RAD51 filament formation, dynamics, and regulation.

Abstract

DNA damage can arise through various mechanisms such as exogenous sources (UV radiation) or endogenous sources (replication fork collapse). Damaged DNA poses a significant risk to the cell, as unrepaired damage can lead to mutations, gross chromosomal rearrangements, or even cell death. RAD51 is a highly conserved recombinase which serves crucial functions in the cell through its role in DNA damage repair (homologous recombination, HR) and in replication fork protection. In HR, RAD51 (with the help of mediators and paralogs) forms a nucleoprotein filament on the single-stranded DNA produced via resection of a double-stranded break. This nucleoprotein filament then invades the homologous sequence which will serve as a template for accurate repair of the damaged DNA. The mechanism by which the RAD51 nucleoprotein filament protects the replication fork is poorly understood. Slight deviations towards either a more or a less stable RAD51 filament may cause aberrant recombination, and/or failure to protect DNA replication forks by changing the filament assembly, stability and/or dynamics. Using a combination of single-molecule and bulk biochemical assays, this study examines filament dynamics of RAD51 in varying contexts: different physiological conditions, varying mutated residues, and with and without small molecule inhibitors known to alter RAD51 function. We use dual-optical tweezers combined with confocal microscopy to examine RAD51 filament formation in real time, with a microfluidic chip set up that allows for introduction of varying conditions for the same single DNA molecule. Following characterization of filament formation at the single-molecule level, DNA strand exchange assays are used to determine how variations on filament assembly and stability alter the primary recombinase function of RAD51. We show here that alterations to RAD51 filament formation, stability, or dynamics can alter the critical recombinase function of RAD51.