Expression of cancer related BRCA1 missense variants decreases MMS-induced recombination in Saccharomyces cerevisiae without altering its nuclear localization

Abstract

BRCA1 tumor suppressor gene is found mutated in familial breast and ovarian cancer. Most cancer related mutations were found located at the RING (Really Interesting New Gene) and at the BRCT (BRca1 C-Terminal) domain. However, 20 y after its identification, the biological role of BRCA1 and which domains are more relevant for tumor suppression are still being elucidated. We previously reported that expression of BRCA1 cancer related variants in the RING and BRCT domain increases spontaneous homologous recombination in yeast indicating that BRCA1 may interact with yeast DNA repair/recombination. To finally demonstrate whether BRCA1 interacts with yeast DNA repair, we exposed yeast cells expressing BRCA1wt, the cancer-related variants C-61G and M1775R to different doses of the alkylating agent methyl methane-sulfonate (MMS) and then evaluated the effect on survival and homologous recombination. Cells expressing BRCA1 cancer variants were more sensitive to MMS and less inducible to recombination as compared to cell expressing BRCA1wt. Moreover, BRCA1-C61G and -M1775R did not change their nuclear localization form as compared to the BRCA1wt or the neutral variant R1751Q indicating a difference in the DNA damage processing. We propose a model where BRCA1 cancer variants interact with the DNA double strand break repair pathways producing DNA recombination intermediates, that maybe less repairable and decrease MMS-induced recombination and survival. Again, this study strengthens the use of yeast as model system to characterize the mechanisms leading to cancer in humans carrying the BRCA1 missense variant.

Keywords: BRCA1; Methyl methanesulfonate; Saccharomyces cerevisiae; homologous recombination; nuclear localization.

Figure 1.

Expression of BRCA1 wild type and missense variants in the strain RS112 of Saccharomyces cerevisiae. (A) Schematic representation of full length BRCA1 and the most relevant domains. Arrows indicate the missense variants we have studied. (B) Description of the BRCA1 missense variants; in the table, the localization domain, nucleotide and amino-acid change is reported. IARC classification is also listed; Class 5 highly pathogenic and Class 1 not pathogenic or neutral. (C) BRCA1 was detected in the total protein extracts from yeast strains grown with galactose by Western blot analysis with anti BRCA1 antibody. Extracts were prepared from yeast expressing BRCA1wt, BRCA1-C61G, BRCA1-M1775R, BRCA1-mCherry, BRCA1- K45Q mCherry, BRCA1-C61G mCherry, BRCA1- R1751Q mCherry or BRCA1-M1775R mCherry, and loaded as indicated. Protein extract from yeast carrying empty pYES2 was loaded as control. In the lower part of the figure, the loading control was evaluated by detecting the 3PGK band is shown. The lower tables represent the densitometry calculated as ratio between intensity of BRCA1and PGK. Data are reported as mean of 3 independent experiments ± error bar. (D) RNA was exacted from yeast cells expressing BRCA1 wt, BRCA1-C61G, BRCA1-M1775R, BRCA1-mCherry, BRCA1- K45Q mCherry, BRCA1-C61G mCherry, BRCA1- R1751Q mCherry or BRCA1-M1775R mCherry, reverse transcribed and amplified for quantitative analysis as described in the Materials and Methods. Results are reported as mean of 4 experiments ± standard deviation.

Figure 2.

Localization of BRCA1wt and missense variants in the strain RS112 of Saccharomyces cerevisiae after exposure with MMS. Yeast cells co-expressing BRCA1-mCherry fusion proteins and Nup133 were exposed to 100µg/ml MMS for 17 hours. Thereafter, cells were spotted onto glass slides and analyzed under the fluorescence microscope as described in the Materials and Methods. Merged images indicate that BRCA1 wt and all variants localized in the nucleus. In nontreated cells (control) BRCA1wt and all the missense variants localized mainly as “diffuse signal.” Some single nuclear focus was visible also in untreated cells expressing BRCA1 wt or missense variants; these single focused cells were not shown in the image because the number of cells with diffuse signal was more relevant; after MMS treatment, BRCA1wt and the neutral R1751Q localized as single nuclear focus (arrows).