Kinetic Analysis of Pairing and Strand Exchange Catalyzed by RecA

Abstract

RecA is a 38-kDa protein from Escherichia coli that polymerizes on single-stranded DNA, forming a nucleoprotein filament that pairs with homologous duplex DNA and carries out strand exchange in vitro. In this study, we measured RecA-catalyzed pairing and strand exchange in solution by energy transfer between fluorescent dyes on the ends of deoxyribo-oligonucleotides. By varying the position of the dyes in separate assays, we were able to detect the pairing of single-stranded RecA filament with duplex DNA as an increase in energy transfer, and strand displacement as a decrease in energy transfer. With these assays, the kinetics of pairing and strand displacement were studied by stopped-flow spectrofluorometry. The data revealed a rapid, second order, reversible pairing step that was followed by a slower, reversible, first order strand exchange step. These data indicate that an initial unstable intermediate exists which can readily return to reactants, and that a further, rate-limiting step (or steps) is required to effect or complete strand exchange.RecA is a 38-kilodalton protein from Escherichia coli, which has been shown to be necessary for conjugal homologous recombination in vivo (1). In vitro, RecA protein polymerizes on single-stranded DNA in the 5Ј to 3Ј direction to form a righthanded helical structure in which the DNA is extended to 1.5 times its original length (2, 3). Pairing with homologous duplex DNA results in a rapid uptake of the double-stranded DNA into a three-stranded complex, which can be kilobases in length (4 -6). Strand exchange results in displacement of the strand of duplex DNA that has the same sequence as the filament strand; the strand is displaced in the 5Ј to 3Ј direction (7-9). Homologs of RecA exist in eukaryotes from yeast to man and have been found to hydrolyze ATP, to form nucleoprotein filaments, to pair homologous DNA, and to carry out strand exchange in ways that are qualitatively similar to RecA protein (10 -15). Studies of the mechanisms of E. coli RecA may help to shed light on eukaryotic as well as prokaryotic recombination.Most studies on the kinetics of RecA-catalyzed strand exchange have used phage DNA that is several kilobases in length (16 -20). RecA filaments formed on long single-stranded DNA generate coaggregates with duplex DNA that concentrate the DNA but also limit diffusion (21). Despite this complication, joint molecule formation displayed the saturation of rates with increasing substrate concentration that is typical of MichaelisMenten kinetics (17). This observation suggested the existence of a reversible pairing step and a second, rate-limiting step in the reaction. Yancey-Wrona and Camerini-Otero (22) developed a solution assay for pairing and stable synapsis of a single-stranded oligonucleotide with duplex DNA in which a RecA filament formed in the presence of ATP␥S 1 protects a restriction site in the duplex target molecule. With this assay, they found that pairing was second order, reversible, and independent of the complexity of the target. They were...

Roles of Bacillus subtilis DprA and SsbA in RecA-mediated Genetic Recombination

“…The RecA⅐dATP transitional states resemble those described previously for RecA Eco , designated as O, Ac, Ao, and P states ( Fig. 1) (15)(16)(17)(18). In short, RecA in the O state is largely inactive, but RecA⅐dATP induces a high affinity DNA binding state, which leads to cooperative polymerization of RecA⅐dATP onto SSB-coated ssDNA ( Fig.…”

The synaptic activity of HsDmc1, a human recombination protein specific to meiosis

“…The (Ϫ) and (ϩ) strands contained a six-carbon primary amine linker at the 3Ј and the 5Ј ends, respectively. The labeling of these oligonucleotides with fluorescein and rhodamine was done as described before (30).…”

“…The His-tagged Dmc1 protein hydrolyzed ATP in a DNA-dependent manner with a catalytic rate constant (k cat ) of 0.7 mols͞mol͞min, similar to the k cat for nontagged protein, described before (27). In previous studies, we have used assays based on FRET to distinguish and kinetically characterize two phases of recombination reactions catalyzed by members of the RecA family of proteins (20,30,31). These phases are the recognition of homology and the exchange of strands.…”

DNA replication meets genetic exchange: Chromosomal damage and its repair by homologous recombination

“…Charles Radding reviewed the current understanding of the most critical and still mysterious step of recombinational repair, the RecA-catalyzed homology search with subsequent strand exchange. Fluorescence energy transfer between strands of a DNA duplex reveals that the RecA-catalyzed reaction is subdivided into two, possibly three phases: (i) a rapid, second order step, reflecting initial interactions of the ssDNA with the complementary strand of the duplex; (ii) a rapid (and cryptic) first order step, perhaps reflecting subsequent proximity of the ssDNA to the homologous strand of the duplex; and (iii) a slower first order strand exchange step (56). All of the steps are reversible, suggesting that the observed high specificity of the RecA-promoted homology search is the product of several steps of moderate specificity.…”