Summary
The RecA protein ofEscherichia coli is essential for genetic recombination and postreplicational repair of DNA. In vitro, RecA protein promotes strand transfer reactions between full length linear duplex and single stranded circular DNA of ϕX174 to form heteroduplex replicative form II-like structures (Cox and Lehman 1981a). In a similar way, it transfers one strand of a short duplex restriction fragment to a single stranded circle. Both reactions require RecA and single strand binding protein (SSB) in amounts sufficient to saturate the ssDNA. The rate and extent of strand transfer is enhanced considerably when SSB is added after preincubation of the DNA with RecA protein. In contrast, SSB protein is not required for RecA protein catalysed reciprocal strand exchanges between regions of duplex DNA. These results indicate that while SSB is necessary for efficient transfer between linear duplex and ssDNA to form a single heteroduplex, it is not required for branch migration reactions between duplex molecules that form two heteroduplexes.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Abbreviations
- SSB:
-
single strand binding protein
- ssDNA:
-
single stranded DNA
- ϕX:
-
phage ϕX174
- bp:
-
base pairs
- ATP[S]:
-
adenosine 5′-O-(gamma-thiotriphosphate)
References
Cassuto E, West SC, Mursalim J, Conlon S, Howard-Flanders P (1980) Initiation of genetic recombination: Homologous pairing between duplex DNA molecules promoted by RecA protein. Proc Natl Acad Sci USA 77:3962–3966
Cox MM, Lehman IR (1981a) RecA protein promotes branch migration, a kinetically distinct phase of DNA strand exchange. Proc Natl Acad Sci USA 78:3433–3437
Cox MM, Lehman IR (1981b) Directionality and polarity in RecA protein-promoted branch migration. Proc Natl Acad Sci USA 78:6018–6022
Cox MM, McEntee K, Lehman IR (1981c) A simple and rapid procedure for the large scale purification of the RecA protein ofEscherichia coli. J Biol Chem 256:4676–4678
Cunningham RP, DasGupta C, Shibata T, Radding CM (1980) Homologous pairing in genetic recombination: RecA protein makes joint molecules of gapped circular DNA and closed circular DNA. Cell 20:223–235
Cunningham RP, Wu AM, Shibata T, DasGupta C, Radding CM (1981) Homologous pairing and topological linkage of DNA molecules by combined action ofE. coli RecA protein and topoisomerase I. Cell 24:213–223
DasGupta C, Shibata T, Cunningham RP, Radding CM (1980) The topology of homologous pairing promoted by RecA protein. Cell 22:437–446
Godson GN (1974) Evolotion of ϕX174: Isolation and characterization of four new ϕX-like phages and comparison with ϕX174. Virology 58:272–289
Godson GN, Boyer H (1974) Susceptibility of the ϕX-like phages G4 and G14 to R. EcoRI endonuclease. Virology 62:270–275
McEntee K, Weinstock GM, Lehman IR (1979) Initiation of genetic recombination catalysedin vitro by the RecA protein ofEscherichia coli. Proc Natl Acad Sci USA 76:2615–2619
McEntee K, Weinstock GM, Lehman IR (1980) RecA protein catalysed strand assimilation: Stimulation byEscherichia coli singlestranded DNA binding protein. Proc Natl Acad Sci USA 77:857–861
Ross P, Howard-Flanders P (1977) Initiation ofrecA +-dependent recombination inEscherichia coli. J Mol Biol 117:137–158
Rupp WD, Wilde CE, Reno DL, Howard-Flanders P (1971) Exchanges between DNA strands in ultraviolet-irradiatedEscherichia coli. J Mol Biol 61:25–44
Sancar A, Stachelek C, Konigsberg W, Rupp WD (1980) Sequences of therecA gene and protein. Proc Natl Acad Sci USA 77:2611–2615
Scott JF, Eisenberg S, Bertsch LL, Kornberg A (1977) A mechanism of duplex DNA replication revealed by enzymatic studies of phage ϕX174: Catalytic strand separation in advance of replication. Proc Natl Acad Sci USA 74:193–197
Shibata T, DasGupta C, Cunningham RP, Radding CM (1979) PurifiedEscherichia coli RecA protein catalyses homologous pairing of superhelical DNA and single stranded fragments. Proc Natl Acad Sci USA 76:1638–1642
Shibata T, DasGupta C, Cunningham RP, Radding CM (1980) Homologous pairing in genetic recombination: Formation of D loops by combined action of RecA protein and a helix-destabilizing protein. Proc Natl Acad Sci USA 77:2606–2610
Sigal N, Delius H, Kornberg T, Gefter M, Alberts B (1972) A DNA-unwinding protein isolated fromEscherichia coli: Its interaction with DNA and with DNA polymerases. Proc Natl Acad Sci USA 69:3537–3541
Weiner JH, Bertsch LL, Kornberg A (1975) The deoxyribonucleic acid unwinding protein ofEscherichia coli. J Biol Chem 250:1972–1980
Weinstock GM, McEntee K, Lehman IR (1979) ATP-dependent renaturation of DNA catalysed by the RecA protein ofEscherichia coli. Proc Natl Acad Sci USA 76:126–130
West SC, Cassuto E, Mursalim J, Howard-Flanders P (1980) Recognition of duplex DNA containing single stranded regions by RecA protein. Proc Natl Acad Sci USA 77:2569–2573
West SC, Cassuto E, Howard-Flanders P (1981a) RecA protein promotes homologous pairing and strand exchange reactions between duplex DNA molecules. Proc Natl Acad Sci USA 78:2100–2104
West SC, Cassuto E, Howard-Flanders P (1981b) Heteroduplex formation by RecA protein: Polarity of strand exchanges. Proc Natl Acad Sci USA 78:6149–6153
West SC, Cassuto E, Howard-Flanders P (1981c) Mechanism ofE. coli RecA protein directed strand exchanges in postreplication repair of DNA. Nature 294:659–662
Whittier RF, Chase JW (1981) DNA repair inE. coli strains deficient in single-strand DNA binding protein. Mol Gen Genet 183:341–347
Yarranton GT, Das RH, Gefter ML (1979) Enzyme catalysed DNA unwinding. J Biol Chem 254:12002–12006
Author information
Authors and Affiliations
Additional information
Communicated by B.A. Bridges
Rights and permissions
About this article
Cite this article
West, S.C., Cassuto, E. & Howard-Flanders, P. Role of SSB protein in RecA promoted branch migration reactions. Molec. Gen. Genet. 186, 333–338 (1982). https://doi.org/10.1007/BF00729451
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00729451