Functional and mutational landscapes of BRCA1 for homology-directed repair and therapy resistance

Abstract

BRCA1 plays a critical role in homology-directed repair (HDR) of DNA double strand breaks, and the repair defect of BRCA1-mutant cancer cells is being targeted with platinum drugs and poly (ADP-ribose) polymerase (PARP) inhibitors. We have employed relatively simple and sensitive assays to determine the function of BRCA1 variants or mutants in two HDR mechanisms, homologous recombination (HR) and single strand annealing (SSA), and in conferring resistance to cisplatin and olaparib in human cancer cells. Our results define the functionality of the top 22 patient-derived BRCA1 missense variants and the contribution of different domains of BRCA1 and its E3 ubiquitin ligase activity to HDR and drug resistance. Importantly, our results also demonstrate that the BRCA1-PALB2 interaction dictates the choice between HR and SSA. These studies establish functional and mutational landscapes of BRCA1 for HDR and therapy resistance, while revealing novel insights into BRCA1 regulatory mechanisms and HDR pathway choice.DOI: http://dx.doi.org/10.7554/eLife.21350.001

BRCA1 Mutation-Specific Responses to 53BP1 Loss-Induced Homologous Recombination and PARP Inhibitor Resistance

“…We investigated the importance of the BRCA1-PALB2 interaction for HR and PARPi resistance in the setting of BRCT-less BRCA1 protein expression and 53BP1 depletion. The BRCA1 L1407P missense mutation, previously shown to abrogate the BRCA1-PALB2 interaction (Anantha et al, 2017; Sy et al, 2009), was introduced into the BRCA1-ΔBRCT constructs and expressed in MDA-MB-436 cells. We confirmed that full-length BRCA1+L1407P was unable to interact with PALB2 and consequently BRCA2 and RAD51 in co-immunoprecipitation experiments.…”

“…BRCA1 promotes the displacement of 53BP1 from chromatin surrounding DSBs and consequently activates DNA end resection (Chapman et al, 2012; Densham et al, 2016). After DNA end resection, BRCA1 directly interacts with and recruits PALB2 to DSB sites, where BRCA2-RAD51 form s a com plex with PALB2, and mutations that block the BRCA1-PALB2 interaction have been shown to reduce the efficiency of RAD51 foci formation and HR (Anantha et al, 2017; Foo et al, 2017; Sy et al, 2009). …”

Homology-Directed Repair and the Role of BRCA1, BRCA2, and Related Proteins in Genome Integrity and Cancer

“…BRCA1 also interacts with the PALB2 protein (Sy et al 2009, Zhang et al 2009) (Figure 2), which in turn binds BRCA2 to facilitate RAD51 filament formation, a later step in HDR. Patient-derived mutations of BRCA1 that impair HDR typically also reduce SSA, another DSB repair pathway that requires end resection (Anantha et al 2017, Stark et al 2004). The exceptions are missense mutations that specifically disrupt BRCA1 interaction with PALB2, which result in reduced HDR but increased SSA, similar to BRCA2 deficiency (Anantha et al 2017, Stark et al 2004).…”

“…Through these interactions, BRCA1 may have a role in fine-tuning the length of resected DNA through distinct BRCT complexes for optimal HDR function. Several missense and truncating mutations in the BRCT domain have been identified in patients, compromising multiple aspects of BRCA1 function, including damage foci localization, protein stability, and resection-dependent HDR and SSA (Anantha et al 2017, Chen et al 2017, Lee et al 2010, Li & Yu 2013, Nelson & Holt 2010). Mice carrying a Brca1 S1598F point mutation in the BRCT domain, which is analogous to the patient-derived mutation S1655F, show accelerated tumorigenesis similar to complete inactivation of Brca1 (Shakya et al 2011).…”

“…Germ-line tumor-predisposing mutations, mostly truncating but also missense, occur throughout both genes, with common founder mutations in some populations. Variants of unknown significance have often been identified, leading to the development of functional assays for HDR to evaluate whether the variants may be pathogenic (Anantha et al 2017, Bouwman et al 2013, Kuznetsov et al 2008, Millot et al 2012). Tumor formation is effectively suppressed in the heterozygous state; thus, a primary requirement for tumorigenesis in heterozygous individuals is loss of the wild-type allele in pretumorigenic cells.…”