Homologous recombination and human health: the roles of BRCA1, BRCA2, and associated proteins

Abstract

Homologous recombination (HR) is a major pathway for the repair of DNA double-strand breaks in mammalian cells, the defining step of which is homologous strand exchange directed by the RAD51 protein. The physiological importance of HR is underscored by the observation of genomic instability in HR-deficient cells and, importantly, the association of cancer predisposition and developmental defects with mutations in HR genes. The tumor suppressors BRCA1 and BRCA2, key players at different stages of HR, are frequently mutated in familial breast and ovarian cancers. Other HR proteins, including PALB2 and RAD51 paralogs, have also been identified as tumor suppressors. This review summarizes recent findings on BRCA1, BRCA2, and associated proteins involved in human disease with an emphasis on their molecular roles and interactions.

Figure 1.

Simplified schemes of double-strand break (DSB) repair by homologous recombination (HR) and nonhomologous end joining (NHEJ). Once a DSB is generated, it can be processed for HR by end resection proteins, leading to ssDNA. The RAD51 strand-exchange protein forms a nucleoprotein filament with ssDNA that invades an unbroken homologous DNA, typically the sister chromatid, as shown. The 3′ end primes DNA synthesis from the homologous DNA; using the sister chromatid, the repair can be precise to restore the original sequence before damage. To complete HR, the newly synthesized strand can dissociate to anneal to the other end. Other outcomes are also possible, for example, in which Holliday junctions are formed and either dissolved or resolved. Alternatively, DNA ends are protected from end resection by NHEJ proteins; subsequent steps in NHEJ can result in mutagenic repair with deletions and insertions (Δ, +).

Figure 2.

Competition and collaboration between double-strand break (DSB) repair pathways. (A) Schematic of the interactions between the homologous recombination (HR) and nonhomologous end joining (NHEJ) pathways for DSB repair. (B) Competition for repair of a DSB. In the direct repeat green fluorescent protein (DR-GFP) reporter assay, a DSB repaired through HR restores a functional GFP gene, as detected by flow cytometry. NHEJ-deficient Ku70^−/− cells show substantially elevated HR, showing how HR and NHEJ can act on the same DSB, such that NHEJ suppresses HR (modified from Pierce et al. 2001). The DSB is generated by I-SceI endonuclease. (C) Collaboration between HR and NHEJ, as illustrated in the embryonic brain. (i,ii) Apoptosis is rare in the wild-type embryonic brain (E13.5), as indicated by the lack of TdT-mediated dUTP-biotin nick end labeling (TUNEL) staining in either the proliferating ventricular zone (VZ) or the postmitotic subventricular (SV) zone (marked with Tuj1). (iii) HR-deficient Xrcc2^−/− cells show substantial apoptosis in the VZ. (iv) In contrast, NHEJ-deficient Lig4^−/− cells predominantly have elevated apoptosis in the SV. Therefore, HR and NHEJ both contribute to the integrity of the embryonic brain (modified from Orii et al. 2006).

Figure 3.

Protein interactions for a functional homologous recombination (HR) pathway. BRCA1 executes its various functions using the RING, coiled-coil, and BRCT domains. BRCA1 and BARD1 interact at their respective RING domains. BRCA1 BRCT motif mediates interaction through a pSXXF motif in Abraxas, BRIP1, and CtIP in the A, B, and C complexes, respectively. The coiled-coil domain of BRCA1 recognizes the coiled-coil domain of PALB2, which, in turn, binds BRCA2 through its WD40 domain. BRCA2 has mediator activity for loading RAD51 onto replication protein A (RPA)-coated ssDNA (BRC repeats) and for the stabilization of the RAD51 presynaptic filament carboxy terminal (C-ter) domain. RAD51 paralog complexes also interact with RAD51 and may promote/stabilize RAD51 filaments. BRCA2 also has a DSS1 and DNA-binding domain that, although not required for HR, likely is required for optimal HR levels. This domain consists of four globular domains and a helical tower domain with a three-helix bundle at its apex.

Figure 4.

BRCA1 and BRCA2 have distinct roles in HR. BRCA1 acts at an early HR step to promote end resection and at a later step to recruit PALB2 and, hence, promote BRCA2 chromatin localization. BRCA1 acts by antagonizing the resection inhibitor 53BP1. It may further regulate resection by recruiting CtIP (gold ball) in the BRCA1-C complex, while inhibiting end resection in the BRCA1-A complex containing Abraxas and RAP80 (black and gray balls); alternatively, Abraxas-RAP80 may act independently of BRCA1 to suppress resection. BRCA2 promotes loading of RAD51 recombinase onto the resection product to form an RAD51-ssDNA filament, which is essential for HR and prevents the engagement of the 3′-ssDNA into the deleterious single-strand annealing (SSA) pathway. SSA acts when homologous repeats are present and leads to a deletion of sequences between the repeats.