Abstract
In the Escherichia coli, RecA plays a central role in the recombination and repair of the DNA. For homologous recombination, RecA binds to ssDNA forming a nucleoprotein filament. The RecA-ssDNA filament searches for a homologous sequence on a dsDNA and, subsequently, RecA mediates strand exchange between the ssDNA and the dsDNA. In vitro, RecA binds to both ssDNA and dsDNA. Despite a wide range of studies of the polymerization of RecA on dsDNA, both at the single molecule level and by means of biochemical methods, important aspects of this process are still awaiting a better understanding. Specifically, a detailed, quantitative description of the nucleation and growth dynamics of the RecA-dsDNA filaments is still lacking. Here, we use Optical Tweezers together with a single molecule analysis approach to measure the dynamics of the individual RecA domains on dsDNA and the corresponding growth rates for each of their fronts. We focus on the regime where the nucleation and growth rate constants, k n and k g, are comparable, leading to a coverage of the dsDNA molecule that consists of a small number of RecA domains. For the case of essentially irreversible binding (using ATPγS instead of ATP), we find that domain growth is highly asymmetric with a ratio of about 10:1 between the fast and slow fronts growth rates.
Highlights
The primary function of the RecA protein is to exchange strands as part of the homologous recombination process in Escherichia Coli (Radding, 1988; Cox, 1999; Kowalczykowski, 2000; Cox, 2007b; Prentiss et al, 2015; Bell and Kowalczykowski, 2016)
We present a new approach to measuring the growth rates of individual RecA domains on dsDNA that allows obtaining the growth rate for each of the two fronts of individual domains
Within our interpretation of the data, the growth velocity of one front is about 12 times faster than the other. We suggest that this strong asymmetry between the growth rates of the individual domain fronts represents a general feature in the RecA-dsDNA system in the presence of ATPγS
Summary
The primary function of the RecA protein is to exchange strands as part of the homologous recombination process in Escherichia Coli (Radding, 1988; Cox, 1999; Kowalczykowski, 2000; Cox, 2007b; Prentiss et al, 2015; Bell and Kowalczykowski, 2016). Single molecule methods were used to study the effect of force applied to the DNA on the rate of RecA polymerization and the efficiency of the homology search (Leger et al, 1998; van Loenhout et al, 2009; Danilowicz et al, 2012; De Vlaminck et al, 2012; Fu et al, 2013a; Fu et al, 2013b). We choose the experimental parameters such that the nucleation and growth rates are comparable, kg/kn ≈ 1 In this regime, we obtain a small number of RecA domains on each dsDNA molecule that, in turn, allows extracting their dynamics from the dsDNA contour length variation. We find that domain growth is strongly asymmetric whereby the rate of growth is about 10 times larger for the fast front than for the slow one
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