Abstract
Efficient bacterial recombinational DNA repair involves rapid cycles of RecA filament assembly and disassembly. The RecX protein plays a crucial inhibitory role in RecA filament formation and stability. As the broken ends of DNA are tethered during homologous search, RecA filaments assembled at the ends are likely subject to force. In this work, we investigated the interplay between RecX and force on RecA filament formation and stability. Using magnetic tweezers, at single molecular level, we found that Mycobacterium tuberculosis (Mt) RecX could catalyze stepwise de-polymerization of preformed MtRecA filament in the presence of ATP hydrolysis at low forces (<7 pN). However, applying larger forces antagonized the inhibitory effects of MtRecX, and a partially de-polymerized MtRecA filament could re-polymerize in the presence of MtRecX, which cannot be explained by previous models. Theoretical analysis of force-dependent conformational free energies of naked ssDNA and RecA nucleoprotein filament suggests that mechanical force stabilizes RecA filament, which provides a possible mechanism for the observation. As the antagonizing effect of force on the inhibitory function of RecX takes place in a physiological range; these findings broadly suggest a potential mechanosensitive regulation during homologous recombination.
Highlights
In a cell, a variety of factors affect the stability and integrity of the genome resulting in structural damage to the DNA molecule
To determine how a preformed MtRecA filament is regulated when MtRecX is added to the MtRecA reaction solution, we investigated the effects of MtRecX on the dynamics of disassembly of preformed MtRecA filaments
We found that de-polymerization of MtRecA filament by MtRecX is dependent on ATP hydrolysis: when ATP was replaced by its non-hydrolysable homologue, ATP␥ S, inhibition of MtRecA de-polymerization was sustained over a long period of time (>2000 s) (Figure 2D)
Summary
A variety of factors affect the stability and integrity of the genome resulting in structural damage to the DNA molecule. Various proteins are involved in the regulation of the polymerization and de-polymerization of RecA to avoid either insufficient or unlimited formation of the RecA filament One such protein, single-stranded DNA binding protein (SSB), outcompetes RecA to bind to ssDNA, inhibiting nucleation and polymerization of the RecA filament, both in vivo and in vitro [2,7,8,9]. Single-stranded DNA binding protein (SSB), outcompetes RecA to bind to ssDNA, inhibiting nucleation and polymerization of the RecA filament, both in vivo and in vitro [2,7,8,9] It promotes net RecA depolymerization in an ATP hydrolysis- and force-dependent manner [9]. Besides SSB, the RecX protein strongly inhibits RecA filament nucleation and polymerization, though much less is understood about the RecX protein’s regulatory mechanisms
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