Summary
Interaction of the RecA protein with single-stranded DNA (ssDNA) was analyzed by challenge with the hydroxyl radical, which can cleave the DNA backbone. We found that RecA protein induces cleavage by the radical at a defined distance from the 5′ end. The cleavage was at the 11th nucleotide in many oligodeoxynucleotides. Cleavage may be intermittent since a second cleavage was induced at the 22nd or 21st site. This specific cleavage was observed under optimal conditions for filament formation, homologous pairing and strand exchange. Specificity in cleavage was, however, decreased by replacement of ATP by adenosine 5′-(γ-thio)triphosphate (ATPγS), replacement of RecA protein by a mutant (RecAl) protein, or an increase in Mg2– concentration. We propose that RecA protein induces a special structural alteration, such as bending, perhaps sequentially, on ssDNA and that this altered site plays an important role in homologous pairing and strand exchange.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Abbreviations
- ssDNA:
-
single-stranded DNA
- dsDNA:
-
doublestranded DNA
- ATPγS:
-
adenosine 5′-(γ-thio)triphosphate
References
Cox MM, Lehman IR (1981) recA protein of Escherichia coli promotes branch migration, a kinetically distinct phase of DNA strand exchange. Proc Natl Acad Sci USA 78:3433–3437
Cox MM, Lehman IR (1987) Enzymes of general recombination. Annu Rev Biochem 56:229–262
Cox MM, McEntee K, Lehman IR (1981) A simple and rapid procedure for the large scale purification of the recA protein of Escherichia coli. J Biol Chem 256:4676–4678
Di Capua E, Müller B (1987) The accessibility of DNA to dimethyl sulfate in complexes with recA protein. EMBO J 6:2493–2498
Dunn K, Chrysogelos S, Griffith J (1982) Electron microscopic visualization of recA-DNA filaments: Evidence for a cyclic extension of duplex DNA. Cell 28:757–765
Egelman EH, Stasiak A (1986) Structure of helical RecA-DNA complexes. Complexes formed in the presence of ATP-gammaS or ATP. J Mol Biol 191:677–697
Flory J, Radding CM (1982) Visualization of recA protein and its association with DNA: A priming effect of single-strand-binding protein. Cell 28:747–756
Flory J, Tsang SS, Muniyappa K (1984) Isolation and visualization of active presynaptic filaments of recA protein and single-stranded DNA. Proc Natl Acad Sci USA 81:7026–7030
Hatfull GF, Noble SM, Grindley NDF (1987) The γδ resolvase induces an unusual DNA structure at the recombinational crossover point. Cell 49:103–110
Hertzberg RP, Dervan PB (1984) Cleavage of DNA with methidiumpropyl-EDTA-Iron(II): reaction conditions and product analysis. Biochemistry 23:3934–3945
Honigberg SM, Gonda DK, Flory J, Radding CM (1985) The pairing activity of stable nucleoprotein filaments made from recA protein, single-stranded DNA, and adenosine 5′-(γ-thio)triphosphate. J Biol Chem 260:11845–11851
Howard-Flanders P, West SC, Stasiak A (1984) Role of RecA protein spiral filaments in genetic recombination. Nature 309:215–220
Koller Th, Di Capua E, Stasiak A (1983) Complexes of recA protein with single stranded DNA. In: Cozzarelli NR (ed) Mechanisms of DNA replication and recombination, vol 10. Alan R. Liss, New York, pp 723–729
Leahy MC, Radding CM (1986) Topography of the interaction of recA protein with single-stranded deoxyoligonucleotides. J Biol Chem 261:6954–6960
Maxam AM, Gilbert W (1977) A new method for sequencing DNA. Proc Natl Acad Sci USA 74:560–564
Maxam AM, Gilbert W (1980) Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol 65:499–560
McEntee K, Weinstock GM, Lehman IR (1980) recA protein-catalyzed strand assimilation: Stimulation by Escherichia coli single-stranded DNA-binding protein. Proc Natl Acad Sci USA 77:857–861
McEntee K, Weinstock GM, Lehman IR (1981) Binding of the recA protein of Escherichia coli to single- and double-stranded DNA. J Biol Chem 256:8835–8844
Riddles PW, Lehman IR (1985) The formation of plectonemic joints by the recA protein of Escherichia coli. J Biol Chem 260:170–173
Stasiak A, Di Capua E, Koller T (1981) Elongation of duplex DNA by recA protein. J Mol Biol 151:557–564
Travers AA (1989) DNA conformation and protein binding. Annu Rev Biochem 58:427–452
Tsang SS, Chow SA, Radding CM (1985) Networks of DNA and recA protein are intermediates in homologous pairing. Biochemistry 24:3226–3232
Tullius TD, Dombroski BA (1986) Hydroxyl radical “footprinting”: High resolution information about DNA-protein contacts and application to λ repressor and Cro protein. Proc Natl Acad Sci USA 83:5469–5473
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
Author information
Authors and Affiliations
Additional information
Communicated by M. Sekiguchi
Deceased
Rights and permissions
About this article
Cite this article
Akaboshi, E., Howard-Flanders, P. RecA-ssDNA interaction: Induced strand cleavage by hydroxyl radical at a defined distance from the 5′ end. Mol Gen Genet 220, 456–460 (1990). https://doi.org/10.1007/BF00391753
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00391753