The simultaneous binding of two double-stranded DNA molecules by Escherichia coli RecA protein

Eugene N. Zaitsev, Stephen C. Kowalczykowski

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

We have characterized the double-stranded DNA (dsDNA) binding properties of RecA protein, using an assay based on changes in the fluorescence of 4',6-diamidino-2-phenylindole (DAPI)-dsDNA complexes. Here we use fluorescence, nitrocellulose filter-binding, and DNase I-sensitivity assays to demonstrate the binding of two duplex DNA molecules by the RecA protein filament. We previously established that the binding stoichiometry for the RecA protein-dsDNA complex is three base-pairs per RecA protein monomer, in the presence of ATP. In the presence of ATPγS, however, the binding stoichiometry depends on the MgCl2 concentration. The stoichiometry is 3 bp per monomer at low MgCl2 concentrations, but changes to 6 bp per monomer at higher MgCl2 concentrations, with the transition occurring at approximately 5 mM MgCl2. Above this MgCl2 concentration, the dsDNA within the RecA nucleoprotein complex becomes uncharacteristically sensitive to DNase I digestion. For these reasons we suggest that, at the elevated MgCl2 conditions, the RecA-dsDNA nucleoprotein filament can bind a second equivalent of dsDNA. These results demonstrate that RecA protein has the capacity to bind two dsDNA molecules, and they suggest that RecA or RecA-like proteins may effect homologous recognition between intact DNA duplexes.

Original languageEnglish (US)
Pages (from-to)21-31
Number of pages11
JournalJournal of Molecular Biology
Volume287
Issue number1
DOIs
StatePublished - Mar 19 1999

Fingerprint

Rec A Recombinases
Magnesium Chloride
Escherichia coli Proteins
DNA
Nucleoproteins
Deoxyribonuclease I
Fluorescence
Collodion
Base Pairing
Digestion
Adenosine Triphosphate

Keywords

  • DNA binding
  • DNA strand exchange
  • Homologous pairing
  • RecA protein
  • Recombination

ASJC Scopus subject areas

  • Virology

Cite this

The simultaneous binding of two double-stranded DNA molecules by Escherichia coli RecA protein. / Zaitsev, Eugene N.; Kowalczykowski, Stephen C.

In: Journal of Molecular Biology, Vol. 287, No. 1, 19.03.1999, p. 21-31.

Research output: Contribution to journalArticle

Zaitsev, Eugene N. ; Kowalczykowski, Stephen C. / The simultaneous binding of two double-stranded DNA molecules by Escherichia coli RecA protein. In: Journal of Molecular Biology. 1999 ; Vol. 287, No. 1. pp. 21-31.
@article{4c02f503c71045b1ba73f4df0bc620e2,
title = "The simultaneous binding of two double-stranded DNA molecules by Escherichia coli RecA protein",
abstract = "We have characterized the double-stranded DNA (dsDNA) binding properties of RecA protein, using an assay based on changes in the fluorescence of 4',6-diamidino-2-phenylindole (DAPI)-dsDNA complexes. Here we use fluorescence, nitrocellulose filter-binding, and DNase I-sensitivity assays to demonstrate the binding of two duplex DNA molecules by the RecA protein filament. We previously established that the binding stoichiometry for the RecA protein-dsDNA complex is three base-pairs per RecA protein monomer, in the presence of ATP. In the presence of ATPγS, however, the binding stoichiometry depends on the MgCl2 concentration. The stoichiometry is 3 bp per monomer at low MgCl2 concentrations, but changes to 6 bp per monomer at higher MgCl2 concentrations, with the transition occurring at approximately 5 mM MgCl2. Above this MgCl2 concentration, the dsDNA within the RecA nucleoprotein complex becomes uncharacteristically sensitive to DNase I digestion. For these reasons we suggest that, at the elevated MgCl2 conditions, the RecA-dsDNA nucleoprotein filament can bind a second equivalent of dsDNA. These results demonstrate that RecA protein has the capacity to bind two dsDNA molecules, and they suggest that RecA or RecA-like proteins may effect homologous recognition between intact DNA duplexes.",
keywords = "DNA binding, DNA strand exchange, Homologous pairing, RecA protein, Recombination",
author = "Zaitsev, {Eugene N.} and Kowalczykowski, {Stephen C.}",
year = "1999",
month = "3",
day = "19",
doi = "10.1006/jmbi.1998.2580",
language = "English (US)",
volume = "287",
pages = "21--31",
journal = "Journal of Molecular Biology",
issn = "0022-2836",
publisher = "Academic Press Inc.",
number = "1",

