title: Effect of the expression of BRCA2 on spontaneous homologous recombination and DNA damage-induced nuclear foci in Saccharomyces cerevisiae.
abstract: The tumour-suppressor gene BRCA2 has been demonstrated to be involved in maintenance of genome integrity by affecting DNA double-strand break repair and homologous recombination. Protein-truncating mutations in BRCA2 predispose women to early onset breast and ovarian cancers and account for 15-30% of familial breast cancer risk. In contrast, the human cancer risk due to missense mutations, intronic variants, and in-frame deletions and insertions in the BRCA2 gene, called unclassified variants, has not been determined. Here, we want to define if the yeast Saccharomyces cerevisiae is a good model to study the role of BRCA2 in DNA recombination and repair and to characterise the unclassified BRCA2 missense variants. Therefore, we expressed the wild-type BRCA2 in yeast and determined the effect of BRCA2 on yeast homologous recombination, methyl methanesulphonate (MMS)-induced Rad51 and Rad52 foci and MMS sensitivity. The expression of BRCA2 induces a high increase in both intra- and inter-recombination events and confers a higher MMS resistance as compared with the negative control. This may suggest that BRCA2 gets involved in DNA repair pathways in yeast. Moreover, the expression of BRCA2 did not affect the number of cells carrying Rad51 or Rad52 nuclear foci. Finally, we aimed to investigate if yeast could be reliable system to set up a functional assay to distinguish a mutated protein from a neutral polymorphism. Therefore, we have expressed two neutral (M1915T and A2951T) and one pathogenic variant (G2748D) in yeast and checked the effect on recombination. The neutral M1915T variant increased intra-chromosomal recombination by almost 2-fold and the other neutral A2975T variant increased intra-chromosomal recombination 2.5-fold as compared with the control. On the other end, the pathogenic variant G2748D did not increase intra- and inter-chromosomal recombination in yeast and, consequently, confers a phenotype very different from the wild-type BRCA2. Moreover, we have also evaluated whether the expression of the selected unclassified variants affects homologous recombination in yeast. Results indicated that the expression of the selected BRCA2 variants differentially affects yeast recombination suggesting that yeast could be a very promising genetic system to characterise BRCA2 missense variants.
authors: Laboratory of Genome Technology and Integrative Systems Biology, Institute of Clinical Physiology, CNR, Pisa, Italy.
authors: Laura
authors: L
authors: Spugnesi
authors: Cristina
authors: C
authors: Balia
authors: Anita
authors: A
authors: Collavoli
authors: Elisabetta
authors: E
authors: Falaschi
authors: Valentina
authors: V
authors: Quercioli
authors: Maria Adelaide
authors: MA
authors: Caligo
authors: Alvaro
authors: A
authors: Galli
mesh_terms: D024682:BRCA2 Protein / Q000235:genetics* / Q000378:metabolism; D002467:Cell Nucleus / Q000235:genetics* / Q000378:metabolism; D004249:DNA Damage / Q000187:drug effects*; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D005260:Female; D005455:Fluorescent Antibody Technique; D059765:Homologous Recombination; D006801:Humans; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D020125:Mutation, Missense; D010641:Phenotype; D010957:Plasmids; D011110:Polymorphism, Genetic; D011401:Promoter Regions, Genetic; D051721:Rad52 DNA Repair and Recombination Protein / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism
chemical_list: D024682:BRCA2 Protein; D004268:DNA-Binding Proteins; C448305:RAD51C protein, human; C497194:RAD52 protein, human; D051721:Rad52 DNA Repair and Recombination Protein; D008741:Methyl Methanesulfonate