RESUMO
The breast and ovarian cancer susceptibility genes, BRCA1 and BRCA2, are key players in the homologous recombination (HR) repair pathway and act as tumor suppressors by maintaining genome stability. The yeast Saccharomyces cerevisiae has no BRCA1/2 homolog; however, a number of HR genes are evolutionary conserved between human and yeast. Among them, RAD52 is involved in DNA double strand break (DSB) repair by HR, and promotes genome stability. We previously reported that the heterologous expression of cancer-associated BRCA1/2 missense variants in growing yeast cultures affects both spontaneous HR and gene reversion (GR) suggesting that yeast could be a reliable system to assess the functional impact of variants. Because inhibition of Rad52p is lethal in BRCA1/2 mutated tumors, and Rad52p is conserved between humans and yeast, we asked if the effect of BRCA1/2 variants on HR and GR could be affected by loss of RAD52. We found that the rad52∆ mutation predominantly suppressed the stimulation of HR in yeast by pathogenic BRCA1 variants but also facilitated increased GR by pathogenic variants. Conversely, the rad52∆ mutation stimulated HR by a pathogenic BRCA2 variant in yeast but had no effect on GR. These results demonstrate a functional interplay between the pathogenic BRCA1/2 variants and Rad52p in budding yeast, supporting the use of budding yeast as a suitable system for evaluating potential chemotherapeutic strategies.
Assuntos
Proteína Rad52 de Recombinação e Reparo de DNA , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Reparo do DNA , Instabilidade Genômica , Recombinação Homóloga , Humanos , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Evaluation of the functional impact of germline BRCA1 variants that are likely to be associated to breast and ovarian cancer could help to investigate the mechanism of BRCA1 tumorigenesis. Expression of pathogenic BRCA1 missense variants increased homologous recombination (HR) and gene reversion (GR) in yeast. We thought to exploit yeast genetics to shed light on BRCA1-induced genome instability and tumorigenesis. We determined the effect on GR of several neutral and pathogenic BRCA1 variants in the yeast strain RSY6wt and its isogenic DSB repair mutants, such as mre11∆, rad50∆ and rad51∆. In the RSY6wt, four out of five pathogenic and two out of six neutral variants significantly increased GR; rad51∆ strain, the pathogenic variants C61G and A1708E induced a weak but significant increase in GR. On the other hand, in rad50∆ mutant expressing the pathogenic variants localised at the BRCT domain, a further GR increase was seen. The neutral variant N132K and the VUS A1789T induced a weak GR increase in mre11∆ mutant. Thus, BRCA1 missense variants require specific genetic functions and presumably induced GR by different mechanisms. As DNA repair is regulated by cell cycle, we determined the effect on GR of BRCA1 variants in cell cycle-arrested RSYwt cells. GR is highly BRCA1-inducible in S-phase-arrested cells as compared to G1 or G2. Sequence analysis of genomic DNA from ILV1 revertant clones showed that BRCA1-induced ilv1-92 reversion by base substitution when GR is at least 6-fold over the control. Our study demonstrated that BRCA1 may interfere with yeast DNA repair functions that are active in S-phase causing high level of GR. In addition, we confirmed here that yeast could be a reliable model to investigate the mechanism and genetic requirements of BRCA1-induced genome instability. Finally, developing yeast-based assays to characterise BRCA1 missense variants could be useful to design more precise therapies.
Assuntos
Proteína BRCA1/genética , Proteínas de Ligação a DNA/genética , Endodesoxirribonucleases/genética , Exodesoxirribonucleases/genética , Rad51 Recombinase/genética , Proteínas de Saccharomyces cerevisiae/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Carcinogênese , Pontos de Checagem do Ciclo Celular/genética , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Reparo do DNA/genética , Feminino , Instabilidade Genômica/genética , Humanos , Mutação de Sentido Incorreto/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Saccharomyces cerevisiae/genética , Treonina Desidratase/genéticaRESUMO
BRCA1 interacts with several proteins implicated in homologous and non homologous recombination and in mismatch repair. The aim of this study is to determine if MSH2, a well known partner of BRCA1 involved in DNA repair, may contribute to breast and ovarian cancer development and progression. To better understand the functional interaction between BRCA1 and MSH2, we studied the effect of the deletion of MSH2 gene on BRCA1-induced genome instability in yeast. Preliminary results in yeast indicated that MSH2 and BRCA1 may interact to modulate homologous recombination (HR). We also carried out a genetic and epigenetic profiling of MSH2 gene by mutational analysis and promoter methylation evaluation in 9 breast and 2 ovarian tumors from carriers of BRCA1 unknown significance variants (VUS). 2/2 ovarian and 2/9 breast tumors carried MSH2 somatic mutations possible pathogenics (4/11, 36%): a missense mutation in exon 3 (p.G162R), a duplication of exon 1 and a deletion of exon 2. In addition, two germline synonymous variants in exon 11 were identified. None of the tumors showed promoter methylation. In conclusion, a surprisingly high frequency of MSH2 gene mutations has been found in tumor tissues from BRCA1 VUS carrier patients. This result supports the indication deriving from the yeast model that BRCA1 driven tumorigenesis may be modulated by MSH2.