My lab studies two regulatory systems essential in maintaining genome stability and preventing tumor progression in certain types of cancers. We primarily study the üspindle checkpointý in the budding yeast Saccharomyces cerevisiae using a combination of genetic and molecular genetic approaches. The spindle checkpoint is a regulatory mechanism that inhibits the onset of anaphase until all chromosomes achieve bipolar orientation on the spindle. We have a poor understanding about how the spindle signal is generated and two general models that can be considered. The first is that the lack of tension is the initiating event and this model has almost completely dominated the field. The second possibility is that the checkpoint monitors microtubule occupancy and if a kinetochore is unoccupied, then the cells arrest in mitosis.

Both models can accommodate a role for the kinetochore in generating the spindle checkpoint signal. We have provided definitive evidence for a role of the kinetochore in checkpoint signaling in yeast. Several kinetochore mutants lack the spindle checkpoint. We have mapped the checkpoint activity of the kinetochore to the Ndc80 complex of proteins that has a dual role in chromosome segregation and checkpoint signaling. We are currently identifying mutations in the Ndc80 complex that specifically disrupt the spindle checkpoint.

We have recently identified an important phosphorylation event on Mad3 that is required to transmit the signal for the tension branch of the spindle checkpoint. We have raised a phospho-specific antibody to a phosphorylated Mad3 peptide and showed that phosphorylation requires all spindle checkpoint genes and the Ndc80 complex. We are using the antibody to further map the tension checkpoint within the kinetochore.

We have recently discovered that there is cross talk between the spindle checkpoint and the DNA damage checkpoint. We have used genetic analysis to show that DNA damage activates the canonical spindle checkpoint pathway except that DNA damage regulates the pathway in a kinetochore-independent fashion. This suggests that the kinetochore is not obligatory for spindle checkpoint proteins to become inhibitors.

We have also completed a genome-wide screen for all mutants that activate the spindle checkpoint with the goal of identifying every protein that is a component of the mitotic spindle. We identified 128 different mutants, many of which encode novel proteins of unknown function. We are characterizing the mutants and have identified new regulators of spindle assembly and function.

Finally, we are collaborating with Drs. Dan Theodorescu, Department of Urology, Jae Lee, Department of Public Health Science and Stefan Bekiranov, Department of Biochemistry and Molecular Genetics to use a combination of computational biology and yeast genetics to discover new anti-cancer drugs and treatments for a variety of cancers In summary, we are using a combination of genetics, cell biology and biochemistry to understand the role that the kinetochore plays in generating the spindle checkpoint signal. We are using modern genetic approaches and genome wide screens to identity potential lesions that activate the checkpoint and to identify potential regulators of checkpoint activity. All of these are important approaches to understanding this fundamental and important aspect of genetics and cell biology and has significant applications for cancer biology.

Selected References

Chi A, Huttenhower C, Geer LY, Coon JJ, Syka JE, Bai DL, Shabanowitz J, BurkeDJ, Troyanskaya OG, Hunt DF. (2007) "Analysis of phosphorylation sites on proteins from Saccharomyces cerevisiae by electron transfer dissociation (ETD) mass spectrometry." Proc Natl Acad Sci U S A. Feb 104:2193-8. Epub 2007 Feb 7. [PubMed]

Yellman CM, Burke DJ. (2006) "The role of Cdc55 in the spindle checkpoint is through regulation of mitotic exit in Saccharomyces cerevisiae." Mol Biol Cell. 17:658-66. Epub 2005 Nov 28. [PubMed]

Emanuele M, Burke DJ, Stukenberg PT. (2007) "A Hec of a microtubule attachment." Nat Struct Mol Biol. 14:11-3. [PubMed]

Daniel JA, Keyes BE, Ng YP, Freeman CO, Burke DJ. (2006) "Diverse functions of spindle assembly checkpoint genes in Saccharomyces cerevisiae." Genetics. 172:53-65. Epub 2005 Sep 12. [PubMed]

Poddar A, Stukenberg PT, Burke DJ. (2005) "Two complexes of spindle checkpoint proteins containing Cdc20 and Mad2 assemble during mitosis independently of the kinetochore in Saccharomyces cerevisiae." Eukaryot Cell. 4:867-78. [PubMed]