Reversibility, Equilibration, and Fidelity of Strand Exchange Reaction between Short Oligonucleotides Promoted by RecA Protein from Escherichia coli and Human Rad51 and Dmc1 Proteins

Abstract

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. Homologous recombination (HR)2 is a fundamental genetic process with a great variety of biological implications spanning from microbial evolution and the development of pathogens to genome instability and carcinogenesis in higher eukaryotes. It also is an example of an intricate macromolecular choreography. HR has been finding a widening scope of practical applications such as DNA recombineering, eukaryotic gene targeting etc. (for recent reviews see Refs. 1-3 and references therein).The central event of HR is based on a peculiar type of recognition of homology between DNA molecules and until now the mechanism of this recognition remains enigmatic (for recent reviews see Refs. 4 -9). This process is accompanied by drastic changes in DNA conformation (10, 11) (for recent reviews see Refs. 5, 9, and 12-14) and allosteric effects (15-18) in the proteins that promote DNA strand exchange, the central step of HR.The main prerequisite for the thermodynamically correct description of a chemical reaction is finding conditions where the reaction is reversible and reaches equilibrium. However, enzymatic processes are often too complex to separate reversible phases and do not reach equilibrium. In the case of RecApromoted DNA strand exchange the complexity is the result of the multistep nature of the reaction; the filamentous structure and high tendency to aggregate of the RecA protein and its complexes with long DNA molecules; the tendency of ssDNA to form extensive...

Tolerance of DNA Mismatches in Dmc1 Recombinase-mediated DNA Strand Exchange

“…Notably, in vitro, eukaryotic recombinases usually exhibit a less robust strand exchange activity and higher tolerance to interruptions of homology than RecA protein (15). At present, it is not clear if the ability of the eukaryotic recombinases to tolerate DNA mismatches during the catalysis of homologous DNA pairing and strand exchange is subject to regulation by their accessory factors.…”

Linker histone H1 stimulates DNA strand exchange between short oligonucleotides retaining high sensitivity to heterology

“…In the present work we asked whether a strongly basic protein—the linker histone H1—influences DNA strand exchange in a model system of short oligonucleotides. Earlier we demonstrated the applicability of this system for the characterization of the strand exchange reaction promoted by enzymes of homologous recombination from bacteria and human 9. We demonstrate here that human linker histone H1(0) facilitates strand exchange between short oligonucleotides and this reaction exhibits a low tolerance to the interruption of homology resulting from a point base substitutions in the oligonucleotide substrates of the reaction.…”

“…Earlier we described an experimental system for the study of strand exchange between short oligonucleotides promoted by recombinases of the RecA family 9. In the present work this approach was applied to characterize the strand exchange activity of the linker histone.…”

“…In contrast to the RecA‐like enzymes of homologous recombination9, 10 the CCC promoted strand exchange reaction exhibits low tolerance to interruptions of homology, comparable to the tolerance inherent to the spontaneous strand exchange that proceeds at elevated temperatures 5, 11, 12. Most probably, the interaction of the cationic copolymers with DNA is mainly electrostatic in character and it may be expected than many other strongly cationic agents can stimulate DNA strand exchange.…”

Equilibrious Strand Exchange Promoted by DNA Conformational Switching

“…In this process, the release of RecA from the heteroduplex product required ATP hydrolysis28. A contrast was performed in the absence of ATP and no SER product was observed, demonstrating the energy requirement in protein mediated SERs.…”