RuvC protein resolves Holliday junctions via cleavage of the continuous (noncrossover) strands.

Bennett, Richard J.; West, Stephen C.

doi:10.1073/pnas.92.12.5635

Cited by 65 publications

(54 citation statements)

References 22 publications

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“…The two "continuous" strands have a B-DNA-like backbone conformation at the branch point, whereas the two "crossing" strands have a sharp kink at the branch point, similar to that seen in the Cre⅐HJ complex (39). The continuous strands of the HJ substrate are positioned near the enzyme active sites, consistent with the established strand cleavage preference for RuvC (40). Overall, the enzyme-bound DNA junction adopts a conformation that is distinct from the planar, 2-fold symmetric form found in the Cre⅐HJ complex (39), the stacked-X form found in the T7 endonuclease I⅐HJ complex (41), and the 2-fold symmetric unstacked X form found in the T4 endonuclease VII⅐HJ complex (42).…”

Section: Resultssupporting

confidence: 53%

Structure and Metal Binding Properties of a Poxvirus Resolvase

Hwang

Perry

et al. 2016

Journal of Biological Chemistry

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Poxviruses replicate their linear genomes by forming concatemers that must be resolved into monomeric units to produce new virions. A viral resolvase cleaves DNA four-way junctions extruded at the concatemer junctions to produce monomeric genomes. This cleavage reaction is required for viral replication, so the resolvase is an attractive target for small molecule inhibitors. To provide a platform for understanding resolvase mechanism and designing inhibitors, we have determined the crystal structure of the canarypox virus (CPV) resolvase. CPV resolvase is dimer of RNase H superfamily domains related to Escherichia coli RuvC, with an active site lined by highly conserved acidic residues that bind metal ions. There are several intriguing structural differences between resolvase and RuvC, and a model of the CPV resolvase⅐Holliday junction complex provides insights into the consequences of these differences, including a plausible explanation for the weak sequence specificity exhibited by the poxvirus enzymes. The model also explains why the poxvirus resolvases are more promiscuous than RuvC, cleaving a variety of branched, bulged, and flap-containing substrates. Based on the unique active site structure observed for CPV resolvase, we have carried out a series of experiments to test divalent ion usage and preferences. We find that the two resolvase metal binding sites have different preferences for Mg 2؉ versus Mn 2؉ . Optimal resolvase activity is maintained with 5 M Mn 2؉ and 100 M Mg 2؉ , concentrations that are well below those required for either metal alone. Together, our findings provide biochemical insights and structural models that will facilitate studying poxvirus replication and the search for efficient poxvirus inhibitors.Poxviruses infect a broad range of vertebrate and invertebrate hosts, including mammals, birds, reptiles, and insects (1). The best known example is variola, the virus responsible for smallpox. Although smallpox has been eradicated, there is still significant interest in poxvirus therapeutics. Part of this interest stems from the potential use of variola as a biological weapon, but other poxvirus strains also cause significant morbidity and mortality even today (2).All poxviruses have long, linear, double-stranded DNA genomes that are replicated in the cytoplasm of infected cells. The vaccinia virus (VV), 3 used for many years as a naturally attenuated vaccine to eliminate smallpox has served as the prototype poxvirus for laboratory research (3). The VV genome is 200 kb in length and encodes ϳ200 genes.During poxvirus replication (Fig. 1), a viral DNA polymerase and several auxiliary factors generate concatemers of the linear genome using a rolling hairpin replication mechanism (1). The concatemer junctions between unit genomes contain inverted repeat sequences that can be extruded as four-way DNA junctions (4). These cruciform structures are cleaved by a viral resolvase, resulting in monomeric genomes that can ultimately be packaged into new virions (5, 6). The VV resolvase (named A22)...

