange model. Also, topo-isomerase II isolated from calf thymus has the ability to mediate recombination between two phage DNA molecules (Bae et al. 1988), indicating that this recombinational activity occurs via the subunit exchange model. Our objective was to determine the involvement of eukaryotic topo-isomerases in recombination, by using the in vifro system of Symington et al. (1983). To this end, cell-free extracts from topo-isomerase mutants of Saccharomyces cerevisiae were assayed for their ability to catalyse recombination between homologous plasmids containing different mutations in the gene for tetracycline resistance. Recombinants were detectable following the transformation of E. coli DH5.

The substrate plasmids, derivatives of pBR322, contained different mutations in the gene for tetracycline resistance. The plasmid pUW1 (3.7 kb) carries a gene for ampicillin resistance and has a tet-10 mutation at nucleotide position 26. The homologous pUW4 (5.6 kb) carries ampicillinand neomycin-resistance genes and contains a tet-14 mutation at nucleotide position 1268. The assay procedure also followed that of Symington et al. (1983). Each extract was thawed on ice and subjected to lo-fold dilutions in the protein buffer, up to 10e3. Assays were carried out in 1.5-ml microcentrifuge tubes in 50-~1 volumes containing 26 ~1 of H,O, 10 ~1 of recombination buffer (35 IIIM Hepes, pH 7.8; 10 mM MgCI,; 1 mM DTT; 2 IIIM spermidine; 0.01 ITIM each of dATP, dCTP, dGTP, TTP; 100 mg/ml BSA; and 5 mM ATP), and 10 ~1 of the appropriate extract dilution. ATP was excluded from the recombination buffer in certain recombination assays. For controls, 10 ~1 of protein buffer were added instead of extract. Then, 2 ~1 (0.5 pg) of each substrate plasmid were added to the top of each tube and the reaction was initiated by spinning for 10 s in the microcentrifuge. The mixture was incubated at 30°C or 37°C (as indicated) for 2 h without agitation. Reactions were terminated either by phenol extraction (Maniatis et al. 1982) or by proteinase K digestion. Transformations of competent E. coli DH5 (obtained from Atomic Energy of Canada, Ltd) were carried out in duplicate in 1.5-m] screw-cap microtubes using 10 ~1 of recombinant DNA added to 100 ~1 of competent cells. Cells were incubated at 0°C for 10 min, heat shocked at exactly 42°C for 45 s, and placed on ice for 2 min. SOC medium (900 ~1) (Hanahan 1983) was added

and the culture was incubated for 2 h at 37°C with gyrotation

Materials

and Methods

Cell-free extracts from four strains of S. cerevisiae were used: the wild-type strain; a temperature-sensitive topo-isomerase II mutant (top&1) which shows

Genetic recombination of homologous plasmids catalysed by cell-free extracts of topo-isomerase mutant strains of Saccharomyces cerevisiae.

Cell-free extracts of the yeast Saccharomyces cerevisiae can be used to catalyse the recombination of bacterial plasmids in vitro. Recombination betwe...
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