Our laboratory studies two related aspects of cell cycle regulation in yeast. We study (I) the DNA damage checkpoint and the way in which cell cycle arrest facilitates DNA repair. (II) the Anaphase Promoting Complex (APC) .
(I) When eukaryotic cells have DNA damage they arrest their progression through the cell cycle. The machinery that monitors DNA damage and elicits this arrest (the checkpoint) is conserved between humans and yeast. We are now examining the mechanisms by which the checkpoint is activated and subsequently inactivated by DNA damage. We are also using tools that we have generated in these studies to explore the behavior of broken chromosomes during mitosis.
We have GFP tagged two different complexes required for the DNA damage checkpoint, the kinase Mec1/Ddc2 (ATR/ATRIP in humans) complex and the PCNA-like Ddc1 (RAD9 in humans) complex, and shown that these complexes are recruited to a double stranded break. Interestingly, Ddc2 localization requires Mec1 but no other known checkpoint proteins, while Ddc1 localization requires other members of the PCNA-like group, but not Mec1 or Ddc2. Hence, Ddc1 and Ddc2 recognize DNA damage by independent mechanisms. These data support a model in which assembly of multiple checkpoint complexes at DNA damage sites stimulates checkpoint activation. We are also using checkpoint protein localization to damage sites as a tool for analysis of DNA damage in living cells. We have found, for example, that when a break is made in the middle of a chromosome only one focus is seen, even after many hours, suggesting that the two halves of a broken chromosome stay together. Preliminary genetic analysis has suggested a mechanism by which chromosomes are tethered, and we are exploring this further. In addition, we are extending these studies by using lacI-GFP fusions to visualize specific chromosomal loci. Using these tools, we can determine, for example, whether the acentric half of a broken chromosome is correctly segregated after checkpoint adaptation.
(II) In addition to our work on the DNA damage checkpoint, we are also investigating the regulation of the cell cycle by the targeted degradation of proteins. Proteolytic targeting is accomplished, in part, by a 13 subunit ubiquitin ligase called the Anaphase Promoting Complex (APC). Thus far, at least a dozen proteins have been identified as APC targets. We have found that after targeted deletion/inhibition of two targets, the securin PDS1 and the Cdk/B-cyclin kinase respectively, we are able to delete otherwise essential APC genes. This has allowed us to pinpoint the obligatory targets of the APC. The generation of such strains allows us to analyze the biology and mechanisms of the APC enzyme without the constraints normally imposed by its essential nature.
Paulovich, A., Toczyski, D.P., and Hartwell, L.H. (1997) "When checkpoints fail". Cell 88 : 315-321.
Toczyski,D.P., Galgoczy, D.J., and Hartwell, L.H. (1997) " CDC5 and CKII Control Adaptation to the Yeast DNA Damage Checkpoint". Cell 90 : 1097-1106.
Galgoczy, D.J., and Toczyski, D.P. (2001) "Checkpoint adaptation precedes spontaneous and DNA damage-induced genomic instability in S. cerevisiae" MCB 21 : 1710-1718
Melo, J., Cohen, J. and Toczyski, D.P. (2001) "Two checkpoint complexes are independently recruited to sites of DNA damage in vivo " Genes and Development 15 : 2809-2821
Melo, J. and Toczyski, D. P. (2002) "The DNA damage checkpoint response" Current Opinions in Cell Biology 14: 237-245
Thornton, B. and Toczyski, D. P. (2003) "Cycling with the cyclosome: Securin and B cyclins are the only obligatory targets of the APC/C." Nature Cell Biology 5(12): 1090-4.
Thornton , B.R., Chen, K.C., Cross, F.R., Tyson, J.J., and Toczyski, D.P. (2004) "Cycling without the cyclosome: Modeling a yeast strain lacking the APC." The Cell Cycle 3(5): 629-33.
Kaye, J., Melo, J. , Cheung, S.K., Vaze, M.B., Haber, J. and Toczyski, D. P. " DNA breaks promote genomic instability by impeding proper chromosome segregation " under review.