RecA protein assisted selection reveals a low fidelity of recognition of homology in a duplex DNA by an oligonucleotide.
We have developed an in vitro selection procedure to elucidate the specificity of RecA assisted oligonucleotide recognition of double stranded DNA. The procedure was based on formation of a synaptic complex between an oligonucleotide-RecA filament and a supercoiled plasmid bearing a homologous partially degenerate region. The specificity of the selection depended on the reaction conditions: starting with a population that had, on average, 2.8 randomly distributed mismatches out of 27 bp, a population selected in the presence of 100 mM KCl had on average 1.0 mismatches, while a population selected at low ionic strength was less specific and had, on average, 2.0 mismatches. From the distributions of mismatches observed we calculated that the average destabilization free energy for one mismatch is 1.7(+/-0.5) kcal/mol. This is substantially less than the free energy for the incorporation of one mismatch in naked DNA duplex or a Py-Pu-Py triplex. Thus, RecA has an ability to decrease the fidelity of the homologous pairing reaction and minimize the cost of pairing between similar but not identical sequences. This "antiproofreading" activity of RecA protein does not require ATP hydrolysis.
Malkov
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Genetics and Biochemistry Branch, National Institutes of Health (NIDDK), Building 10 Room 9D15, 10 Center Drive MSC 1810, Bethesda, MD, 20892-1810, USA.
Sastry
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Camerini-Otero
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D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D015723:Gene Library; D008962:Models, Theoretical; D009838:Oligodeoxyribonucleotides / Q000737:chemistry* / Q000378:metabolism*; D009994:Osmolar Concentration; D010957:Plasmids; D011189:Potassium Chloride; D011336:Probability; D011938:Rec A Recombinases / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D013816:Thermodynamics
D009838:Oligodeoxyribonucleotides; D011189:Potassium Chloride; D004247:DNA; D011938:Rec A Recombinases