A 5′-to-3′ strand exchange polarity is intrinsic to RecA nucleoprotein filaments in the absence of ATP hydrolysis

Abstract

RecA is essential to recombinational DNA repair in which RecA filaments mediate the homologous DNA pairing and strand exchange. Both RecA filament assembly and the subsequent DNA strand exchange are directional. Here, we demonstrate that the polarity of DNA strand exchange is embedded within RecA filaments even in the absence of ATP hydrolysis, at least over short DNA segments. Using single-molecule tethered particle motion, we show that successful strand exchange in the presence of ATP proceeds with a 5′-to-3′ polarity, as demonstrated previously. RecA filaments prepared with ATPγS also exhibit a 5′-to-3′ progress of strand exchange, suggesting that the polarity is not determined by RecA disassembly and/or ATP hydrolysis. RecAΔC17 mutants, lacking a C-terminal autoregulatory flap, also promote strand exchange in a 5′-to-3′ polarity in ATPγS, a polarity that is largely lost with this RecA variant when ATP is hydrolyzed. We propose that there is an inherent strand exchange polarity mediated by the structure of the RecA filament groove, associated by conformation changes propagated in a polar manner as DNA is progressively exchanged. ATP hydrolysis is coupled to polar strand exchange over longer distances, and its contribution to the polarity requires an intact RecA C-terminus.

The regulation mechanism of the C-terminus of RecA proteins during DNA strand-exchange process

“…In previous studies, the synapse lifetime depended on the buffer (pH, ionic strength, ion species, etc.) and the length and sequence of the DNA (59,60). A synapse lifetime with a range of seconds to approximately a hundred seconds was reported, similar to our experimental results here.…”

“…The C-terminus of RecA affects the stability of synaptic complexes in the homologous sequence searching process Our previous single-molecule analysis showed that >80% of E. coli RecA nucleoprotein filaments participate in futile interactions with dsDNA, and only <20% of collisions lead to successful DNA strand exchange reactions (35,59). For those molecules that failed to proceed into DNA strand exchange, tethers appeared only for seconds and then disappeared (Fig.…”

Isothermal amplifications – a comprehensive review on current methods

“…The nucleoprotein filament assembles, disassembles, and promotes strand exchange with a predominant 5 0 -to-3 0 polarity. The observed polarity of RecAmediated DNA strand exchange and filament disassembly from DNA relies on ATP hydrolysis to a large extent (Lin Y-H et al 2019). RecA associates more tightly with DNA bound to ATP than with ADP.…”

RecA balances genomic stability and evolution using many successive mismatch tolerant homology tests

“…To study how accepting mismatches in heteroduplex products might influence DSB repair accuracy in vivo , we developed a highly simplified model of homology recognition (see Methods and Materials). The model is informed by experimental results, simulations, modeling, and in vitro experiments (Danilowicz et al, 2015, Sagi et al, 2006, Yang et al, 2015, Bazemore et al, 1997, Cox, 2007, Cox and Lehman, 1981, Lin et al, 2019, Qi et al, 2015, Hsieh et al, 1992, Prentiss et al, 2015, Lee et al, 2015). The model is designed to demonstrate how effectively a highly mismatch tolerant RecA could target allelic pairing.…”