Research

Genomic Instability in Yeast and Cancer

Alison Adams
AZCC/NAU

Ted Weinert
AZCC/UA

Jason Tidwell
NAU

Danita Davis
NAU

Patricia Chan
NAU

Marlene Begay
NAU

Obed Adarkwah
NAU

Rachel Bodamer
NAU

Sabrina Yazzie
NAU

Julia Soap
UA

Abstract:

The long-term goal of this study is to understand how environmental exposure and genetic factors influence the risk of genomic instability in cancer. During cancer development,

chromosomes undergo breakages that lead to rearrangements (e.g., translocations), which are often unstable and consequently result in further breakage and rearrangements (see figure). Such events may result in altered gene products and/or changes in gene expression, and may be so unstable as to lead to chromosome loss . Such changes can affect growth control, and consequently lead to cancer. We are using yeast as a model organism to study genomic instability. In particular, we are using the extremely powerful genetics possible with this yeast system to identify genes that are normally required to stabilize the genome. To this end, we are looking for genes that, when over-expressed, cause increased genomic instability (as judged by a colony-sectoring assay). So far, we have identified two genes (SRS2 and MSS11) and a plasmid insert (containing four genes), whose over-expression leads to increased instability. We are currently testing the three most likely candidates on the plasmid insert (HST3, BUB3 and AHC1) to determine which of these genes causes the observed instability when over-expressed. In addition, we are looking for more genes that similarly cause increased levels of genomic instability. We will then conduct experiments to identify the roles of genes of interest in stabilizing the genome in yeast and (eventually) in humans.

Cancer cells have a high frequency of chromosome rearrangements, such as translocations, seen below as multi-colored chromosomes. WHY????

Translocations cause cancer because the junctions of two chromosomes can lead to mutations that interfere with growth control

Why use yeast to study unstable translocations?

• Great genetics! Easy to work with. A simple assay for instability already identified.
• Yeast and human cells have similar mechanisms of DNA replication, repair etc, so lessons learned in yeast will likely apply to human cells
• Great precedent - yeast has already proven invaluable in understanding cancer (e.g., colorectal cancer)
• Identifying Yeast Genes required for Genomic Stability
• Identifying Genes that, when over-expressed, cause increased levels of genomic instability (colony sectoring)

Rationale:
Over-expression of genes is often as deleterious to a cell as is under-expression or the presence of a mutation. We wish to identify genes required to stabilize genomes by looking for those that, when over-expressed, cause increased levels of genomic instability (in the same way that mutations in genes of interest would).

Experimental approach:
Use a ‘2 micron’ plasmid that is present in yeast cells at 50-100 copies per cell. Any gene carried on such plasmids will be expressed at 50-100X normal levels. Test known genes and ‘fish out’ unknown genes from a 2-micron library to see which ones cause increased levels of genomic instability (assayed by colony sectoring).

Testing known genes to see if over-expression causes increased levels of genomic instability: We are testing a gene (RRM3) that, when mutant, cause increased levels of instability (see Jason Tidwell’s poster).

Looking for unknown genes:
We are using a library of yeast genes in a 2 micron plasmid to transform yeast and ask which genes, when over-expressed, increase instability.

Results:
In a screen of ~750 plasmids, we have identified THREE that increase colony sectoring. Colonies with cells containing one of these plasmids is shown below.

Genes that we identified on these three plasmids are:

• SRS2, which encodes a DNA helicase
• MSS11, which encodes a transcriptional regulatory factor.
• AHC1, BUB3, or HST3. These three genes are all present on one plasmid. We are currently testing each gene to see which of the three causes the increased instability seen when over-expressed.

Ongoing and future studies:

1. Look for more genes that, when over-expressed, increased genomic instability
2. Identify the roles of SRS2, MSS11, and other genes in stabilizing the genome.
3. Determine the roles of genes identified in this study in stabilizing the genomes of human cells.