Crucial roles of the BRCA1-BARD1 E3 ubiquitin ligase activity in homology-directed DNA repair.

The tumor-suppressor breast cancer 1 (BRCA1) in complex with BRCA1-associated really interesting new gene (RING) domain 1 (BARD1) is a RING-type ubiquitin E3 ligase that modifies nucleosomal histone and other substrates. The importance of BRCA1-BARD1 E3 activity in tumor suppression remains highly controversial, mainly stemming from studying mutant ligase-deficient BRCA1-BARD1 species that we show here still retain significant ligase activity. Using full-length BRCA1-BARD1, we establish robust BRCA1-BARD1-mediated ubiquitylation with specificity, uncover multiple modes of activity modulation, and construct a truly ligase-null variant and a variant specifically impaired in targeting nucleosomal histones. Cells expressing either of these BRCA1-BARD1 separation-of-function alleles are hypersensitive to DNA-damaging agents. Furthermore, we demonstrate that BRCA1-BARD1 ligase is not only required for DNA resection during homology-directed repair (HDR) but also contributes to later stages for HDR completion. Altogether, our findings reveal crucial, previously unrecognized roles of BRCA1-BARD1 ligase activity in genome repair via HDR, settle prior controversies regarding BRCA1-BARD1 ligase functions, and catalyze new efforts to uncover substrates related to tumor suppression.

High-Efficiency Plasmid DNA Transformation in Yeast.

Transformation of DNA into cells of the budding yeast Saccharomyces cerevisiae and other industrially important yeasts is most commonly performed using chemical-based methods. Current protocols typically involve exposure of the cells to lithium ions in a solution containing the crowding agent polyethylene glycol (PEG), often in conjunction with other reagents such as dimethyl sulfoxide (DMSO) that promote destabilization of the cell wall and/or cell envelope. Recent work has demonstrated that it is possible to achieve high transformation efficiencies with early stationary phase cells, i.e., small overnight liquid cell cultures, using methods that are rapid and readily scalable for high-throughput projects. Herein, we describe carrier DNAs, chemical reagents, and cell growth media that permit transformation of yeast cells with either plasmids or linear DNA fragments with high efficiency.