Cells' ability to efficiently replicate their genomes is essential for regulating chromosomal division and maintaining chromosome integrity.  Defects in any of these cellular processes may cause genomic instability, potentially leading to cancer.  The Chaos3 allele in the yeast Saccharomyces cerevisiae is a single base pair change causing an amino acid substitution in the Mcm4 protein.  Mcm4, a component of the replicative helicase, is recruited to replication origins to unwind double stranded DNA and initiate replication.  Chaos3 is in a region of MCM4 that is highly conserved across eukaryotes; while mutations in conserved regions are generally non-viable, Chaos3 is a viable allele that causes genomic instability, leading to elevated cancer rates in mice.  In S. cerevisiae, Chaos3 decreases early firing of the autonomously replicating sequences (ARS) where DNA replication begins.  Chaos3 does not affect all early firing ARSs in the genome; rather, a large proportion of origins near centromeres, thereby delaying replication of those centromeres.  Essential for chromosome segregation, the centromere is the location where spindle fibers attach to pull apart sister chromatids during cell division.  I hypothesize that this delay in centromere replication results in chromosomal instability, including the loss of a chromosome.  I am using CRISPR guided cutting directed by a customizable guide RNA to replace centromeric adjacent ARS510, that has decreased firing levels in Chaos3, with unaffected, early firing ARS305, to see if firing levels in the mutants are affected based on ARS chromosome location (i.e., proximity to a centromere) or ARS sequence.  If replacing ARS510 with ARS305 restores early origin firing in this region this will confirm the Chaos3 mutation affects specific ARS sequences rather than ARS location on the chromosome.  Furthermore, if centromere replication delays are the cause of genomic instability in Chaos3, this ARS replacement should rescue chromosome loss.

Budding yeast cultures grown in limited sulfate conditions are overtaken by cells with an inverted triplication of the gene SUL1, which encodes for a sulfate transporter. The extra copies of the sulfate transporter provide a selective advantage because these cells outcompete other yeast cells for the limiting resource. To explain the mechanism behind this type of amplification the Brewer and Dunham Labs proposed a model (Origin Dependent Inverted Repeat Amplification or ODIRA), which requires both a DNA replication origin and inverted repeats flanking SUL1. ODIRA starts with a DNA replication error involving replication fork regression that leads to an extrachromosomal DNA intermediate. This intermediate then replicates and recombines into the genome, producing the observed amplification. Because similar triplications are observed in the human genome, including in human disorders, the mechanism of ODIRA offers insights into human genome evolution and disease. While the yeast research is consistent with ODIRA, we still do not know which proteins are responsible for the process. I am testing whether the genes RAD5 and RAD54 — involved in fork regression and strand switching, respectively — are involved in ODIRA. To do so, I am measuring the ODIRA frequency in strains with each gene deleted compared to a wild-type control. If either gene deletion leads to a statistically significant change in ODIRA frequency compared to the wild-type strain, I can conclude this gene is involved in ODIRA. To measure ODIRA frequency, I grow the deletion strains under selection for DNA recombination events and use whole chromosome gel electrophoresis and Southern blotting to detect ODIRA events. Preliminary data analysis suggests that there is a reduction in ODIRA events when either RAD5 or RAD54 is deleted, indicating that these genes are likely needed for ODIRA. These results may provide insight into how inverted triplications may arise.