RecA protein assisted selection reveals a low fidelity of recognition of homology in a duplex DNA by an oligonucleotide

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...

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“…In agreement with previously reported data (38,62) this shift for complexes with RecA protein is lower than for free DNA.…”

Section: Tablesupporting

confidence: 93%

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

Volodin

Bocharova²,

Smirnova³

et al. 2009

Journal of Biological Chemistry

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“…RecA phasing with respect to open-reading frames and the ability of RecA to significantly decrease the fidelity of heteroduplex formation (16,17) compared with that of heteroduplex formation in the absence of protein might have an important role in permitting recombination between homologous but not identical protein encoding DNAs with different codon usage. If, for example, the phasing allows this "anti-proofreading" decrease in fidelity to be mostly at the third position in a codon, the wobble position could be more easily assimilated in genetic crosses.…”

Section: Discussionmentioning

confidence: 99%

Influence of DNA Sequence on the Positioning of RecA Monomers in RecA-DNA Cofilaments

Volodin

Camerini‐Otero

2002

Journal of Biological Chemistry

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We show that certain DNA sequences have the ability to influence the positioning of RecA monomers in RecA-DNA complexes. A tendency for RecA monomers to be phased was observed in RecA protein complexes with several oligonucleotides containing a recombinational hotspot sequence, the chi-site from Escherichia coli. This influence was observed in both the 5 to 3 and 3 to 5 directions with respect to chi. A 5-end phosphate group and probably some other features in DNA also influence the phasing of RecA monomers. We conclude that natural DNAs contain a number of features that influence the positioning of RecA monomers. The ability of specific DNA sequences to influence the positioning of RecA monomers demonstrates some specificity in the binding of individual bases at different sites within a RecA monomer and, most likely, reflects the stereochemical non-equivalence of these sites. The possible biological implications of the phasing of RecA monomers in presynaptic DNA-protein cofilaments are discussed.RecA protein is the central component of homologous recombination in Escherichia coli. In the last few years a number of RecA protein analogues from different sources have been characterized. That these proteins exhibit rather similar properties suggests that the fundamental features of homologous recombination are common in prokaryotes and eukaryotes (for review, see Refs. 1 and 2).In vitro, the first step of RecA-promoted strand exchange reaction is the formation of a RecA protein complex with singlestranded DNA (presynaptic complex). RecA protein monomers bind cooperatively, completely cover the DNA, and thus form a close-packed DNA-protein co-filament. The most widely accepted binding stoichiometry is that each RecA monomer interacts with 3 nucleotides of single-stranded (ss) 1 DNA (3, 4). Thus, the repeat unit of the presynaptic complex should correspond to 3 nucleotides interacting with one RecA monomer.Earlier we demonstrated that when RecA protein binding is initiated on a fluorescent dye molecule attached to the 5Ј-end of an oligonucleotide there is a modulation of the DMS reactivity of a guanine residue along the oligonucleotide with a period of 3 bases (5). This result indicated that there was a strictly ordered (phased) arrangement of RecA monomers on the dyetagged oligonucleotides. In the present study we have asked the question of whether natural DNA contains some features that can influence the positioning of RecA monomers.Homologous recombination is considered to proceed mainly in a sequence-independent manner. At the same time, however, it has been demonstrated that there are some preferred DNA binding and pairing sequences for RecA and the chi-site sequence is among these (6). The chi-site is a recombination hot spot in E. coli (7) with the sequence -GCTGGTGG (8) that contains two 3-base repeats that conform with the RecA protein-DNA binding stoichiometry and might provide a physical basis for the ordered arrangement of RecA monomers in RecA protein complexes with chi-containing DNA. For these reasons ...

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“…(Bottom) RecA-mediated realignment kinetics of mono-, di-, and trinucleotide repeat testers is similar. Three types of testers, namely, A 30 (b, O), (AC) 15 (1,3), and (CTG) 10 (9, 0), were paired with T-template, GT-template, and CAG-template, respectively. TL assay was done in the presence of a common labeled up-tether.…”

Section: Realignment Kinetics Of Mono- Di- and Trinucleotide Repeatmentioning

confidence: 99%

RecA Realigns Suboptimally Paired Frames of DNA Repeats through a Process That Requires ATP Hydrolysis

2000

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Microsatellite repeats such as mono-, di-, and trinucleotides are highly abundant and viable targets for homologous recombination in the genome. However, if recombination ensues in such repetitive regions, they are intrinsically prone to frame misalignments during pairing and might eventually give rise to genetic instabilities. Suboptimally paired frames lead to an abrogation of branch migration at the junctions of mixed sequences and repeats, due to a heterologous register. If so, can recombination machinery rectify such misalignments in order to avoid subsequent arrest in branch migration? We analyzed Escherichia coli RecA, the universal prototype of a recombinase, for its pairing abilities across repeats. We used a complementary pairing assay to test whether RecA can mediate realignments of stochastically paired suboptimal frames to a maximally aligned register. Here, we demonstrate that RecA-single stranded DNA filament indeed facilitates such a realignment, probably by sliding the paired strands across mono- and di- as well as trinucleotide repeats. These realignments apparently have no net directional bias. Such a putative "motor" function of RecA seems to be ATP hydrolysis-dependent.

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RecA protein assisted selection reveals a low fidelity of recognition of homology in a duplex DNA by an oligonucleotide

Cited by 20 publications

References 32 publications

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

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

Influence of DNA Sequence on the Positioning of RecA Monomers in RecA-DNA Cofilaments

RecA Realigns Suboptimally Paired Frames of DNA Repeats through a Process That Requires ATP Hydrolysis

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