polymerase I large fragment, and the amino-terminusencoding part of the recA gene was subcloned as a Sac II/EcoRI fragment in plasmid pUC18, which had been cut with EcoRI and Sma I, yielding plasmid pRecA-1. The same plasmid was digested with Hinfl, the ends were rendered blunt, and the carboxyl-terminus-encoding part of the recA gene was subcloned in pUC19 (EcoRI/Sma I) as a HinflI/EcoRI fragment, yielding plasmid pRecA-2. The amino-terminusencoding part was further modified.

We have purified a cruciform DNA resolving endonuclease (Endo X3) greater than 1000‐fold from crude extracts of mitotically growing Saccharomyces cerevisiae. The enzyme shows high specificity for DNAs with secondary structures and introduces characteristic patterns of staggered ‘nicks’ in the immediate vicinity of the structure. The following substrates were analyzed in detail: (i) naturally occurring four‐way X junctions in cruciform DNA of a supercoiled plasmid; (ii) synthetic four‐way X junctions with arms of 9 bp; (iii) synthetic three‐way Y junctions with arms of 10 bp; and (iv) heteroduplex loops with 19 nucleotides in the loop. Cleavages were always found in the double stranded portion of the DNA, located immediately adjacent to the junction of the respective structure. The Endo X3 induced cleavage patterns are identical or very similar to the cleavage patterns induced in the same substrates by endonuclease VII (Endo VII) from phage T4. Furthermore, the activity of Endo X3 is completely inhibited in the presence of anti‐Endo VII antiserum. Endo X3 has an apparent mol. wt of 43,000 daltons, determined by gel filtration and of approximately 18,000 daltons in SDS‐‐polyacrylamide gels. Maximum activity of the enzyme was obtained in the presence of 10 mM MgCl2 at 31 degrees C in Tris‐HCl buffer over a broad pH range with a maximum approximately 8.0. About 70% of maximal activity was obtained when Mg2+ was replaced by equimolar amounts of Mn2+ or Ca2+.

Endonuclease VII is the product of gene 49 of phage T4 and was the first enzyme shown to resolve Holliday structures in vitro [Mizuuchi, K. et al. (1982) Cell 29, 357-3651, Low amounts of the enzyme were originally purified from phage-infected cells Eur. J . Biochem. 115, 123 -1311. We now report a purification procedure for milligram amounts of cloned endonuclease VII expressed in Escherichia coli with gene 49 under the control of a temperature-inducible promoter on a plasmid system [Tomaschewski, J. (1988) PhD Thesis, University of Bochum, FRG]. The protein was purified 500-fold from crude extracts in five steps with a recovery of 15%. The steps include (a) poly(ethyleneglycol)/dextran two-phase separation; (b) DEAEcellulose; (c) single-stranded DNA-agarose; (d) Mono-Q and (e) Mono-S chromatography. The final protein was more than 98% pure as estimated from SDSjPAGE analysis. The protein has an apparent molecular mass of 17.8 kDa on SDS-containing polyacrylamide gels and 36 kDa when determined by gel filtration or sedimentation through sucrose gradients in the presence of high salt (600 mM NaCl). In the absence of additional salt, the enzyme has a tendency to aggregate and products of molecular masses differing in steps of about 18 kDa appear on SDS-containing polyacrylamide gels.Resolution of Holliday structures made during genetic recombination is a vital function for cells to maintain the integrity of their chromosomes [l]. The idea that this step might be enzymatically controlled was supported when endonuclease VII from phage T4 was shown to resolve Holliday structures in vitro, giving genetically sensible products [2, 31. Branched DNA substrates were used to investigate the specificity of the enzyme. These included four-armed X structures, three-armed Y structures, heteroduplex loops, mismatching bases, single-stranded overhangs and cruciforms in supercoiled DNA with palindromes [4-101. In summary, the results show that endonuclease VII has a broad range of specificity for DNA with secondary structures. The enzyme introduces ligatable nicks which flank within a few nucleotides the 3' sides of the structural perturbations in double-stranded DNA. Single-stranded DNA without secondary structures is not attacked by endonuclease VII [8]. These findings make the enzyme a versatile tool in the analyses of DNA secondary structures [8].Endonuclease VII is the product of gene 49 of phage T4. The amount of enzyme in phage-infected cells is small and did not allow its purification to homogeneity [2, 111. Recent cloning and the over-expression of gene 49 in Escherichia coli opened a way for obtaining large quantities of the enzyme; we describe here a procedure for large-scale preparations of cloned endonuclease VII (endonuclease VIIcl) made from an inducible expression system in E. coli [12-141. The purified protein, which contains less than 2% contaminating proteins, were used for raising antibodies.

We have characterised a RecA protein fused to the simian virus 40 large T nuclear-localisation signal. The fusion protein was targeted to the nucleus in transgenic tobacco plants with high efficiency. By contrast, authentic RecA was not enriched in the nuclei of plant cells expressing comparable amounts of protein. For detailed characterisation of the strand-exchange activity of the nuclear-targeted RecA protein, a nearly identical protein was expressed in Escherichia coli and purified to homogeneity. This protein was found to bind to single-stranded DNA with the same stoichiometry and to promote the exchange of homologous DNA strands with the same kinetics as authentic RecA. It was concluded that the amino-terminal modification did not alter any of the essential properties of RecA and that the fusion protein is a fully functional strand-exchange protein. However, the ATPase activity of this protein was 20 times greater than that of RecA in the absence of single-stranded DNA. As with RecA, this activity was further stimulated by the addition of single-stranded DNA. Since ATPase activity is correlated with the ability of RecA to assume its high affinity state for DNA, the nuclear-targeted RecA protein might be regarded as a constitutively stimulated RecA variant, fully functional in promoting homologous recombination.