Gene amplification can be associated with genetic disorders such as cancer and varying types of autism. One such form of amplification is a neochromosome where a chromosomal segment is amplified and these two segments are joined at an inverted repeat. There are two models to explain how this event could occur: double stranded break and repair and origin dependent inverted repeat amplification (ODIRA). ODIRA hypothesizes that a replication fork error occurring near short inverted repeats could cause the leading strands to erroneously become ligated to the lagging strands and produce an extrachromosomal palindromic DNA intermediate. Because both models produce identical neochromosomes, I have worked to find which pathway produces them. To do so I utilized CRISPR/cas9 to induce breaks at commonly observed junctions, and analyzed the resulting intermediates using gel analysis of whole yeast chromosomes. If the neochromosome were to occur via the double stranded break and repair model, I expected that providing the break should increase the frequency with which these neochromosomes arise. Alternatively, if the neochromosomes were occurring due to the ODIRA model, I expected the frequency to remain unchanged. I followed this initial analysis with DNA sequencing to give further support to either outcome. In particular I verified that the CRISPR system was working by sequencing across the junctions where cas9 was cutting. If cas9 was indeed cutting, then when comparing the sequence from the cas9 survivors to that of the yeast genome, the survivors had an altered cut site. My work provides insight to the etiology to this fascinating class of chromosome rearrangements.

We are using yeast to investigate how the DNA helicase mutation mcm4^Chaos3 interferes with the early events leading to DNA replication and how these replication defects may lead to its role in cancer development. Mcm4^Chaos3 encodes a mutation in a subunit of the MCM helicase, an essential complex required for unwinding double stranded DNA during replication. Mcm4^Chaos3 mice exhibit genomic instability and more than 80% of female mice homozygous for Mcm4^Chaos3 developed mammary tumors (Shima et. al 2007). Further work in yeast identified an origin-specific minichromosome loss phenotype, suggesting the mcm4^Chaos3 mutation may have particular sequence requirements at origins, where DNA replication initiates. To determine the basis of origin sequence specificity in mcm4^Chaos3 function, we had previously performed a plasmid maintenance competition assay using a mutARS317-seq library, containing 500+ plasmids with random single mutations within the ARS317 origin sequence. This assay identified 5 origin sequence variants that performed better in mcm4^Chaos3 yeast than wild type ARS317. To investigate mcm4^Chaos3 interaction with ARS317 variants, I first measured the loss rate for wild type ARS317 plasmids in mcm4^chaos3 vs wild type yeast. As predicted, wild type cells maintained the plasmids better than the mcm4^Chaos3 mutants (16.8% loss rate/generation compared to 5% in wild type). I am currently characterizing the 5 sequence variants, and based on the competition assay data, am predicting to see differences in plasmid loss rates across the ARS317 variants. Understanding the cause for this origin specificity could help us develop a greater understanding of the mechanics involved in DNA replication, genome stability, and cancer-causing mutations.

Gene amplifications are an often-overlooked source of genetic variation frequently associated with genetic disorders, including cancer and developmental delays. However, the mechanism by which they arise is still unclear. There are currently two competing models for how gene amplifications arise. The first model requires a double-stranded break in DNA that results in chromosome recombination. In the second model, an error in DNA replication results in the formation of an extra-chromosomal DNA intermediate that can reinsert into the genome. To distinguish between these two models, I am using CRISPR/Cas9 to induce double stranded breaks in a strain of yeast with two fragments of the URA3 gene on separate chromosomes. I then select for strains with restored URA3 function, indicating that the two fragments have recombined to form the full URA3 gene. If the genome rearrangements are occurring through the double-stranded break mechanism, inducing these breaks should increase the frequency of recombination events that result in functional URA3 genes. However, the preliminary results suggest that these events are occurring through the replication-error mechanism rather than through double-stranded breaks. Clarifying the mechanism of gene amplifications is a significant step toward understanding the how these genetic disorders arise and suggests further research in preventing disease-causing gene amplifications.