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.

• MeSH
• Adenine Nucleotides metabolism   MeSH
• Adenosine Triphosphate metabolism   MeSH
• Biological Evolution MeSH
• Cryoelectron Microscopy MeSH
• DNA, Single-Stranded metabolism   MeSH
• Kinetics MeSH
• Humans MeSH
• Models, Molecular MeSH
• Mutation MeSH
• Rad51 Recombinase genetics   metabolism   ultrastructure   MeSH
• Binding Sites MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenine Nucleotides MeSH
• Adenosine Triphosphate MeSH
• DNA, Single-Stranded MeSH
• RAD51 protein, human MeSH Browser
• Rad51 Recombinase MeSH

Successful and accurate completion of the replication of damage-containing DNA requires mainly recombination and RAD18-dependent DNA damage tolerance pathways. RAD18 governs at least two distinct mechanisms: translesion synthesis (TLS) and template switching (TS)-dependent pathways. Whereas TS is mainly error-free, TLS can work in an error-prone manner and, as such, the regulation of these pathways requires tight control to prevent DNA errors and potentially oncogenic transformation and tumorigenesis. In humans, the PCNA-associated recombination inhibitor (PARI) protein has recently been shown to inhibit homologous recombination (HR) events. Here, we describe a biochemical mechanism in which PARI functions as an HR regulator after replication fork stalling and during double-strand break repair. In our reconstituted biochemical system, we show that PARI inhibits DNA repair synthesis during recombination events in a PCNA interaction-dependent way but independently of its UvrD-like helicase domain. In accordance, we demonstrate that PARI inhibits HR in vivo, and its knockdown suppresses the UV sensitivity of RAD18-depleted cells. Our data reveal a novel human regulatory mechanism that limits the extent of HR and represents a new potential target for anticancer therapy.

• MeSH
• Amino Acid Motifs MeSH
• DNA-Binding Proteins chemistry   metabolism   physiology   MeSH
• DNA Polymerase III antagonists & inhibitors   MeSH
• DNA biosynthesis   MeSH
• HEK293 Cells MeSH
• Humans MeSH
• Recombinational DNA Repair * MeSH
• Ubiquitin-Protein Ligases physiology   MeSH
• Ultraviolet Rays MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA-Binding Proteins MeSH
• DNA Polymerase III MeSH
• DNA MeSH
• PARPBP protein, human MeSH Browser
• RAD18 protein, human MeSH Browser
• Ubiquitin-Protein Ligases MeSH

Most mitotic homologous recombination (HR) events proceed via a synthesis-dependent strand annealing mechanism to avoid crossing over, which may give rise to chromosomal rearrangements and loss of heterozygosity. The molecular mechanisms controlling HR sub-pathway choice are poorly understood. Here, we show that human RECQ5, a DNA helicase that can disrupt RAD51 nucleoprotein filaments, promotes formation of non-crossover products during DNA double-strand break-induced HR and counteracts the inhibitory effect of RAD51 on RAD52-mediated DNA annealing in vitro and in vivo. Moreover, we demonstrate that RECQ5 deficiency is associated with an increased occupancy of RAD51 at a double-strand break site, and it also causes an elevation of sister chromatid exchanges on inactivation of the Holliday junction dissolution pathway or on induction of a high load of DNA damage in the cell. Collectively, our findings suggest that RECQ5 acts during the post-synaptic phase of synthesis-dependent strand annealing to prevent formation of aberrant RAD51 filaments on the extended invading strand, thus limiting its channeling into potentially hazardous crossover pathway of HR.

• MeSH
• Cell Line MeSH
• Rad52 DNA Repair and Recombination Protein metabolism   MeSH
• DNA metabolism   MeSH
• DNA Breaks, Double-Stranded * MeSH
• RecQ Helicases metabolism   MeSH
• DNA, Single-Stranded metabolism   MeSH
• Humans MeSH
• Recombinational DNA Repair * MeSH
• Rad51 Recombinase metabolism   MeSH
• Sister Chromatid Exchange MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Rad52 DNA Repair and Recombination Protein MeSH
• DNA MeSH
• RecQ Helicases MeSH
• DNA, Single-Stranded MeSH
• RECQL5 protein, human MeSH Browser
• Rad51 Recombinase MeSH