Bhupinder Bhullar

Cells have evolved mechanisms to monitor the integrity of their genome and repair damaged DNA. DNA is the only molecule in the cell that is repaired, whereas all other molecules are replaced. The mechanism that monitors for damaged DNA is intimately linked with cell cycle events and has been termed "checkpoint" (Weinert and Hartwell, Science 1988).

Defects in checkpoint function are an important indicator in the prediction of cancer prognosis. Loss of checkpoint function predisposes a cell to acquire selective oncogenic mutations that give rise to cancer. Individuals with genetic instability disorders (such as ataxia telangiectasia (AT), Bloom's syndrome, Fanconi anemia (FA), xeroderma pigmentosum (XP), and Nijmegen breakage syndrome) have defects in checkpoint function and an increased rate in the incidence of many cancers1.

The conservation of checkpoint mechanisms through evolution from yeast to mammals reflects the universal importance of these pathways2. Genetic studies have identified many of the components of checkpoint in the yeast S. cerevisiae and S. pombe. Different checkpoint pathways exist in the cell that monitor different types of DNA damage that can be incurred at different stages of the cell cycle. In the G1 stage, cells accumulate primarily oxidative damage to DNA, S phase cells risk incomplete replication and nucleotide incorporation, and cells undergoing mitosis risk chromosome breakage during segregation of sister chromatids. Checkpoint proteins arrest cells at these stages to repair and/or replicate the DNA.

We are using a high throughput approach to screen for genes that can negatively regulate the checkpoint function. These genes may be potential markers for early prediction of cancer development, and or targets for therapeutic intervention.

To screen for checkpoint inhibitors, we have constructed a library of full length sequence validated yeast ORFs. The Yeast FLEXGene library of individually arrayed and annotated clones was generated by recombination cloning using the Gateway cloning system. To generate expression ready clones, we subsequently transferred the clones by recombination cloning into an yeast vector pBY011 (CEN, URA3) under the control of the inducible Gal1-10 promoter. All DNA and cell manipulations are done in high-throughput using protocols that incorporate liquid handling robots.

Figure 1. YeastExpression Plasmid pBY011  Enlarge

Methods

Figure 2. Overview of assay for checkpoint inhibitors  Enlarge

Results

Sample data

Figure 3. Clone #57 inhibits cell growth in the presence of hydroxyurea. PSY580 cells were transformed with 81 genes from an array of yeast FLEXGene expression clones and plated on the indicated SD-URA plates. The yellow squares indicate a clone that hinders growth of cells in the presence of 100 mM hydroxyurea.  Enlarge

Figure 4. Gene #5324 inhibits cell growth after exposure to UV radiation. PSY580 cells transformed with partial yeast FLEXGene library and plated on SD-URA with 2% Gal or 2% Glu (as indicated) and treated with or without 100u of UV radiation. Clone #5324 confers UV sensitivity to cells (panel 4). ( UV units x100 uJoules/cm2)  Enlarge

References

1. Khanna, K, (2000) J. Natl. Cancer Inst. May 17;92(10):795-802.
2. Zhou and Elledge (2000) Nature 408: 433-439