Cruciform cutting endonucleases from Saccharomyces cerevisiae and phage T4 show conserved reactions with branched DNAs.

Abstract

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+.

Structure and dynamics of three-way DNA junctions: atomic force microscopy studies

“…Such a DNA structure-dependent electrostatic effect may contribute to structural recognition of the junctions by junction-specific enzymes, e.g. resolvases (41,42), and low molecular weight ligands (43).…”

A Shared, Non-canonical DNA Conformation Detected at DNA/Protein Contact Sites and Bent DNA in the Absence of Supercoiling or Cognate Protein Binding

“…T7 endonuclease I cleaves cruciform DNA 2-4 bp away from the single-stranded loop and base regions (25). 2 In branched dsDNA, cleavage by T7 endonuclease I and other singlestranded endonucleases occurs 1-10 bp away from the branch point at sites that do not conform to a strict pattern with respect to distance from the branch point, strand, or sequence preference (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35). The same pattern emerges from probing synthetic, mismatched DNA, where strand scission occurs at variable lengths from the mismatch (36).…”

Role ofRAD52Epistasis Group Genes in Homologous Recombination and Double-Strand Break Repair

“…No homologues of RuvC or the bacteriophage-encoded resolvases have been identified among sequenced eukaryotic genomes, except for the mitochondrial activity, Cce1 (13). Biochemical approaches have identified three Holliday junction-resolving activities from fractionated extracts of mitotic yeast cells (154,380,418). Of these, Cce1 (Mgt1) is mitochondrial and is important for the segregation of mitochondrial genomes (92,175,208,380).…”