Abstract
RAD51 mediated homologous recombinational repair (HRR) of DNA double-strand breaks (DSBs) is essential to maintain genomic integrity. RAD51 forms a nucleoprotein filament (NPF) that catalyzes the fundamental homologous pairing and strand exchange reaction (recombinase) required for HRR. Based on structural and functional homology with archaeal and yeast RAD51, we have identified the human RAD51 (HsRAD51) subunit interface residues HsRad51(F129) in the Walker A box and HsRad51(H294) in the L2 ssDNA binding region as potentially important participants in salt-induced conformational transitions essential for recombinase activity. We demonstrate that the HsRad51(F129V) and HsRad51(H294V) substitution mutations reduce DNA dependent ATPase activity and are largely defective in the formation of a functional NPF, which ultimately eliminates recombinase catalytic functions. Our data are consistent with the conclusion that the HsRAD51(F129) and HsRAD51(H294) residues are important participants in the cation-induced allosteric activation of HsRAD51.
Highlights
Failure to repair DNA double strand breaks (DSBs) leads to tumorigenesis and genomic instability
Previous studies have confirmed a small but reproducible salt induced ATPase catalytic rate enhancement in the presence of single-stranded DNA (ssDNA) compared to double stranded DNA (dsDNA) [15]
RAD51(F129V) displayed a similar salt (150 mM KCl) induced catalytic rate enhancement in the presence of ssDNA (Fig. 2E). Together these observations are consistent with the conclusion that HsRAD51(H294) residue and to a significantly lesser extent the HsRAD51(F129) residue are required for appropriate ATP catalysis
Summary
Failure to repair DNA double strand breaks (DSBs) leads to tumorigenesis and genomic instability. Homologous recombination (HR) is an evolutionary conserved repair pathway utilized to restore DSBs. HR mediated DSB repair is initiated by resection of the 59-end of the break to leave a 39-single-stranded DNA (ssDNA) overhang [1,2]. RAD51 forms a nucleoprotein filament (NPF) on the newly formed ssDNA region aided by other recombination mediators such as RAD52 in yeast and BRCA2 in vertebrates [3,4,5,6,7,8]. No mutations of RAD51 have been found in cancers, its expression is elevated in many cancer cell lines; perhaps to provide an advantage to rapidly dividing cells by repairing DSBs that would lead to replication fork collapse [11,12,13]
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