Daniel J. Burke
Professor of Biochemistry & Molecular Genetics
Ph.D., Johns Hopkins University
Genome Instability in Spindle Checkpoint Mutants

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My lab studies two regulatory systems essential in maintaining genome stability and preventing tumor progression in certain types of cancers. We study the spindle assembly checkpoint (SAC) in the budding yeast Saccharomyces cerevisiae using a combination of genetic and molecular genetic approaches. The SAC 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 SAC signal is generated but it is clear that the signal originates in the kinetochore, where microtubules bind to orchestrate chromosome segregation at mitosis.

The current models propose that the kinetochore catalyzes the assembly of SAC proteins into complexes that inhibit mitosis when microtubule binding is impaired. The model is simple and is supported by multiple lines of experimental evidence. However, the molecular details of how this happens in the kinetochore remain elusive. We have provided genetic evidence for a role of the kinetochore in SAC signaling in yeast as several kinetochore mutants lack the checkpoint. We mapped the SAC activity of the kinetochore to a complex of proteins that has a dual role in chromosome segregation and checkpoint signaling. We are performing comprehensive mutagenesis on the genes that encode these kinetochore proteins that will systematically insert all 20 amino acids at every position in the polypeptide and we will use deep sequencing to identify those mutations that eliminate spindle checkpoint but not microtubule binding.

We identified an Ipl1/Aurora B phosphorylation site on the spindle checkpoint protein Mad3 that is required to transmit the signal. We used the comprehensive barcoded deletion collection of yeast mutants in a genome-wide screen to identify novel mutants in the SAC that are sensitive to Mad3 phosphorylation. One of the genes is a different kinetochore protein and we collaborated with Todd Stukenberg's lab to show that this response to Aurora B phosphorylation is evolutionarily conserved from yeast to human cells. We are currently characterizing several other genes that we identified to find their role in the SAC.

We discovered that there is cross talk between the SAC and the DNA damage checkpoint. We have used genetic analysis to show that DNA damage activates the SAC except that DNA damage regulates the pathway in a kinetochore-independent fashion. The kinetochore is not obligatory for spindle checkpoint proteins to become inhibitors. We showed that SAC activation by DNA damage requires phosphorylation of the SAC protein Mad1 and we are characterizing the cellular response. We identified conditions where the SAC protects cells from DNA damage when the cells lack the DNA damage checkpoint. We are using these conditions and the comprehensive deletion collection of yeast mutants in a novel genome-wide screen, dependent on deep sequencing, to identify novel genes in the SAC response to DNA damage. Finally, we collaborated with Dr. David Amberg (SUNY Upstate Medical School) who has developed a novel method for identifying genetic interactions through complex haplo-insufficiencies. Diseases are often attributable to situations where the individual is heterozygous for two (or more) mutations in different genes. The complex haplo-insufficiencies can be explored in a systematic way in yeast and we are using this approach to identify novel targets of the Tor kinase that plays an integral role in cell growth and cancer.

Selected References

Keyes BE, Sykes KD, Remington CE, Burke DJ. (2012) "Sister chromatids segregate at mitosis without mother-daughter bias in Saccharomyces cerevisiae." Genetics. 192:1553-7. doi: 10.1534/genetics.112.145680. Epub 2012 Oct10. [PubMed]

Demirel PB, Keyes BE, Chaterjee M, Remington CE, Burke DJ. (2012) "A redundant function for the N-terminal tail of Ndc80 in kinetochore-microtubule interaction in Saccharomyces cerevisiae." Genetics. 192:753-6. doi: 10.1534/genetics.112.143818. Epub 2012 Jul 30. [PubMed]

Matson DR, Demirel PB, Stukenberg PT, Burke DJ. (2012) "A conserved role for COMA/CENP-H/I/N kinetochore proteins in the spindle checkpoint." Genes Dev. Mar 26:542-7. doi: 10.1101/gad.184184.111. [PubMed]

Haarer B, Aggeli D, Viggiano S, Burke DJ, Amberg DC. (2011) "Novel interactions between actin and the proteasome revealed by complex haploinsufficiency." PLoS Genet. 7:e1002288. doi: 10.1371/journal.pgen.1002288. Epub 2011 Sep 22. [PubMed]

Smith SC, Havaleshko DM, Moon K, Baras AS, Lee J, Bekiranov S, Burke DJ,Theodorescu D. (2011) "Use of yeast chemigenomics and COXEN informatics in preclinical evaluation of anticancer agents." Neoplasia. 13:72-80. [PubMed]

Burke DJ. (2009) "Interpreting spatial information and regulating mitosis in response to spindle orientation." Genes Dev. Jul 23(14):1613-8. doi: 10.1101/gad.1826409. [PubMed]

Keyes BE, Burke DJ. (2009) "Irc15 Is a microtubule-associated protein that regulates microtubule dynamics in Saccharomyces cerevisiae." Curr Biol. Mar 19:472-8. doi: 10.1016/j.cub.2009.01.068. Epub 2009 Mar12. [PubMed]

Kim EM, Burke DJ. (2008) "DNA damage activates the SAC in an ATM/ATR-dependent manner, independently of the kinetochore." PLoS Genet. Feb 4:e1000015. doi: 10.1371/journal.pgen.1000015. [PubMed]

Keyes BE, Yellman CM, Burke DJ. (2008) "Differential regulation of anaphase promoting complex/cyclosome substrates by the spindle assembly checkpoint in Saccharomyces cerevisiae." Genetics. 178:589-91. doi: 10.1534/genetics.107.083642. [PubMed]