Crystal Structure of DNA Recombination Protein RuvA and a Model for Its Binding to the Holliday Junction

Rafferty, John B.; Sedelnikova, Svetlana E.; Hargreaves, David; Artymiuk, Peter J.; Baker, Patrick J.; Sharples, Gary J.; Mahdi, Akeel A.; Lloyd, Robert G.; Rice, David W.

doi:10.1126/science.274.5286.415

Cited by 165 publications

(165 citation statements)

References 63 publications

(15 reference statements)

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“…Crystallographic studies have revealed a three-dimensional structure of a RuvA tetramer in which four monomers of RuvA are related by a fourfold symmetry (Rafferty et al 1996;Nishino et al 1998). A model of the RuvA-Holliday junction complex which was constructed from the RuvA crystal structure is consistent with the above branch migration mechanism hypothesis (Rafferty et al 1996).…”

Section: Introductionmentioning

confidence: 59%

Mutational analysis on structure–function relationship of a Holliday junction specific endonuclease RuvC

et al. 1998

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Section: Introductionmentioning

confidence: 59%

Mutational analysis on structure–function relationship of a Holliday junction specific endonuclease RuvC

et al. 1998

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“…1B). On the extensive basic surface of the RuvA tetramer, eight residues, corresponding to Glu-55 and Asp-56 in each subunit, form an acidic central pin (9) (Fig. 2D).…”

Section: Resultsmentioning

confidence: 99%

“…Crystallographic and biochemical studies revealed that RuvA adopts a unique tetrameric architecture formed by identical subunits with three distinct domains (9,10). Proteolytic and mutational analyses demonstrated that domain III plays a major role in the ATP-dependent branch migration through direct contact with RuvB whereas the remaining major core (I and II) is responsible for Holliday junction binding (10,11).…”

mentioning

confidence: 99%

Crystal structure of the Holliday junction DNA in complex with a single RuvA tetramer

Ariyoshi

Nishino

Iwasaki

et al. 2000

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

122

146

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In the major pathway of homologous DNA recombination in prokaryotic cells, the Holliday junction intermediate is processed through its association with RuvA, RuvB, and RuvC proteins. Specific binding of the RuvA tetramer to the Holliday junction is required for the RuvB motor protein to be loaded onto the junction DNA, and the RuvAB complex drives the ATP-dependent branch migration. We solved the crystal structure of the Holliday junction bound to a single Escherichia coli RuvA tetramer at 3.1-Å resolution. In this complex, one side of DNA is accessible for cleavage by RuvC resolvase at the junction center. The refined junction DNA structure revealed an open concave architecture with a four-fold symmetry. Each arm, with B-form DNA, in the Holliday junction is predominantly recognized in the minor groove through hydrogen bonds with two repeated helix-hairpin-helix motifs of each RuvA subunit. The local conformation near the crossover point, where two base pairs are disrupted, suggests a possible scheme for successive base pair rearrangements, which may account for smooth Holliday junction movement without segmental unwinding.

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“…RuvA has four acidic pins that are probably involved in strand separation, although they do not take up space in the center of the HJ (41). Peptide WRWYCR does not prevent a RuvA tetramer from binding to HJ substrates (K.V.K.…”

Section: Discussionmentioning

confidence: 99%

Holliday junction-binding peptides inhibit distinct junction-processing enzymes

Kepple

Boldt

Segall

2005

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

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Holliday junctions (HJ) are the central intermediates in both homologous recombination and site-specific recombination performed by tyrosine recombinases such as the bacteriophage Integrase (Int) protein. Previously, our lab identified peptide inhibitors of Int-mediated recombination that prevent the resolution of HJ intermediates. We now show that two of these inhibitors bind HJ DNA in the square-planar conformation even in the absence of Int protein. The peptides prevent unwinding of branched DNA substrates by the RecG helicase of Escherichia coli and interfere with the resolution of HJ substrates by the RuvABC complex. Our results suggest that these peptides target all proteins that process HJ in the square-planar conformation. These inhibitors should be extremely useful for dissecting homologous recombination and recombination-dependent repair in vitro and in vivo.homologous recombination ͉ recombination-dependent repair ͉ RecG helicase ͉ RuvABC resolvasome ͉ tyrosine recombinase H olliday junctions (HJ), or four-way junctions, are central intermediates in homologous recombination, repair of collapsed replication forks, and reactions performed by the tyrosine recombinase family of site-specific enzymes (1-5). The first two processes are important in all organisms and are involved in the maintenance of chromosome integrity and repair of DNA damage. In the case of diploid organisms, faithful chromosome segregation depends on homologous recombination (6). Sitespecific recombination reactions performed by tyrosine recombinases are also very widespread and control gene expression, regulate plasmid and bacterial chromosome copy number, and mediate lysogeny (3). The presence and disappearance of HJ, their level within cells, and the enzymes that both generate and resolve them are of intense interest. More tools would be extremely useful for both in vitro and in vivo dissection of homologous recombination and repair processes in all organisms.Phage integrase (Int) binds to and mediates strand exchange between pairs of att sites. During recombination, one round of DNA cleavage, strand exchange, and ligation of the top strands of each partner DNA molecule generates a HJ intermediate, which is resolved by a second round of the same catalytic steps (3). These reactions are both rapid and highly reversible, making intermediates very difficult to study.We have identified peptides that inhibit recombination by trapping the protein-bound HJ intermediate and preventing its resolution either to substrates or to products (7-9). The most potent inhibitory peptide, WRWYCR, traps virtually all HJ formed during a reaction and has an IC 50 of 5-20 nM (9). A related peptide, KWWCRW, is very similar to WRWYCR in potency (8). These peptides also inhibit the mechanistically related Cre, XerC and D, and Flp proteins (ref. 9; A.M.S., unpublished results; A. Conway and P. A. Rice, personal communication). Because these proteins share little primary sequence identity, we reasoned that these peptides might interact with free HJ.HJ adopt one...

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Crystal Structure of DNA Recombination Protein RuvA and a Model for Its Binding to the Holliday Junction

Cited by 165 publications

References 63 publications

Mutational analysis on structure–function relationship of a Holliday junction specific endonuclease RuvC

Mutational analysis on structure–function relationship of a Holliday junction specific endonuclease RuvC

Crystal structure of the Holliday junction DNA in complex with a single RuvA tetramer

Holliday junction-binding peptides inhibit distinct junction-processing enzymes

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