Homologous recombination in budding yeast expressing the human RAD52 gene reveals a Rad51-independent mechanism of conservative double-strand break repair

Abstract

RAD52 is a homologous recombination (HR) protein that is conserved from bacteriophage to humans. Simultaneously attenuating expression of both the RAD52 gene, and the HR and tumor suppressor gene, BRCA2, in human cells synergistically reduces HR – indicating that RAD52 and BRCA2 control independent mechanisms of HR. We have expressed the human RAD52 gene (HsRAD52) in budding yeast strains lacking the endogenous RAD52 gene and found that HsRAD52 supports repair of DNA double-strand breaks (DSB) by a mechanism of HR that conserves genome structure. Importantly, this mechanism of HR is independent of RAD51, which encodes the central strand exchange protein in yeast required for conservative HR. In contrast, BRCA2 exerts its effect on HR in human cells together with HsRAD51, potentially explaining the synergistic effect of attenuating the expression of both HsRAD52 and BRCA2. This suggests that multiple mechanisms of conservative DSB repair may contribute to tumor suppression in human cells.

Variants of the human RAD52 gene confer defects in ionizing radiation resistance and homologous recombination repair in budding yeast

“…FLAG-tagged recombinants also were produced in order to facilitate immunological detection and selection of the proteins. As observed previously with strains expressing the adh1::HsRAD52-FLAG allele [29], Western blots of whole cell extracts from strains expressing the adh1::HsRAD52-G59R-FLAG allele produced a single, stable 49 kDa protein, with the steady-state level of the mutant protein appearing to exceed that of wild-type (Figure 1). Strains expressing the adh1::HsRAD52-S346X-FLAG allele displayed a single 38 kDa protein consistent with the deletion of 72 amino acids from the C-terminus of HsRAD52.…”

“…Expression of adh1::HsRAD52-FLAG in rad52 mutant cells suppressed the MTI defect by 69-fold indicating that HsRAD52-FLAG possesses substantial functionality in this heterologous system ( Figure 3B; Table S4). Importantly, adh1::HsRAD52-FLAG also suppressed the inability of rad52 mutant cells to repair a DSB at the HIS3 locus by gene conversion using an unlinked, defective copy of the HIS3 gene ( Figure S1, Table S4) [29]. This indicates that HsRAD52-FLAG can support the repair of HO-catalyzed DSBs by HRR in multiple genomic contexts in budding yeast.…”

“…Accordingly, we examined the effects of HsRAD52-G59R and HsRAD52-S346X, two variants identified in a screen of African-American women with breast cancer, for their effects on the function of HsRAD52 in budding yeast cells. Utilizing our previous strategy for obtaining robust expression of HsRAD52 in yeast [29], we inserted cDNAs of the HsRAD52-G59R and HsRAD52-S346X alleles into the ADH1 locus such that their expression was controlled by the ADH1 promoter and terminator sequences. Strains expressing C-terminally Whole cell extracts from strains ABX3684-12B (adh1::HsRAD52-FLAG), ABX3782-2D (adh1::HsRAD52-G59R-FLAG) and ABX3974-11C (adh1::HsRAD52-S346X-FLAG) were run on SDS-PAGE gels, blotted to a nylon membrane and probed with anti-FLAG and anti-GAPDH antibodies.…”

“…Advancing the development of effective inhibitors of RAD52 would benefit from a more complete understanding of its function at the molecular level during DSB repair in living cells. Studying the function of human RAD52 (hereafter referred to as HsRAD52) in IR resistance and HRR in budding yeast cells has provided a compelling model as our previous investigation showed that expressing the HsRAD52 gene in rad52 mutant yeast strains suppresses both their IR sensitivity and HRR defects [29]. Further, HsRAD52-dependent HRR and association of HsRAD52 with DSBs during repair are independent of the central HR factor, Rad51 and other members of the canonical HRR apparatus of yeast, paralleling observations in mammalian cells.…”

Mechanistic basis for increased human gene targeting by promoterless vectors—roles of homology arms and Rad54 paralogs

“…It may be that alternative end‐joining or homeologous recombination (e.g., single‐strand annealing between two Alu sequences ) could account for the gene‐targeting events that we observed in cells lacking Rad54/Rad54B in this study. It is also worth mentioning that, in addition to its role in single‐strand annealing , human Rad52 likely contributes to HR in a Rad51‐independent manner . Clearly, further work is required to clarify the mechanism of Rad54/Rad54B‐independent gene targeting in human cells, the occurrence of which is demonstrated in our present study.…”