The A-Like Faker Assay for Measuring Yeast Chromosome III Stability

Abstract

The ability to rapidly assess chromosome instability (CIN) has enabled profiling of most yeast genes for potential effects on genome stability. The A-like faker (ALF) assay is one of several qualitative and quantitative marker loss assays that indirectly measure loss or conversion of genetic material using a counterselection step. The ALF assay relies on the ability to count spurious mating events that occur upon loss of the MATα locus of haploid Saccharomyces cerevisiae strains. Here, we describe the deployment of the ALF assay for both rapid and simple qualitative, and more in-depth quantitative analysis allowing determination of absolute ALF frequencies.

Exonuclease domain mutants of yeast DIS3 display genome instability

“…To test CIN on endogenous chromosomal markers, and extend this to include the dis3 E729K allele, we measured instability of chr III using the A-like faker (ALF) assay that measures loss of the native MAT locus [31,32]. We observed a significant increase in the frequency of ALF colonies for both dis3-ts and dis3 E729K ( Figure 5(a,b)).…”

“…While it is unclear why CIN rates are this high in dis3-EXO, we note that this allele completely ablates the exonuclease activity and shows slow growth at all tested temperatures, whereas dis3-ts and dis3 E729K only show strong growth defects at temperatures above 34°C. ALF phenotypes can reflect multiple forms of CIN, including whole chromosome loss, local deletions or chromosome arm loss, and gene conversion [31,32]. One mechanism of CIN in RNA processing mutants that could lead to an ALF phenotype is the accumulation of DNA:RNA hybrids in genomic DNA called R-loops [33].…”

“…The next day, 100 μl of the saturated overnight cultures was mixed with 300 μL of a MATα mating tester strain, spun down, and resuspended in 100 μl distilled water. These MATα mixtures were then spotted on synthetic media lacking all supplements and only permitting prototrophic mated progeny to form colonies [31]. As well, the saturated overnight cultures were diluted by 1:100,000 and plated on complete media to ascertain colony viability.…”

The yeast core spliceosome maintains genome integrity through R-loop prevention and α-tubulin expression

“…We did not detect enhanced benomyl sensitivity in yhc1-1 using this assay, possibly because of limited allelic penetrance at 30°C. By quantifying the rate of endogenous chromosome III loss using the A-like faker assay (Novoa et al, 2018) in the single and double mutants at permissive temperature of 25°C, we found a dramatic synergy between disruption of SNU114 or HSH155 and loss of the SAC ( Figure 3C ). Recurrently, yhc1-1 phenotype was not enhanced, consistent with the lack of benomyl sensitivity under the same conditions and likely a limitation of the ts-allele.…”

“…Growth curves were conducted in a Tecan M200 plate reader and compared using the area under the curve as previously described (Chang et al, 2017). The CTF and ALF assays were performed as described (Novoa et al, 2018; Stirling et al, 2011). For plasmid loss, strains carrying pRS313::HIS3 were grown overnight in SC-histidine, then plated on YPD and allowed to form colonies without selection before replica plating onto SC-histidine.…”