Reversibility, equilibration, and fidelity of strand exchange reaction between short oligonucleotides promoted by RecA protein from escherichia coli and human Rad51 and Dmc1 proteins.
We demonstrate the reversibility of RecA-promoted strand exchange reaction between short oligonucleotides in the presence of adenosine 5'-O-(thiotriphosphate). The reverse reaction proceeds without the dissociation of RecA from DNA. The reaction reaches equilibrium and its yield depends on the homology between the reaction substrates. We estimate the tolerance of the RecA-promoted strand exchange to individual base substitutions for a comprehensive set of possible base combinations in a selected position along oligonucleotide substrates for strand exchange and find, in agreement with previously reported estimations, that this tolerance is higher than in the case of free DNA. It is demonstrated that the short oligonucleotide-based approach can be applied to the human recombinases Rad51 and Dmc1 when strand exchange is performed in the presence of calcium ions and ATP. Remarkably, despite the commonly held belief that the eukaryotic recombinases have an inherently lower strand exchange activity, in our system their efficiencies in strand exchange are comparable with that of RecA. Under our experimental conditions, the human recombinases exhibit a significantly higher tolerance to interruptions of homology due to point base substitutions than RecA. Finding conditions where a chemical reaction is reversible and reaches equilibrium is critically important for its thermodynamically correct description. We believe that the experimental system described here will substantially facilitate further studies on different aspects of the mechanisms of homologous recombination.
Volodin
Alexander A
AA
Institute of Molecular Genetics of the Russian Academy of Sciences, Kurchatov Square, 123182 Moscow, Russia.
Bocharova
Tatiana N
TN
Smirnova
Elena A
EA
Camerini-Otero
R Daniel
RD
D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D009841:Oligonucleotides / Q000737:chemistry* / Q000378:metabolism; D051135:Rad51 Recombinase / Q000737:chemistry* / Q000378:metabolism; D011938:Rec A Recombinases / Q000737:chemistry* / Q000378:metabolism; D011995:Recombination, Genetic / Q000502:physiology*; D013873:Thionucleotides / Q000737:chemistry
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D009841:Oligonucleotides; D013873:Thionucleotides; C017817:adenosine 5'-(1-thio)triphosphate; C495153:RAD51 protein, human; D051135:Rad51 Recombinase; D011938:Rec A Recombinases; C074556:DMC1 protein, human; D002118:Calcium