FIGURE 4: Schematic representation of the yeast functional assays for BRCA1/2 (A) Transcription activation assay (TA): The BRCT domain of BRCA1 is cloned in frame with the GAL4 or LexA DNA binding domain (DBD). WT BRCT fused to DBD is able to activate transcription of the reporter gene LacZ under control of the minimal promoter (white) containing the binding sequences (violet) recognized by the GAL4 or LexA DBD. Variants affecting BRCT domain activity are not able to activate LacZ transcription. (B) Small colony phenotype assay (SCP): yeast cells expressing BRCA1 (from pADH1 fused to the activation domain of GAL4, or from inducible pGAL1) form colonies considerably smaller than controls after incubation at 30°C. Yeast colonies re resuspended in water, and the number of cells per colony are determined by counting. Small colonies correlate with slow growth, therefore the inhibition of growth determined by BRCA1 expression can be performed also in liquid medium. (C) Liquid Medium assay: this assay monitors the growth defect of yeast cells expressing BRCA1 as in the SCP, but in liquid instead of solid medium. (D) Yeast localization phenotype assay (YLP): this assay is performed in yeast cells expressing BRCA1 cloned in frame with m-Cherry at C-terminus (BRCA1-mCherry) under control of the inducible GAL promoter. Yeast cells expressing WT or BRCA1 variants fused to mCherry are induced for 4 h with galactose before live fluorescent microscopy analyses. The nucleus is identified by the expression of the nuclear protein Nup133 fused to GFP (Nup133-GFP, green). Whereas the WT BRCA1 protein shows mainly nuclear localization (Red spot), pathogenic variants show prevalent cytoplasmic localization. (E) Intra- chromosomal recombination: the yeast strain carries the two his3 alleles separated by the LEU2 marker and by the plasmid DNA sequence, one with a deletion at the 3' end and the other with a deletion at the 5' end, which share 400 bp of homology (gray box). An intra-chromosomal recombination event leads to HIS3 reversion and loss of LEU2 determined by counting colonies grown in medium lacking histidine (HIS3+). (F) Inter-chromosomal recombination: the yeast strain contains the two alleles ade2-40 and ade2-101, located in two homologous chromosomes. An inter-chromosomal recombination event leads to WT ADE2 determined directly by counting colonies grown in medium lacking adenine (ADE2+). (G) Gene reversion (GR): the yeast strain carries the ilv1-92 that allows the assessment of gene reversion to ILV1 by direct counting colonies grown in medium lacking isoleucine (ILE+).