Novel insights into ATP-stimulated cleavage of branched DNA and RNA substrates through structure-guided studies of the Holliday junction resolvase RuvX

AbstractElectrophoresis in polyacrylamide gels provides a simple yet powerful means of analyzing the relative disposition of helical arms in branched nucleic acids. The electrophoretic mobility of DNA or RNA with a central discontinuity is determined by the angle subtended between the arms radiating from the branchpoint. In a multi-helical branchpoint, comparative gel electrophoresis can provide a relative measure of all the inter-helical angles and thus the shape and symmetry of the molecule. Using the long–short arm approach, the electrophoretic mobility of all the species with two helical arms that are longer than all others is compared. This can be done as a function of conditions, allowing the analysis of ion-dependent folding of branched DNA and RNA species. Notable successes for the technique include the four-way (Holliday) junction in DNA and helical junctions in functionally significant RNA species such as ribozymes. Many of these structures have subsequently been proved correct by crystallography or other methods, up to 10 years later in the case of the Holliday junction. Just as important, the technique has not failed to date. Comparative gel electrophoresis can provide a window on both fast and slow conformational equilibria such as conformer exchange in four-way DNA junctions. But perhaps the biggest test of the approach has been to deduce the structures of complexes of four-way DNA junctions with proteins. Two recent crystallographic structures show that the global structures were correctly deduced by electrophoresis, proving the worth of the method even in these rather complex systems. Comparative gel electrophoresis is a robust method for the analysis of branched nucleic acids and their complexes.

Download Full-text

Recognition and manipulation of branched DNA by the RusA Holliday junction resolvase of Escherichia coli

Nucleic Acids Research ◽

10.1093/nar/26.7.1560 ◽

1998 ◽

Vol 26 (7) ◽

pp. 1560-1566 ◽

Cited By ~ 36

Author(s):

S. N. Chan ◽

S. D. Vincent ◽

R. G. Lloyd

Keyword(s):

Escherichia Coli ◽

Holliday Junction ◽

Branched Dna ◽

Holliday Junction Resolvase

Download Full-text

Fission Yeast Mus81·Eme1 Holliday Junction Resolvase Is Required for Meiotic Crossing Over but Not for Gene Conversion

Genetics ◽

10.1093/genetics/165.4.2289 ◽

2003 ◽

Vol 165 (4) ◽

pp. 2289-2293 ◽

Cited By ~ 1

Author(s):

Gerald R Smith ◽

Michael N Boddy ◽

Paul Shanahan ◽

Paul Russell

Keyword(s):

Homologous Recombination ◽

Fission Yeast ◽

Gene Conversion ◽

Protein Complex ◽

Holliday Junctions ◽

Holliday Junction ◽

Crossing Over ◽

Dna Structures ◽

Branched Dna ◽

Holliday Junction Resolvase

Abstract Most models of homologous recombination invoke cleavage of Holliday junctions to explain crossing over. The Mus81·Eme1 endonuclease from fission yeast and humans cleaves Holliday junctions and other branched DNA structures, leaving its physiological substrate uncertain. We report here that Schizosaccharomyces pombe mus81 mutants have normal or elevated frequencies of gene conversion but 20- to 100-fold reduced frequencies of crossing over. Thus, gene conversion and crossing over can be genetically separated, and Mus81 is required for crossing over, supporting the hypothesis that the fission yeast Mus81·Eme1 protein complex resolves Holliday junctions in meiotic cells.

Download Full-text

Faculty Opinions recommendation of Mammalian BTBD12/SLX4 assembles a Holliday junction resolvase and is required for DNA repair.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1163988.624638 ◽

2009 ◽

Author(s):

Johannes Walter

Keyword(s):

Dna Repair ◽

Holliday Junction ◽

Holliday Junction Resolvase

Download Full-text

The site-specific cleavage of synthetic Holliday junction analogs and related branched DNA structures by bacteriophage T7 endonuclease I.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)47870-2 ◽

1987 ◽

Vol 262 (30) ◽

pp. 14826-14836

Author(s):

P Dickie ◽

G McFadden ◽

A R Morgan

Keyword(s):

Holliday Junction ◽

Bacteriophage T7 ◽

Site Specific ◽

Dna Structures ◽

Branched Dna

Download Full-text

A Test of the Double-Strand Break Repair Model for Meiotic Recombination in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/144.1.27 ◽

1996 ◽

Vol 144 (1) ◽

pp. 27-41 ◽

Cited By ~ 7

Author(s):

Larry A Gilbertson ◽

Franklin W Stahl

Keyword(s):

Meiotic Recombination ◽

Strand Break ◽

Double Strand Break ◽

Double Strand Break Repair ◽

Holliday Junction ◽

Single Event ◽

Heteroduplex Dna ◽

Holliday Junction Resolvase ◽

Break Repair ◽

Two Sides

Abstract We tested predictions of the double-strand break repair (DSBR) model for meiotic recombination by examining the segregation patterns of small palindromic insertions, which frequently escape mismatch repair when in heteroduplex DNA. The palindromes flanked a well characterized DSB site at the ARC4 locus. The “canonical” DSBR model, in which only 5′ ends are degraded and resolution of the four-stranded intermediate is by Holliday junction resolvase, predicts that hDNA will frequently occur on both participating chromatids in a single event. Tetrads reflecting this configuration of hDNA were rare. In addition, a class of tetrads not predicted by the canonical DSBR model was identified. This class represented events that produced hDNA in a “trans” configuration, on opposite strands of the same duplex on the two sides of the DSB site. Whereas most classes of convertant tetrads had typical frequencies of associated crossovers, tetrads with trans hDNA were parental for flanking markers. Modified versions of the DSBR model, including one that uses a topoisomerase to resolve the canonical DSBR intermediate, are supported by these data.

Download Full-text