Crystal structure of RuvC resolvase in complex with Holliday junction substrate

Gorecka, Karolina M.; Komorowska, Weronika; Nowotny, Marcin

doi:10.1093/nar/gkt769

Cited by 71 publications

(104 citation statements)

References 45 publications

(63 reference statements)

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“…2B) (24,25). Although substrate binding occurs in a sequenceindependent manner, cleavage occurs preferentially at the sequence 5′-A / T TT G / C -3′ and generates a pair of nicked duplexes (26).…”

Section: Significancementioning

confidence: 99%

“…GEN1/Yen1 belong to the XPG/Rad2 family of 5′-flap endonucleases (28,29), whereas RuvC is related to the retroviral integrase superfamily (25). Like RuvC, GEN1 promotes HJ resolution by a coordinated nick and counternick mechanism, but it is currently unknown whether the eukaryotic HJ resolvases exhibit a defined sequence specificity (30,31).…”

Section: Significancementioning

confidence: 99%

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Resolution of single and double Holliday junction recombination intermediates by GEN1

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Martín

Wyatt

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Genetic recombination provides an important mechanism for the repair of DNA double-strand breaks. Homologous pairing and strand exchange lead to the formation of DNA intermediates, in which sister chromatids or homologous chromosomes are covalently linked by four-way Holliday junctions (HJs). Depending on the type of recombination reaction that takes place, intermediates may have single or double HJs, and their resolution is essential for proper chromosome segregation. In mitotic cells, double HJs are primarily dissolved by the BLM helicaseTopoisomeraseIIIα-RMI1-RMI2 (BTR) complex, whereas single HJs (and double HJs that have escaped the attention of BTR) are resolved by structure-selective endonucleases known as HJ resolvases. These enzymes are ubiquitous in nature, because they are present in bacteriophage, bacteria, archaea, and simple and complex eukaryotes. The human HJ resolvase GEN1 is a member of the XPG/Rad2 family of 5′-flap endonucleases. Biochemical studies of GEN1 revealed that it cleaves synthetic DNA substrates containing a single HJ by a mechanism similar to that shown by the prototypic HJ resolvase, Escherichia coli RuvC protein, but it is unclear whether these substrates fully recapitulate the properties of recombination intermediates that arise within a physiological context. Here, we show that GEN1 efficiently cleaves both single and double HJs contained within large recombination intermediates. Moreover, we find that GEN1 exhibits a weak sequence preference for incision between two G residues that reside in a T-rich region of DNA. These results contrast with those obtained with RuvC, which exhibits a strict requirement for the consensus sequence 5′-A / T TT G / C -3′.

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Section: Significancementioning

confidence: 99%

Section: Significancementioning

confidence: 99%

Resolution of single and double Holliday junction recombination intermediates by GEN1

Punatar

Martín

Wyatt

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…A Model of the CPV Resolvase⅐HJ Complex-The crystal structure of T. thermophilus RuvC bound to a DNA four-way junction has provided a framework for considering how the bacterial enzyme recognizes this branched substrate (18). Given the common features of the dimeric E. coli (and T. thermophilus) RuvC and CPV resolvase structures, we reasoned that the modes of substrate recognition are also likely to be similar.…”

Section: Resultsmentioning

confidence: 99%

“…Despite the substantial biochemical data now available for the VV and FPV resolvases, there are no structural models available for these proteins that could facilitate interpretation of existing data, guide new experiments, or enable structure-based poxvirus inhibitor design. Although crystal structures of bacterial RuvC and a yeast mitochondrial resolvase are known (17)(18)(19)(20), the limited sequence similarities and distinct biochemical activities of the poxvirus enzymes have made it difficult to utilize homology-based models.…”

mentioning

confidence: 99%

“…Using the Thermus thermophilus RuvC⅐HJ complex crystal structure (18) and biochemical data (21) as a guide, we constructed a model of CPV resolvase bound to an HJ substrate. The model provides insights into the functional importance of the structural differences between the two enzymes, including an explanation for why the poxvirus enzymes can efficiently cleave such a broad range of branched DNA substrates.…”

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confidence: 99%

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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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Crystal structures of the structure-selective nuclease Mus81-Eme1 bound to flap DNA substrates

Gwon

Baek

et al. 2014

The EMBO Journal

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The Mus81-Eme1 complex is a structure-selective endonuclease with a critical role in the resolution of recombination intermediates during DNA repair after interstrand cross-links, replication fork collapse, or double-strand breaks. To explain the molecular basis of 3 0 flap substrate recognition and cleavage mechanism by Mus81-Eme1, we determined crystal structures of human Mus81-Eme1 bound to various flap DNA substrates. Mus81-Eme1 undergoes gross substrate-induced conformational changes that reveal two key features: (i) a hydrophobic wedge of Mus81 that separates pre-and post-nick duplex DNA and (ii) a 5 0 end binding pocket that hosts the 5 0 nicked end of post-nick DNA. These features are crucial for comprehensive protein-DNA interaction, sharp bending of the 3 0 flap DNA substrate, and incision strand placement at the active site. While Mus81-Eme1 unexpectedly shares several common features with members of the 5 0 flap nuclease family, the combined structural, biochemical, and biophysical analyses explain why Mus81-Eme1 preferentially cleaves 3 0 flap DNA substrates with 5 0 nicked ends.

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Crystal structure of RuvC resolvase in complex with Holliday junction substrate

Cited by 71 publications

References 45 publications

Resolution of single and double Holliday junction recombination intermediates by GEN1

Resolution of single and double Holliday junction recombination intermediates by GEN1

Structure and Metal Binding Properties of a Poxvirus Resolvase

Crystal structures of the structure-selective nuclease Mus81-Eme1 bound to flap DNA substrates

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