Cleavage of symmetric immobile DNA junctions by Escherichia coli RuvC

R. Sha, H. Iwasaki, F. Liu, H. Shinagawa, Nadrian Seeman

Research output: Contribution to journalArticle

Abstract

The Holliday junction is a key DNA intermediate in the process of genetic recombination. It consists of two double-helical domains composed of homologous strands that flank a branch point; two of the strands are roughly helical, and two form the crossover between the helices. RuvC is a Holliday junction resolvase that cleaves the helical strands at a symmetric sequence, leading to the production of two recombinant molecules. We have determined the position of the cleavage site relative to the crossover point by the use of symmetric immobile junctions; these are DNA molecules containing two crossover points, one held immobile by sequence asymmetry and the second a symmetric sequence, but held immobile by torsional coupling to the first junction. We have built five symmetric immobile junctions, in which the tetranucleotide recognition site is moved stepwise relative to the branch point. We have used kinetic analysis of catalysis, gel retardation, and hydroxyl radical hypersensitivity to analyze this system. We conclude that the internucleotide linkage one position 3' to the crossover point is the favored site of cleavage.

Original languageEnglish (US)
Pages (from-to)11982-11988
Number of pages7
JournalBiochemistry
Volume39
Issue number39
DOIs
StatePublished - Oct 3 2000

Fingerprint

Escherichia coli
Holliday Junction Resolvases
Cruciform DNA
Molecules
DNA
Catalysis
Hydroxyl Radical
Genetic Recombination
Hypersensitivity
Gels
Kinetics

ASJC Scopus subject areas

  • Biochemistry

Cite this

Cleavage of symmetric immobile DNA junctions by Escherichia coli RuvC. / Sha, R.; Iwasaki, H.; Liu, F.; Shinagawa, H.; Seeman, Nadrian.

In: Biochemistry, Vol. 39, No. 39, 03.10.2000, p. 11982-11988.

Research output: Contribution to journalArticle

Sha, R, Iwasaki, H, Liu, F, Shinagawa, H & Seeman, N 2000, 'Cleavage of symmetric immobile DNA junctions by Escherichia coli RuvC', Biochemistry, vol. 39, no. 39, pp. 11982-11988. https://doi.org/10.1021/bi001037z
Sha, R. ; Iwasaki, H. ; Liu, F. ; Shinagawa, H. ; Seeman, Nadrian. / Cleavage of symmetric immobile DNA junctions by Escherichia coli RuvC. In: Biochemistry. 2000 ; Vol. 39, No. 39. pp. 11982-11988.
@article{33d28da71f55419cb935cdd9134f4d97,
title = "Cleavage of symmetric immobile DNA junctions by Escherichia coli RuvC",
abstract = "The Holliday junction is a key DNA intermediate in the process of genetic recombination. It consists of two double-helical domains composed of homologous strands that flank a branch point; two of the strands are roughly helical, and two form the crossover between the helices. RuvC is a Holliday junction resolvase that cleaves the helical strands at a symmetric sequence, leading to the production of two recombinant molecules. We have determined the position of the cleavage site relative to the crossover point by the use of symmetric immobile junctions; these are DNA molecules containing two crossover points, one held immobile by sequence asymmetry and the second a symmetric sequence, but held immobile by torsional coupling to the first junction. We have built five symmetric immobile junctions, in which the tetranucleotide recognition site is moved stepwise relative to the branch point. We have used kinetic analysis of catalysis, gel retardation, and hydroxyl radical hypersensitivity to analyze this system. We conclude that the internucleotide linkage one position 3' to the crossover point is the favored site of cleavage.",
author = "R. Sha and H. Iwasaki and F. Liu and H. Shinagawa and Nadrian Seeman",
year = "2000",
month = "10",
day = "3",
doi = "10.1021/bi001037z",
language = "English (US)",
volume = "39",
pages = "11982--11988",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "39",

}

TY - JOUR

T1 - Cleavage of symmetric immobile DNA junctions by Escherichia coli RuvC

AU - Sha, R.

AU - Iwasaki, H.

AU - Liu, F.

AU - Shinagawa, H.

AU - Seeman, Nadrian

PY - 2000/10/3

Y1 - 2000/10/3

N2 - The Holliday junction is a key DNA intermediate in the process of genetic recombination. It consists of two double-helical domains composed of homologous strands that flank a branch point; two of the strands are roughly helical, and two form the crossover between the helices. RuvC is a Holliday junction resolvase that cleaves the helical strands at a symmetric sequence, leading to the production of two recombinant molecules. We have determined the position of the cleavage site relative to the crossover point by the use of symmetric immobile junctions; these are DNA molecules containing two crossover points, one held immobile by sequence asymmetry and the second a symmetric sequence, but held immobile by torsional coupling to the first junction. We have built five symmetric immobile junctions, in which the tetranucleotide recognition site is moved stepwise relative to the branch point. We have used kinetic analysis of catalysis, gel retardation, and hydroxyl radical hypersensitivity to analyze this system. We conclude that the internucleotide linkage one position 3' to the crossover point is the favored site of cleavage.

AB - The Holliday junction is a key DNA intermediate in the process of genetic recombination. It consists of two double-helical domains composed of homologous strands that flank a branch point; two of the strands are roughly helical, and two form the crossover between the helices. RuvC is a Holliday junction resolvase that cleaves the helical strands at a symmetric sequence, leading to the production of two recombinant molecules. We have determined the position of the cleavage site relative to the crossover point by the use of symmetric immobile junctions; these are DNA molecules containing two crossover points, one held immobile by sequence asymmetry and the second a symmetric sequence, but held immobile by torsional coupling to the first junction. We have built five symmetric immobile junctions, in which the tetranucleotide recognition site is moved stepwise relative to the branch point. We have used kinetic analysis of catalysis, gel retardation, and hydroxyl radical hypersensitivity to analyze this system. We conclude that the internucleotide linkage one position 3' to the crossover point is the favored site of cleavage.

UR - http://www.scopus.com/inward/record.url?scp=0034601829&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0034601829&partnerID=8YFLogxK

U2 - 10.1021/bi001037z

DO - 10.1021/bi001037z

M3 - Article

C2 - 11009612

AN - SCOPUS:0034601829

VL - 39

SP - 11982

EP - 11988

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 39

ER -