Abstract
The RecA filament formed on double-stranded (ds) DNA is proposed to be a functional state analogous to that generated during the process of DNA strand exchange. RecA polymerization and de-polymerization on dsDNA is governed by multiple physiological factors. However, a comprehensive understanding of how these factors regulate the processes of polymerization and de-polymerization of RecA filament on dsDNA is still evolving. Here, we investigate the effects of temperature, pH, tensile force, and DNA ends (in particular ssDNA overhang) on the polymerization and de-polymerization dynamics of the E. coli RecA filament at a single-molecule level. Our results identified the optimal conditions that permitted spontaneous RecA nucleation and polymerization, as well as conditions that could maintain the stability of a preformed RecA filament. Further examination at a nano-meter spatial resolution, by stretching short DNA constructs, revealed a striking dynamic RecA polymerization and de-polymerization induced saw-tooth pattern in DNA extension fluctuation. In addition, we show that RecA does not polymerize on S-DNA, a recently identified novel base-paired elongated DNA structure that was previously proposed to be a possible binding substrate for RecA. Overall, our studies have helped to resolve several previous single-molecule studies that reported contradictory and inconsistent results on RecA nucleation, polymerization and stability. Furthermore, our findings also provide insights into the regulatory mechanisms of RecA filament formation and stability in vivo.
Highlights
In Escherichia coli, the formation of a RecA filament on singlestranded DNA is a crucial step in the processing of DSB ends during recombinational DNA repair [1]
RecA filament is a dynamic structure under conditions of ATP hydrolysis, which is subject to competition between polymerization and de-polymerization processes [5,8,9]
During RecA-catalyzed DNA strand exchange, RecA binds to ssDNA through its primary ssDNA binding site to form a nucleoprotein filament, which interacts with dsDNA weakly via its secondary site
Summary
In Escherichia coli, the formation of a RecA filament on singlestranded DNA (ssDNA) is a crucial step in the processing of DSB ends during recombinational DNA repair [1]. The formation of the RecA filament encompasses two distinct steps: a slower nucleation step and a faster polymerization step. The latter was shown to occur primarily in a 5’ to 3’ direction on ssDNA [1,2,3], while recent studies suggest that 3’ to 5’ polymerization can occur under certain conditions [4,5,6,7]. Once sequence homology is found, RecA aligns homologous sequences, the strands invade each other and begin the process of strand exchange [1,10]
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