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Section: Resultssupporting

confidence: 53%

Structure and Metal Binding Properties of a Poxvirus Resolvase

Hwang

Perry

et al. 2016

Journal of Biological Chemistry

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“…The RuvC protein is able to resolve synthetic Holliday junctions (Dunderdale et al, 1991(Dunderdale et al, , 1994Bennett et al, 1993Bennett et al, , 1995, recombination intermediates generated by RecA (Dunderdale et al, 1991, Shah et al, 1994 and cruciform structures extruding from supercoiled plasmids (Iwasaki et al, 1991). Recently the atomic structure of RuvC and thereby the amino acids possibly representing the active site were determined.…”

Section: Discussionmentioning

confidence: 99%

The ParB protein encoded by the RP4 par region is a Ca2+-dependent nuclease linearizing circular DNA substrates

Grohmann¹,

Stanzer

Schwab

1997

Microbiology

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The parCBA operon, which together with the parDE operon constitutes an efficient stabilization system of the broad-host-range plasmid RP4, encodes a 20 kDa polypeptide (ParB), which exhibits sequence homology to nucleases. The ParB protein was overexpressed by means of an inducible tac-promoter system. Plate assays with herring sperm DNA as substrate provided evidence for nuclease activity. The ParB nuclease shows specificity for circular DNA substrates and linearizes them regardless of the presence in cis of parts of the RP4 partitioning region. The nuclease activity in witro is stimulated by the presence of Ca2+ ions. EDTA (5 mM) completely inhibits nuclease activity. By restriction analysis of the ParB-linearized products, cleavage of circular DNA substrates taking place preferentially at specific sites was demonstrated. Run-off sequencing and primer extension analysis of ParB-linearized plasmid DNA revealed a specific target for ParB action adjacent to an AT-rich region containing palindromic sequence elements on a pBR322-derived plasmid.

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“…In plasmid 2295, the inserted DNA fragment was prepared by annealing of oligonucleotides (attI-AD3 to attI-AD4). Annealing was carried out as described by Bennett and West (1995 in pSU18. The effect of the deletions was monitored by the mobilization rates with R388⌬1.…”

Section: Construction Of Deletion Mutationsmentioning

confidence: 99%

Non‐palindromic attI sites of integrons are capable of site‐specific recombination with one another and with secondary targets

Hansson

Sköld

Sundström

1997

Molecular Microbiology

108

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SummaryGenes borne on cassettes are mobile owing to sitespecific recombination systems called integrons, which have created various combinations of antibiotic resistance genes in R-plasmids. In these processes, the palindromic site, attC (59-base element), at cassette junctions has been proposed as being essential. Excised and circularized cassettes have been found to integrate with preference for an attI site at one end of the conserved sequence in integrons. In this work, we give evidence that recombination is possible in the absence of the highly organized attC sites between the more simply organized attI sites. Furthermore, at a very low frequency representing the background in our recombination assay, we observed cross-overs between attI and secondary sites. To characterize recombination excluding the attC sites, we have used naturally occurring attI variants and constructed mutants. The cross-over point was identified between a guanine and a thymine in attI using point mutations. Progressive deletions showed the extent of attI and identified two important regions in the conserved sequence 5Ј of the cross-over point. A region 27-36 bp 5Ј of attI influenced recombination with attC sites only, whereas a sequence 9-14 bp 5Ј of the cross-over point in attI was important for recombination with both attI and attC. Recombination between attI and secondary sites could allow fusion of the conserved sequence encoding the integron site-specific recombinase to new sequences.

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RuvC protein resolves Holliday junctions via cleavage of the continuous (noncrossover) strands.

Cited by 65 publications

References 22 publications

Structure and Metal Binding Properties of a Poxvirus Resolvase

Structure and Metal Binding Properties of a Poxvirus Resolvase

The ParB protein encoded by the RP4 par region is a Ca2+-dependent nuclease linearizing circular DNA substrates

Non‐palindromic attI sites of integrons are capable of site‐specific recombination with one another and with secondary targets

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