• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of pnasPNASInfo for AuthorsSubscriptionsAboutThis Article
Proc Natl Acad Sci U S A. Jan 1990; 87(1): 21–25.
PMCID: PMC53191

Stable DNA heteroduplex formation catalyzed by the Escherichia coli RecA protein in the absence of ATP hydrolysis.

Abstract

A question remaining to be answered about RecA protein function concerns the role of ATP hydrolysis during the DNA-strand-exchange reaction. In this paper we describe the formation of joint molecules in the absence of ATP hydrolysis, using adenosine 5'-[gamma-thio]triphosphate (ATP[gamma S]) as nucleotide cofactor. Upon the addition of double-stranded DNA, the ATP[gamma S]-RecA protein-single-stranded DNA presynaptic complexes can form homologously paired molecules that are stable after deproteinization. Formation of these joint molecules requires both homology and a free homologous end, suggesting that they are plectonemic in nature. This reaction is very sensitive to magnesium ion concentration, with a maximum rate and extent observed at 4-5 mM magnesium acetate. Under these conditions, the average length of heteroduplex DNA within the joint molecules is 2.4-3.4 kilobase pairs. Thus, RecA protein can form extensive regions of heteroduplex DNA in the presence of ATP[gamma S], suggesting that homologous pairing and the exchange of the DNA molecules can occur without ATP hydrolysis. A model for the RecA protein-catalyzed DNA-strand-exchange reaction that incorporates these results and its relevance to the mechanisms of eukaryotic recombinases are presented.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (1.3M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Click on the image to see a larger version.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Gonda DK, Radding CM. By searching processively RecA protein pairs DNA molecules that share a limited stretch of homology. Cell. 1983 Sep;34(2):647–654. [PubMed]
  • Cox MM, Lehman IR. recA protein of Escherichia coli promotes branch migration, a kinetically distinct phase of DNA strand exchange. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3433–3437. [PMC free article] [PubMed]
  • Riddles PW, Lehman IR. The formation of plectonemic joints by the recA protein of Escherichia coli. Requirement for ATP hydrolysis. J Biol Chem. 1985 Jan 10;260(1):170–173. [PubMed]
  • Honigberg SM, Gonda DK, Flory J, Radding CM. The pairing activity of stable nucleoprotein filaments made from recA protein, single-stranded DNA, and adenosine 5'-(gamma-thio)triphosphate. J Biol Chem. 1985 Sep 25;260(21):11845–11851. [PubMed]
  • Uhlin BE, Clark AJ. Overproduction of the Escherichia coli recA protein without stimulation of its proteolytic activity. J Bacteriol. 1981 Oct;148(1):386–390. [PMC free article] [PubMed]
  • Griffith J, Shores CG. RecA protein rapidly crystallizes in the presence of spermidine: a valuable step in its purification and physical characterization. Biochemistry. 1985 Jan 1;24(1):158–162. [PubMed]
  • Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. [PubMed]
  • Lanzetta PA, Alvarez LJ, Reinach PS, Candia OA. An improved assay for nanomole amounts of inorganic phosphate. Anal Biochem. 1979 Nov 15;100(1):95–97. [PubMed]
  • Weinstock GM, McEntee K, Lehman IR. Interaction of the recA protein of Escherichia coli with adenosine 5'-O-(3-thiotriphosphate). J Biol Chem. 1981 Aug 25;256(16):8850–8855. [PubMed]
  • Griffith J, Formosa T. The uvsX protein of bacteriophage T4 arranges single-stranded and double-stranded DNA into similar helical nucleoprotein filaments. J Biol Chem. 1985 Apr 10;260(7):4484–4491. [PubMed]
  • Cox MM, Lehman IR. recA protein-promoted DNA strand exchange. Stable complexes of recA protein and single-stranded DNA formed in the presence of ATP and single-stranded DNA binding protein. J Biol Chem. 1982 Jul 25;257(14):8523–8532. [PubMed]
  • Roman LJ, Kowalczykowski SC. Relationship of the physical and enzymatic properties of Escherichia coli recA protein to its strand exchange activity. Biochemistry. 1986 Nov 18;25(23):7375–7385. [PubMed]
  • Menetski JP, Kowalczykowski SC. Enhancement of Escherichia coli RecA protein enzymatic function by dATP. Biochemistry. 1989 Jul 11;28(14):5871–5881. [PubMed]
  • Stasiak A, Stasiak AZ, Koller T. Visualization of RecA-DNA complexes involved in consecutive stages of an in vitro strand exchange reaction. Cold Spring Harb Symp Quant Biol. 1984;49:561–570. [PubMed]
  • Register JC, 3rd, Christiansen G, Griffith J. Electron microscopic visualization of the RecA protein-mediated pairing and branch migration phases of DNA strand exchange. J Biol Chem. 1987 Sep 15;262(26):12812–12820. [PubMed]
  • Schutte BC, Cox MM. Homology-dependent changes in adenosine 5'-triphosphate hydrolysis during recA protein promoted DNA strand exchange: evidence for long paranemic complexes. Biochemistry. 1987 Sep 8;26(18):5616–5625. [PubMed]
  • Menetski JP, Kowalczykowski SC. Interaction of recA protein with single-stranded DNA. Quantitative aspects of binding affinity modulation by nucleotide cofactors. J Mol Biol. 1985 Jan 20;181(2):281–295. [PubMed]
  • Menetski JP, Kowalczykowski SC. Transfer of recA protein from one polynucleotide to another. Kinetic evidence for a ternary intermediate during the transfer reaction. J Biol Chem. 1987 Feb 15;262(5):2085–2092. [PubMed]
  • Egelman EH, Stasiak A. Structure of helical RecA-DNA complexes. Complexes formed in the presence of ATP-gamma-S or ATP. J Mol Biol. 1986 Oct 20;191(4):677–697. [PubMed]
  • Menetski JP, Varghese A, Kowalczykowski SC. Properties of the high-affinity single-stranded DNA binding state of the Escherichia coli recA protein. Biochemistry. 1988 Feb 23;27(4):1205–1212. [PubMed]
  • Hsieh P, Meyn MS, Camerini-Otero RD. Partial purification and characterization of a recombinase from human cells. Cell. 1986 Mar 28;44(6):885–894. [PubMed]
  • Kolodner R, Evans DH, Morrison PT. Purification and characterization of an activity from Saccharomyces cerevisiae that catalyzes homologous pairing and strand exchange. Proc Natl Acad Sci U S A. 1987 Aug;84(16):5560–5564. [PMC free article] [PubMed]
  • Sugino A, Nitiss J, Resnick MA. ATP-independent DNA strand transfer catalyzed by protein(s) from meiotic cells of the yeast Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1988 Jun;85(11):3683–3687. [PMC free article] [PubMed]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...