}

TY - JOUR

T1 - The simultaneous binding of two double-stranded DNA molecules by Escherichia coli RecA protein

AU - Zaitsev, Eugene N.

AU - Kowalczykowski, Stephen C.

PY - 1999/3/19

Y1 - 1999/3/19

N2 - We have characterized the double-stranded DNA (dsDNA) binding properties of RecA protein, using an assay based on changes in the fluorescence of 4',6-diamidino-2-phenylindole (DAPI)-dsDNA complexes. Here we use fluorescence, nitrocellulose filter-binding, and DNase I-sensitivity assays to demonstrate the binding of two duplex DNA molecules by the RecA protein filament. We previously established that the binding stoichiometry for the RecA protein-dsDNA complex is three base-pairs per RecA protein monomer, in the presence of ATP. In the presence of ATPγS, however, the binding stoichiometry depends on the MgCl2 concentration. The stoichiometry is 3 bp per monomer at low MgCl2 concentrations, but changes to 6 bp per monomer at higher MgCl2 concentrations, with the transition occurring at approximately 5 mM MgCl2. Above this MgCl2 concentration, the dsDNA within the RecA nucleoprotein complex becomes uncharacteristically sensitive to DNase I digestion. For these reasons we suggest that, at the elevated MgCl2 conditions, the RecA-dsDNA nucleoprotein filament can bind a second equivalent of dsDNA. These results demonstrate that RecA protein has the capacity to bind two dsDNA molecules, and they suggest that RecA or RecA-like proteins may effect homologous recognition between intact DNA duplexes.

AB - We have characterized the double-stranded DNA (dsDNA) binding properties of RecA protein, using an assay based on changes in the fluorescence of 4',6-diamidino-2-phenylindole (DAPI)-dsDNA complexes. Here we use fluorescence, nitrocellulose filter-binding, and DNase I-sensitivity assays to demonstrate the binding of two duplex DNA molecules by the RecA protein filament. We previously established that the binding stoichiometry for the RecA protein-dsDNA complex is three base-pairs per RecA protein monomer, in the presence of ATP. In the presence of ATPγS, however, the binding stoichiometry depends on the MgCl2 concentration. The stoichiometry is 3 bp per monomer at low MgCl2 concentrations, but changes to 6 bp per monomer at higher MgCl2 concentrations, with the transition occurring at approximately 5 mM MgCl2. Above this MgCl2 concentration, the dsDNA within the RecA nucleoprotein complex becomes uncharacteristically sensitive to DNase I digestion. For these reasons we suggest that, at the elevated MgCl2 conditions, the RecA-dsDNA nucleoprotein filament can bind a second equivalent of dsDNA. These results demonstrate that RecA protein has the capacity to bind two dsDNA molecules, and they suggest that RecA or RecA-like proteins may effect homologous recognition between intact DNA duplexes.

KW - DNA binding

KW - DNA strand exchange

KW - Homologous pairing

KW - RecA protein

KW - Recombination

UR - http://www.scopus.com/inward/record.url?scp=0033582947&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0033582947&partnerID=8YFLogxK

U2 - 10.1006/jmbi.1998.2580

DO - 10.1006/jmbi.1998.2580

M3 - Article

C2 - 10074404

AN - SCOPUS:0033582947

VL - 287

SP - 21

EP - 31

JO - Journal of Molecular Biology

JF - Journal of Molecular Biology

SN - 0022-2836

IS - 1

ER -