Affinity chromatography of RecA protein and RecA nucleoprotein complexes on RecA protein-agarose columns.
We have analyzed the nature of RecA protein-RecA protein interactions using an affinity column prepared by coupling RecA protein to an agarose support. When radiolabeled soluble proteins from Escherichia coli are applied to this column, only the labeled RecA protein from the extract was selectively retained and bound tightly to the affinity column. Efficient binding of purified 35S-labeled RecA protein required Mg2+, and high salt did not interfere with the binding of RecA protein to the column. Complete removal of the bound enzyme from the affinity column required treatment with guanidine HCl (5 M) or urea (8 M). These and other properties suggest that hydrophobic interactions contribute significantly to RecA protein subunit recognition in solution. Using a series of truncated RecA proteins synthesized in vitro, we have obtained evidence that at least some of the sequences involved in protein recognition are localized within the first 90 amino-terminal residues of the protein. Based on the observation that RecA proteins from three heterologous bacteria are specifically retained on the E. coli RecA affinity column, it is likely that this binding domain is highly conserved and is required for interaction and association of RecA protein monomers. Stable ternary complexes of RecA protein and single-stranded DNA were formed in the presence of the nonhydrolyzable ATP analog adenosine 5'-O-(thiotriphosphate) and applied to the affinity columns. Most of the complexes formed with M13 DNA could be eluted in high salt, whereas a substantial fraction of those formed with the oligonucleotide (dT)25-30 remained bound in high salt and were quantitatively eluted with guanidine HCl (5 M). The different binding properties of these RecA protein-DNA complexes likely reflect differences in the availability of a hydrophobic surface on RecA protein when it is bound to long polynucleotides compared to short oligonucleotides.
Freitag
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N
Laboratory of Biomedical and Environmental Sciences, School of Medicine, University of California, Los Angeles 90024.
McEntee
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K
D002846:Chromatography, Affinity / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005796:Genes; D005798:Genes, Bacterial; D009698:Nucleoproteins / Q000302:isolation & purification*; D010957:Plasmids; D011938:Rec A Recombinases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D012685:Sepharose; D012763:Shigella flexneri / Q000378:metabolism
D009698:Nucleoproteins; D012685:Sepharose; D011938:Rec A Recombinases