Human RAD51 rapidly forms intrinsically dynamic nucleoprotein filaments modulated by nucleotide binding state

Abstract

Formation of RAD51 filaments on single-stranded DNA is an essential event during homologous recombination, which is required for homology search, strand exchange and protection of replication forks. Formation of nucleoprotein filaments (NF) is required for development and genomic stability, and its failure is associated with developmental abnormalities and tumorigenesis. Here we describe the structure of the human RAD51 NFs and of its Walker box mutants using electron microscopy. Wild-type RAD51 filaments adopt an ‘open’ conformation when compared to a ‘closed’ structure formed by mutants, reflecting alterations in helical pitch. The kinetics of formation/disassembly of RAD51 filaments show rapid and high ssDNA coverage via low cooperativity binding of RAD51 units along the DNA. Subsequently, a series of isomerization or dissociation events mediated by nucleotide binding state creates intrinsically dynamic RAD51 NFs. Our findings highlight important a mechanistic divergence among recombinases from different organisms, in line with the diversity of biological mechanisms of HR initiation and quality control. These data reveal unexpected intrinsic dynamic properties of the RAD51 filament during assembly/disassembly, which may be important for the proper control of homologous recombination.

Non-denaturing bisulfite treatment and single-molecule real-time sequencing reveals d-loop footprints, their length, position and distribution

“…Similar to the results from a restriction-digest based assay (Wright & Heyer, 2014), there was an enrichment of D-loops near the 3′-end of ssDNA, while the 5′-end of homology had the lowest D-loop presence. This observation supports the model that Rad51 filament grows preferentially in a 5′-to-3′ direction (Qiu et al, 2013;Spirek et al, 2018), making it more likely for the D-loops to assemble at the 3′end of homology than at the 5′-end. Only D-loops in which the 3′-OH end is incorporated into the hDNA are competent for D-loop extension (Li & Heyer, 2009).…”

Variation in RAD51 details a hub of functions: opportunities to advance cancer diagnosis and therapy

“…Although there were other differences in the proteins from these two studies, it is possible that the N-terminus constrains the filaments to a single conformation at the ATPase interface 8, 10 . More recently, high-resolution structural models of human RAD51 filaments have been obtained from cryo–electron microscopy reconstructions 11, 12 . Here, filaments formed with human RAD51 had only one conformation at the nucleotide-binding site.…”

“…Specifically, active filaments formed with ATP or an analog bound at the interface have a longer helical pitch and an “open” conformation 12, 15 . Recently, RAD51 nucleoprotein filaments with distinct mechanical properties have been defined for the wild-type protein (for example, persistence length, helical pitch, and rise per monomer) by a combination of single-molecule force spectroscopy and crystallography 13 .…”

“…RAD51 K131A does not bind ATP, and RAD51 K113R can bind but not hydrolyze ATP. Both mutants form filaments with a “closed” conformation, as does the wild-type protein with the non-hydrolyzable ATP analog AMP-PNP 12 . It is suggested that these forms, with different helical pitch and rise, could represent intermediate steps in dynamic filament assembly and rearrangement important for function.…”

“…They are T131P and A293T, located at the ATPase active site interface, close to the amino acids F129 and H294, equivalent to F187 and H352 in yeast Rad51. These amino acids are expected to influence the conformation at this interface and its alternating conformations, which they directly contributed to in yeast Rad51 filaments 8, 12 . Mutations creating different chemical environments in close proximity of this interaction might affect conformational dynamics of human RAD51 nucleoprotein filaments.…”