RAD-ical New Insights into RAD51 Regulation

Abstract

The accurate repair of DNA is critical for genome stability and cancer prevention. DNA double-strand breaks are one of the most toxic lesions; however, they can be repaired using homologous recombination. Homologous recombination is a high-fidelity DNA repair pathway that uses a homologous template for repair. One central HR step is RAD51 nucleoprotein filament formation on the single-stranded DNA ends, which is a step required for the homology search and strand invasion steps of HR. RAD51 filament formation is tightly controlled by many positive and negative regulators, which are collectively termed the RAD51 mediators. The RAD51 mediators function to nucleate, elongate, stabilize, and disassemble RAD51 during repair. In model organisms, RAD51 paralogs are RAD51 mediator proteins that structurally resemble RAD51 and promote its HR activity. New functions for the RAD51 paralogs during replication and in RAD51 filament flexibility have recently been uncovered. Mutations in the human RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3, and SWSAP1) are found in a subset of breast and ovarian cancers. Despite their discovery three decades ago, few advances have been made in understanding the function of the human RAD51 paralogs. Here, we discuss the current perspective on the in vivo and in vitro function of the RAD51 paralogs, and their relationship with cancer in vertebrate models.

Keywords: BRCA1; BRCA2; RAD51 paralogs; RAD51B; RAD51C; RAD51D; Shu complex; XRCC2; XRCC3; cancer; homologous recombination.

Figure 1

Schematic of Figure 1 double-strand break repair pathways and homologous recombination (HR). After the formation of a double-strand break (DSB; purple lines), cells can repair the damage through two primary mechanisms, HR using a homologous template (turquoise lines) or nonhomologous end joining (NHEJ). (A) Pathway choice between HR or NHEJ is mediated by BRCA1, which promotes HR and 53BP1, RIF1, and the shieldin complex, which promotes NHEJ. (B) Resection by the MRN (MRE11–RAD51–NBS1) complex, CtIP, EXO1, BLM, and DNA2 creates 3′ ssDNA overhangs, which are coated by the trimeric replication protein A (RPA) [20] complex (green circles). During canonical HR, RPA is displaced by RAD51 (red circles). Alternatively, RAD51-independent repair can occur through single-strand annealing, where complementary DNA sequences anneal, flap endonucleases cleave the overhang, and the DNA ends are ligated together. (C) RAD51 filament formation is regulated by the positive RAD51 regulators, BRCA2, PALB2, and the RAD51 paralogs. At the same time, RAD51 is negatively regulated by FBH2 and RECQL5. (D) RAD51-mediated homology search and strand invasion then occurs, and is regulated by the RAD51 paralogs and RAD54A,B. At the same time, RAD51-mediated D loops are negatively regulated by RTEL and FANCM. (E) The DNA polymerases then copy the missing information from the homologous template (shown in turquoise, a sister chromatid or a homologous chromosome). (F) During synthesis-dependent strand annealing (SDSA), the D loop is displaced, and the DNA is resolved into a noncrossover product. (G) If there is heteroduplex extension and a double Holliday junction formed by second-end capture, then these DNA intermediates can be resolved by dissolution or resolution. (H) Dissolution results in non-crossover products. (I) Resolution results in both crossover and non-crossover products.

Figure 2

Mammalian RAD51 paralog-containing complexes. The BCDX2 complex is a RAD51 paralog heterotetramer consisting of RAD51B, RAD51C, RAD51D, and XRCC2. The CX3 complex is a RAD51 paralog heterodimer consisting of RAD51C and XRCC3. The PALB2–RAD51–RAD51C–BRCA2 complex consists of the RAD51 paralog, RAD51C, RAD51 itself, and two additional RAD51 mediator proteins, BRCA2 and PALB2. PALB2 acts as a scaffold in this complex by interacting with RAD51, RAD51C, and BRCA2. The Shu complex consists of a highly divergent RAD51 paralog, SWSAP1, and its binding partner SWS1. SWS1 is a member of the evolutionarily conserved Shu2/SWS1 family, which contains a SWIM domain, and interacts with RAD51 paralogs throughout eukaryotes. The SWIM domain is a zinc finger-like domain, CXC…X_n…CXHXXA, where X is any amino acid. Blue circles indicate RAD51 or a RAD51 paralog, a green circle indicates a SWIM domain containing a Shu2/SWS1 protein family member, and an orange circle indicates an additional RAD51 mediator protein.

Figure 3

Proposed functions for the RAD51 paralogs. (A) Nucleation and filament formation. Traditionally, the RAD51 paralogs (blue circles) are thought to aid RAD51 (red circle) in filament formation with BRCA2 (pink circle), which nucleates RAD51 monomers onto ssDNA (turquoise lines). The RAD51 paralogs may act by intercalating into the RAD51 filament directly (shown on left) or by binding to the RAD51 filament to stabilize RAD51 (shown on right). Subsequently, BRCA2 is removed as the filament is stabilized and elongated. (B) RAD51 paralog function in RAD51 filament end capping and flexibility were recently proposed [39,40]. These functions would aid in RAD51 downstream activity to promote strand invasion. (C) Replication fork protection function has also been proposed for the RAD51 paralogs [85]. During fork protection, the RAD51 paralogs could potentially intercalate into (top strand) or stabilize (bottom strand) the RAD51 filament at a stalled replication fork.