Chromosome segregation is crucial to the health and survival of all organisms. Accumulating evidence indicates that the vast majority of human cancers display high rates of chromosome missegregation. This phenomenon, termed chromosomal instability (CIN), has been proposed to drive tumorigenesis by facilitating the emergence of aneuploid clones with increased malignant potential. Thus, it is imperative to obtain a better understanding of the molecules and mechanisms that regulate the process of chromosome segregation in human cells and sustain its high fidelity. We have developed novel methods for 'knocking out' genes in human somatic cells, and we have used these methods to characterize the role of a small mitosis-regulating protein termed securin. This protein forms a tight complex with a large cysteine protease termed separase, which digests the physical links that hold sister chromatids together. Based on our preliminary studies, we hypothesize that the securin-separase complex plays a central role in mitotic progression and high-fidelity chromosome segregation in humans. Precise regulation of this complex is proposed to link sister chromatid separation to upstream checkpoints and restrict it to the the appropriate phase of the cell cycle, i.e., anaphase. To test these hypotheses, we will investigate how specific post-translational modifications contribute to the function of the separase-securin complex.
In Aim 1, we use a genetic approach to elucidate the in vivo role of separase auto-cleavage, to determine its relevance to mitotic progression and chromosome dynamics.
In Aim 2, we test a proposed securin-independent mechanism for inhibiting premature sister chromatid separation, taking advantage of the hSecurin- 'knockout' cells developed in our earlier studies.
In Aim 3, we evaluate potential mechanisms for separase-dependent regulation of processes other than sister chromatid separation. Together, these studies will significantly advance our understanding of mitotic chromosome segregation in humans. Such information is essential to developing more effective cancer therapies that attack the CIN phenotype found in most cancer cells.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA107342-03
Application #
7247247
Study Section
Cell Development and Function Integrated Review Group (CDF)
Program Officer
Mietz, Judy
Project Start
2005-07-01
Project End
2010-05-31
Budget Start
2007-06-01
Budget End
2008-05-31
Support Year
3
Fiscal Year
2007
Total Cost
$339,634
Indirect Cost
Name
Sloan-Kettering Institute for Cancer Research
Department
Type
DUNS #
064931884
City
New York
State
NY
Country
United States
Zip Code
10065
Maciejowski, John; George, Kelly A; Terret, Marie-Emilie et al. (2010) Mps1 directs the assembly of Cdc20 inhibitory complexes during interphase and mitosis to control M phase timing and spindle checkpoint signaling. J Cell Biol 190:89-100
Tsou, Meng-Fu Bryan; Wang, Won-Jing; George, Kelly A et al. (2009) Polo kinase and separase regulate the mitotic licensing of centriole duplication in human cells. Dev Cell 17:344-54
Berdougo, Eli; Terret, Marie-Emilie; Jallepalli, Prasad V (2009) Functional dissection of mitotic regulators through gene targeting in human somatic cells. Methods Mol Biol 545:21-37
Ciccia, Alberto; Bredemeyer, Andrea L; Sowa, Mathew E et al. (2009) The SIOD disorder protein SMARCAL1 is an RPA-interacting protein involved in replication fork restart. Genes Dev 23:2415-25
Burkard, Mark E; Maciejowski, John; Rodriguez-Bravo, VerĂ³nica et al. (2009) Plk1 self-organization and priming phosphorylation of HsCYK-4 at the spindle midzone regulate the onset of division in human cells. PLoS Biol 7:e1000111
Terret, Marie-Emilie; Sherwood, Rebecca; Rahman, Sadia et al. (2009) Cohesin acetylation speeds the replication fork. Nature 462:231-4
Berdougo, Eli; Nachury, Maxence V; Jackson, Peter K et al. (2008) The nucleolar phosphatase Cdc14B is dispensable for chromosome segregation and mitotic exit in human cells. Cell Cycle 7:1184-90
Larochelle, Stephane; Merrick, Karl A; Terret, Marie-Emilie et al. (2007) Requirements for Cdk7 in the assembly of Cdk1/cyclin B and activation of Cdk2 revealed by chemical genetics in human cells. Mol Cell 25:839-50
Burkard, Mark E; Randall, Catherine L; Larochelle, Stephane et al. (2007) Chemical genetics reveals the requirement for Polo-like kinase 1 activity in positioning RhoA and triggering cytokinesis in human cells. Proc Natl Acad Sci U S A 104:4383-8
Jallepalli, Prasad V; Pellman, David (2007) Cell biology. Aneuploidy in the balance. Science 317:904-5