Genomic amplification of specific genes is a common mechanism of adaptation that also underpins many human disorders. We use yeast (Saccharomyces cerevisiae) to investigate the mechanism of one such gene amplification. When yeast are grown in sulfate-limited conditions for many generations, the population becomes dominated by cells possessing an inverted triplication of the SUL1 gene, which produces a sulfate transporter. Because of increased transporter levels, these cells have higher fitness in limited sulfate conditions. The Brewer Lab proposed a model — Origin Dependent Inverted Repeat Amplification (ODIRA) — where this gene amplification is initiated via a DNA replication error. In the ODIRA model, DNA replication fork regression at short inverted repeats leads to template switching of the replication machinery and the extrusion of a replication-competent hairpin molecule, which after replication, recombines at the original locus to produce an inverted triplication. An alternative explanation behind the amplification is that the hairpin molecule is generated by double-stranded DNA breaks (DSB). To distinguish between these possibilities, we used an engineered strain in which the selectable marker gene, URA3, is split into overlapping fragments (“ura” and “ra3”) on two different chromosomes. The complete URA3 gene is only present in yeast that undergo rare direct recombination between chromosomes or by recombination of the replicated hairpin formed by ODIRA or DSB. We used CRISPR-Cas9 to induce DSBs upstream of the ura fragment and identify the type of event that restores URA3 function with contour-clamped homogeneous electric field gels (CHEF gels), Southern blots, and polymerase chain reactions (PCR). If DSBs drive hairpin formation, cutting the chromosome upstream of the ura fragment should increase the frequency of URA3 assembly via hairpin intermediate. We demonstrate that double-stranded DNA breaks do not increase frequency of hairpin intermediates, providing further evidence that ODIRA is responsible for the inverted triplications of SUL1 in yeast.

In every eukaryotic genome, there is a cluster of tandemly repeated ribosomal DNA (rDNA) that is present in high copy numbers. Besides encoding ribosomal RNAs, rDNA is also involved in many non-ribosomal cellular functions. It is still not fully understood how this cluster of rDNA is maintained and how variation in its copy number impacts cellular function. Kobayashi et al found that Fob1, a protein that binds to replication fork blocking (RFB) sequences, is involved in the expansion and contraction of the rDNA region, however, the underlying mechanism of this copy number control is unknown. To explore the interactions between FOB1 and rDNA I would like to utilize CRISPR/Cas9-mediated editing to edit specific sequences in each rDNA repeat in wild-type and fob1Δ strains of Saccharomyces cerevisiae. Previous studies in our lab utilizing CRISPR/Cas9-mediated editing of the rDNA found that the rDNA copy number was initially significantly reduced, resulting in very slow cellular growth, and after many cell generations, rDNA copy number would expand through a proposed mechanism of reintegration of excised repeats and unequal sister recombination. These observations raise an important question: if FOB1 is needed for rDNA expansion, would it even be possible to perform rDNA editing and recover rDNA copy number in a fob1Δ strain? To address this, I am performing CRISPR/Cas9 editing of rDNA in fob1Δ cells alongside a wild-type control. I am characterizing viable transformants by studying their growth rate, ploidy, and rDNA copy number expansion. I am expecting to see no rDNA expansion occur in strains that do not have the presence of FOB1. By understanding the phenotypic impact of rDNA copy variation in a fob1Δ strain of Saccharomyces cerevisiae, we can come closer to understanding the interactions between RFB, FOB1, and rDNA copy number along with its effects on cellular processes.