Maintaining the integrity of genetic material and preventing changes over time is essential for normal cellular function. This genomic stability is directly affected by the DNA replication process. Replication must be both accurate and efficient; mutations that affect DNA replication can cause genomic instability and changes in the genetic makeup of the cell. Through a genomic instability screen in mice, researchers discovered a single base pair mutation in a highly conserved gene required for unwinding DNA during DNA replication. The presence of this single base pair substitution, called Chaos3, in both copies of the gene causes female mice to develop mammary tumors. We have found that in the yeast Saccharomyces cerevisiae, the corresponding Chaos3 mutation decreases activation or “firing” of some replication origins—the sites where DNA replication begins. Chaos3 does not affect all early firing origins in the genome; rather, origins near centromeres are specifically affected, thereby delaying replication of those centromeres, causing chromosome loss. We found that when an affected origin is replaced with an unaffected one, firing levels are restored to wild type function and that chromosome loss is rescued. To further understand what components are essential for timely DNA replication, and why only a subset of origin sequences are sensitive to the Chaos3 allele, I am focusing on the origin sequences directly. I am mutating the origin sequence itself and separately deleting different genes whose products have potential interactions with origin sequences. My research aims to advance the understanding of the role these genes play in the activation of origins for timely DNA replication and how the Chaos3 mutation may be interrupting normal function of these processes. This knowledge can help identify key molecular mechanisms that drive cancer development in higher eukaryotes.

Copy number variation (CNV) is associated with genetic disorders in humans. One particular kind of CNV is a gene triplication in which the central copy is inverted. How such structures arise is poorly understood but is of great interest because of their association with cancer and other genetic disorders. We find similar amplified structures in budding yeast Saccharomyces cerevisiae, and therefore, we can use yeast to understand the mechanism that generates them. The Brewer and Dunham labs have proposed Origin Dependent Inverted Repeat Amplification (ODIRA) as a model that explains inverted CNVs. To understand which proteins/enzymes contribute to amplification, we are implementing gene deletions to observe the effect on the production of these ODIRA amplification events. ODIRA events are rare and thus large sample sizes are required to detect them. To streamline their identification, I am developing a system that uses color as a visual indicator of CNV. When a single copy of the bacterial gene, VioA, is expressed in yeast it produces light purple colonies; in multiple copies, the colonies are a darker shade of purple. I am inserting a single copy of the VioA gene into a region of the yeast genome that undergoes inverted triplication events. Simply scanning plates for dark purple colonies will enable me to screen for ODIRA events quickly and measure their frequency. Using this visual indicator will allow me to rapidly screen different yeast mutants and determine the role they play in the ODIRA amplification mechanism.

Gene arrangements are observed in human diseases such as cancer and developmental disorders. In developmental disease, gene triplication with an inverted central copy is observed, while a hallmark of cancer are palindromes, or inverted repeats of DNA. Therefore, testing one mechanism of gene arrangement in an easily studied organism like the  budding yeast Saccharomyces cerevisiae may shed some light on the human genome and disease generation. Yeast cells grown for >200 generations in sulfate limiting media are enriched for triplication of the high affinity sulfate transporter SUL1 with the center copy inverted, as amplification confers a selective advantage. To explain how such a triplication occurs, the Brewer and Dunham Labs proposed a model called Origin Dependent Inverted-Repeat Amplification (ODIRA). The ODIRA mechanism requires a DNA origin of replication and short, inverted repeats flanking the SUL1 gene. During DNA replication, an error of the replication fork, or fork regression, causes annealing of leading and lagging strands to create a hairpin intermediate. The intermediate then replicates and recombines into the genome, forming interstitial triplications, with the middle copy inverted. While the proposed mechanism explains the observed triplication in yeast, the specific proteins involved are not yet known. The genes I've chosen to test are the DNA helicases MPH1 which prevents cross-over between ectopic sequences, and RRM3 which relieves replication fork pauses. Both are predicted to regulate necessary steps of the ODIRA mechanism, making them good candidates for genes that may be involved in these triplications. By deleting each gene, I can then measure the frequency of ODIRA events in those strains and compare them to wild-type strains. Increased ODIRA events in the knockout strains may implicate their role in the ODIRA mechanism and prompt further study of these genes and how they might affect copy number variation in humans.