RAD51 Gene Family Structure and Function

Abstract

Accurate DNA repair and replication are critical for genomic stability and cancer prevention. RAD51 and its gene family are key regulators of DNA fidelity through diverse roles in double-strand break repair, replication stress, and meiosis. RAD51 is an ATPase that forms a nucleoprotein filament on single-stranded DNA. RAD51 has the function of finding and invading homologous DNA sequences to enable accurate and timely DNA repair. Its paralogs, which arose from ancient gene duplications of RAD51, have evolved to regulate and promote RAD51 function. Underscoring its importance, misregulation of RAD51, and its paralogs, is associated with diseases such as cancer and Fanconi anemia. In this review, we focus on the mammalian RAD51 structure and function and highlight the use of model systems to enable mechanistic understanding of RAD51 cellular roles. We also discuss how misregulation of the RAD51 gene family members contributes to disease and consider new approaches to pharmacologically inhibit RAD51.

Keywords: RAD51; RAD51 paralog; Shu complex; double-strand break repair; homologous recombination; replication.

Figure 1

Potential models for the RAD51 gene family roles in mitotic cells. (a) Schematic of the initial steps of DSB repair through HR. Upon DSB formation, the 5 strands of the DNA ends are resected by the MRN complex. Further resection is performed by the exonuclease EXO1 and/or DNA2 together with the BLM helicase. These exposed 3 ssDNA regions are immediately coated by the RPA complex (green circles), thus preventing the formation of secondary structures. RAD51 (orange circles) displaces RPA, aided by the RAD51 mediators (i.e., BRCA2 and the RAD51 paralogs) to form a RAD51 nucleoprotein filament on the ssDNA. The RAD51 filaments perform homology search and strand invasion, leading to D-loop formation. After the D-loop is extended by DNA synthesis, the repair process can be completed by SDSA, GC, or BIR, depending on whether the D-loop is disrupted, the second end is captured, or it is not captured, respectively. (b) Schematic of the canonical hRAD51 paralog subcomplexes BCDX2 (consisting of RAD51B, RAD51C, RAD51D, and XRCC2) and CX3 (consisting of RAD51C and XRCC3). The lines indicate where BCDX2 and CX3 function during HR. (c) Schematic of the roles of RAD51 during replication stress response. Replicative polymerases can be stalled by DNA lesions such as methylation adducts or abasic sites (yellow star). Fork reversal (left) occurs by the annealing of the newly synthetized strands and is dependent on RAD51 and other enzymes such as translocases or helicases (i.e., SMARCL1 and RAD54). This chicken-foot structure protects stalled forks and allows the rescue of the fork by an incoming replication fork or by bypassing the lesion. Protection of the reversed forks from nuclease digestion (orange pacman) prevents ssDNA accumulation and depends on the formation of stable RAD51 filaments at the ssDNA of the reversed arm, which requires BRCA2 (green oval). Finally, reversed forks can be restarted by direct reversal (not shown) or HR. Alternatively (right) polymerase can resume replication by repriming. This leads to the formation of ssDNA gaps behind the fork, which are RPA coated. These gaps can be filled by translesion synthesis or HR-dependent gap filling. During HR-dependent gap filling, RAD51 displaces RPA in the gap and mediates sister chromatid invasion and D-loop formation. DNA synthesis enables the gap to be filled, enabling error-free lesion bypass. (d) Broken forks generated by persistent stalling or encountering of a ssDNA break by the replisome, leading to one-ended DSBs. These breaks can be repaired by RAD51-dependent HR where RAD51 filaments form on the broken DSB end, which then invade the newly synthesized sister chromatid, leading to D-loop formation, which can be extended by BIR. Abbreviations: BIR, break-induced replication; BLM, bloom syndrome protein; D-loop, displacement loop; DSB, double-strand break; GC, gene conversion; HR, homologous recombination; MRN, MRE11-RAD50-NBS1; RPA, replication protein A; SDSA, synthesis-dependent strand annealing; ssDNA, single-stranded DNA.

Figure 2

Sequence alignment of human RAD51 protein and its family, including DMC1, RAD51B, RAD51C, RAD51D, XRCC2, XRCC3, and SWSAP1. Secondary structure of human RAD51 [Protein Data Bank (PDB) ID 5H1B] is shown above the aligned protein sequence with α-helices shown in gray and β-sheets shown in purple. The Walker A and B sequence motifs are in blue boxes, and the RAD51 linker (amino acids 85-GTFF-88) is in a yellow box.

Figure 3

Structural view of human RAD51 presynaptic and postsynaptic filament complexes (PDB IDs 5H1B and 5H1C). RAD51 protomers are shown in dark and light gray, Mg²⁺ ions are in green, AMP-PNP is depicted in stick form, ssDNA is shown in orange, and the dsDNA template is shown in orange and red. (a) AMP-PNP is buried between two RAD51 protomers. The Walker A (pink; amino acids 218-LLIVD-222) and Walker B (blue; amino acids 127-GEFRTGKT-134) motifs are highlighted. (b) Key residues for promoter–protomer interfaces are the linker region (circled; amino acids 85-GFTT-88 in yellow) and Y54 in one protomer with F195 in an adjacent protomer. (c) The ssDNA-bound RAD51 filament viewed from the side highlights the phosphate backbone (orange) directly engaging DNA-binding loops of RAD51 (arrows). (d) The postsynaptic filament highlights the initial bound dsDNA is in a similar conformation as the presynaptic filament shown in panel c. Abbreviations: AMP-PNP, adenylyl-imidodiphosphate; dsDNA, double-stranded DNA; PDB, Protein Data Bank; ssDNA, single-stranded DNA.