Abstract
Viral and bacterial Holliday junction resolvases differ in specificity with the former typically being more promiscuous, acting on a variety of branched DNA substrates, while the latter exclusively targets Holliday junctions. We have determined the crystal structure of a RuvC resolvase from bacteriophage bIL67 to help identify features responsible for DNA branch discrimination. Comparisons between phage and bacterial RuvC structures revealed significant differences in the number and position of positively-charged residues in the outer sides of the junction binding cleft. Substitutions were generated in phage RuvC residues implicated in branch recognition and six were found to confer defects in Holliday junction and replication fork cleavage in vivo. Two mutants, R121A and R124A that flank the DNA binding site were purified and exhibited reduced in vitro binding to fork and linear duplex substrates relative to the wild-type, while retaining the ability to bind X junctions. Crucially, these two variants cleaved Holliday junctions with enhanced specificity and symmetry, a feature more akin to cellular RuvC resolvases. Thus, additional positive charges in the phage RuvC binding site apparently stabilize productive interactions with branched structures other than the canonical Holliday junction, a feature advantageous for viral DNA processing but deleterious for their cellular counterparts.
Highlights
Resolution of Holliday structure joint molecules either completes genetic recombination by severing the strand links between coupled chromosomes or triggers fresh exchanges at regressed replication forks (Atkinson and McGlynn, 2009)
Viral and bacterial Holliday junction resolvases differ in specificity with the former typically being more promiscuous, acting on a variety of branched DNA substrates, while the latter exclusively targets Holliday junctions
Substitutions were generated in phage RuvC residues implicated in branch recognition and six were found to confer defects in Holliday junction and replication fork cleavage in vivo
Summary
Resolution of Holliday structure joint molecules either completes genetic recombination by severing the strand links between coupled chromosomes or triggers fresh exchanges at regressed replication forks (Atkinson and McGlynn, 2009). Cellular Holliday junction resolvases need to be highly selective in the structures they cleave as unwarranted breaks at forks or bubbles in DNA may prove disastrous for replicating cells. This does not hold true for viral resolution endonucleases, which are generally less discriminating than their cellular counterparts, targeting a range of branched DNA forms in addition to Holliday structures (Culyba et al, 2007; Declais and Lilley, 2008). This appears to be an evolutionary adaptation to process any branched intermediates that may potentially interfere with virion assembly
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