Near-simultaneous DNA cleavage by the subunits of the junction-resolving enzyme T4 endonuclease VII

Abstract

junction (Duckett et al., 1988; Mueller et al., 1988), Biochemistry, The University, Dundee DD1 4HN, UK suggesting that each subunit makes a single cleavage. The 1 Corresponding author E.coli resolving enzyme RuvC also tends to introduce pairs of cleavages within the core of a junction that can In common with a number of other DNA junctionundergo a limited degree of branch migration (Bennett resolving enzymes, endonuclease VII of bacteriophage et al., 1993). The crystal structure of this protein has been T4 binds to a four-way DNA junction as a dimer, and solved (Ariyoshi et al., 1994). It is a dimer of subunits, cleaves two strands of the junction. We have used a wherein clusters of acidic residues shown to be important supercoil-stabilized cruciform substrate to probe the for catalytic activity (Saito et al., 1995) are separated by simultaneity of cleavage at the two sites. Active endo-~30 Å between the two subunits. This would indicate that nuclease VII converts the supercoiled circular DNA the two active sites are likely to act independently. One directly into linear product, indicating that the two could envisage this occurring in two extreme ways. First, cleavage reactions must occur within the lifetime of the enzyme might bind, and both strands become cleaved the protein-junction complex. By contrast, a heterowithin a very short interval, i.e. giving effectively simultandimer of active enzyme and an inactive mutant endoeous cleavage of the two sites. Alternatively, the first site nuclease VII leads to the formation of nicked circular might be cleaved, followed later by cleavage at the second product, showing that the subunits operate fully indesite. The latter model might even allow for dissociation pendently.of the enzyme followed some time later by the binding Keywords: cruciform/DNA-protein interaction/DNA of a new molecule that introduces the second cleavage. supercoiling/Holliday junction/recombination Using cloverleaf-type junctions, Mueller et al. (1990) showed that two cleavages are introduced within the same junction molecule by T4 endonuclease VII, but Kemper and colleagues (Pottmeyer and Kemper, 1992) have intro-

Mlh1-Mlh3, a Meiotic Crossover and DNA Mismatch Repair Factor, Is a Msh2-Msh3-stimulated Endonuclease

“…MLH proteins have been shown to bind cooperatively to DNA (52); thus longer DNA substrates may be needed to stabilize MLH binding to promote DNA cleavage. Consistent with this idea, we hypothesize that Msh2-Msh3 stimulates Mlh1-Mlh3 endonuclease activity by binding non-specifically to supercoiled DNA or possibly to loops in supercoiled DNA created by the extrusion of inverted repeat sequences (65). In this model Mlh1-Mlh3 is specifically recruited to Msh2-Msh3 that is stably bound to DNA, thus promoting activation of the Mlh1-Mlh3 endonuclease.…”

Characterization of a Holliday Junction-Resolving Enzyme from Schizosaccharomyces pombe

“…In contrast, T7 endonuclease I is a nondissociable dimer in solution (24). In studies with a cruciform substrate stabilized by negative supercoiling, the subunits of both T4 endonuclease VII and T7 endonuclease I have been shown to act nearly simultaneously (both cleaving within the lifetime of the protein-junction complex) but independently in junction cleavage (12,24).…”

Holliday Junction Resolvases

“…The duplex products contain symmetrically related nicks with ligatable 5 0 -phosphate and 3 0 -hydroxyl termini (Mizuuchi et al 1982;Lilley and Kemper 1984). Like RuvC, the two active sites of T4 endo VII can function independently (Giraud-Panis and Lilley 1997;, although the reaction involves two temporally coordinated cleavages that occur within the lifetime of the protein -DNA complex (Pottmeyer and Kemper 1992;Giraud-Panis and Lilley 1997).…”