Rad52 inactivation is synthetically lethal with BRCA2 deficiency

Abstract

Synthetic lethality is a powerful approach to study selective cell killing based on genotype. We show that loss of Rad52 function is synthetically lethal with breast cancer 2, early onset (BRCA2) deficiency, whereas there was no impact on cell growth and viability in BRCA2-complemented cells. The frequency of both spontaneous and double-strand break-induced homologous recombination and ionizing radiation-induced Rad51 foci decreased by 2-10 times when Rad52 was depleted in BRCA2-deficient cells, with little to no effect in BRCA2-complemented cells. The absence of both Rad52 and BRCA2 resulted in extensive chromosome aberrations, especially chromatid-type aberrations. Ionizing radiation-induced and S phase-associated Rad52-Rad51 foci form equally well in the presence or absence of BRCA2, indicating that Rad52 can respond to DNA double-strand breaks and replication stalling independently of BRCA2. Rad52 thus is an independent and alternative repair pathway of homologous recombination and a target for therapy in BRCA2-deficient cells.DNA repair | genetic instability | chromosomal aberrations D NA double-strand breaks (DSBs) are potentially lethal DNA lesions which may arise spontaneously during DNA replication or result from exposure to ionizing radiation or other DNAdamaging agents (1). To repair DSBs, eukaryotes have developed two DSB repair pathways: nonhomologous end joining and homologous recombination (HR) (2). HR is required for the repair of complex double-strand lesions such as crosslinks or one-ended DSBs that occur with a cleaved replication fork; nonhomologous end joining appears to have little role in the repair of these lesions. The absence of Rad51 in proliferating cells (and therefore any measurable HR) results in cell lethality. The loss of function of proteins involved in HR, such as breast cancer 2, early onset (BRCA2), will be viable only if there is a BRCA2-independent pathway for Rad51 function.In Saccharomyces cerevisiae, the Rad52 protein plays a key role in HR (3). However, in vertebrates, knockouts of the Rad52 gene show little phenotype, with no obvious defect in HR. Rad52 knockout mice exhibit a nearly normal phenotype, and Rad52-deficient embryonic stem cells are not hypersensitive to agents that induce DSBs, either simple or complex (4, 5). In contrast, Rad51 knockout is embryonically lethal (6, 7), and depletion of Rad51 from vertebrate cells results in an accumulation of chromosome aberrations and subsequent cell death (8). These findings indicate the essential role of Rad51 in the maintenance of chromosomal DNA during the mitotic cell cycle, but the role for Rad52 in vertebrate cells is unclear.Accumulating evidence implicates BRCA2 as an integral component of the HR machinery via the direct regulation of the assembly of Rad51 filaments and its subsequent activity in strand exchange (9-11). Biochemical studies showed that the Ustilago maydis BRCA2 ortholog, Brh2, is involved in the recruitment of Rad51 to the sites of HR; Rad51 then mediates the displacement of replication protein A...

Inhibition of Homologous Recombination and Promotion of Mutagenic Repair of DNA Double-Strand Breaks Underpins Arabinoside–Nucleoside Analogue Radiosensitization

“…5D). As reported earlier (39)(40)(41)(42)(43), reduced expression of RAD51 is associated with increased radiosensitivity to killing. Strikingly, while control cells show strong radiosensitization after treatment with araA, treatment with araA of cells treated with siRNA targeting RAD51 fail to show marked radiosensitization (Fig.…”

BRG1 promotes DNA double-strand break repair by facilitating the replacement of RPA with RAD51

“…BRCA2 is known as the main mediator of RPA replacement by RAD51 in human cells (Liu et al, 2010) and, in yeast, Rad52 functions in the same way as BRCA2 (Shinohara and Ogawa, 1998). Recent studies have further reported that mammalian RAD52 shares biochemical activities with yeast Rad52 and compensates for BRCA2 when BRCA2 is deficient (Feng et al, 2011;Lok et al, 2013). RAD52-dependent homologous recombination occurs at a later stage to restart a subset of Fig.…”

Human RAD52 – a novel player in DNA repair in cancer and immunodeficiency

“…Synthetic lethality of RAD52 inactivation in BRCA1, BRCA2 and PALB2 deficient cells has been described, suggesting RAD52 as part of an alternative repair pathway parallel to RAD51-mediated HR. 13,14 We and others propose the alternative RAD52 pathway as being required for DNA stability, and attribute a tumor suppressor function. Additive germline mutations in other DNA repair genes might confer an increased tumor risk.…”