Errors in chromosome segregation are the major source of aneuploidy and a driving force in the development of tumors. The spindle assembly checkpoint (SAC) is one of the mechanisms that prevents aneuploidy and is an important regulator of genomic stability. The SAC is orchestrated by the kinetochore and a major unresolved problem, addressed in this proposal, is to determine how the kinetochore measures the lack of tension and lack of occupancy and how the kinetochore transfers that information to the cell cycle machinery. We will complete high resolution mapping of the tension branch of the SAC within the kinetochore using loss-of- function mutants. We have developed a phospho-specific antibody to Mad3 that is the first biochemical marker for the tension branch of the SAC and we propose to use it to dissect this important component of SAC regulation. We propose a new model for the role of the kinetochore in the tension checkpoint. We will determine if subsets of Mad3-containing proteins are phosphorylated, where Mad3 is phosphorylated in the cell and which kinetochore proteins are needed to transmit the tension signal. We have isolated novel mutants that are specific to the occupancy branch of the SAC. We will dissect the role for the SAC in the DNA damage pathway which requires Mec1 and Tel1 phosphorylation of SAC proteins and represents a novel pathway of mitotic regulation. Together, the experiments in this proposal will provide important insights into how SAC signaling is initiated and integrated into the cell cycle.

Public Health Relevance

Errors in chromosome segregation result in imbalances in chromosome number (aneuploidy), a hallmark of some kinds of birth defects and a major driving force in the development of cancer. The spindle assembly checkpoint is one of the mechanisms that prevents aneuploidy and guards against the diseases resulting from genomic instability. The experiments in this proposal will provide important insights into how the spindle checkpoint is organized, how it is regulated and how it is integrated into the cell cycle. The long term goal is to improve human health by preventing aneuploidy that results in birth defects and cancer.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM086502-04
Application #
8318164
Study Section
Cellular Signaling and Regulatory Systems Study Section (CSRS)
Program Officer
Gindhart, Joseph G
Project Start
2009-08-28
Project End
2014-07-31
Budget Start
2012-08-01
Budget End
2014-07-31
Support Year
4
Fiscal Year
2012
Total Cost
$320,424
Indirect Cost
$109,702
Name
University of Virginia
Department
Biochemistry
Type
Schools of Medicine
DUNS #
065391526
City
Charlottesville
State
VA
Country
United States
Zip Code
22904
Haarer, Brian; Mi-Mi, Lei; Cho, Jessica et al. (2013) Actin dosage lethality screening in yeast mediated by selective ploidy ablation reveals links to urmylation/wobble codon recognition and chromosome stability. G3 (Bethesda) 3:553-61
Matson, Daniel R; Demirel, Pinar B; Stukenberg, P Todd et al. (2012) A conserved role for COMA/CENP-H/I/N kinetochore proteins in the spindle checkpoint. Genes Dev 26:542-7
Haarer, Brian; Aggeli, Dimitra; Viggiano, Susan et al. (2011) Novel interactions between actin and the proteasome revealed by complex haploinsufficiency. PLoS Genet 7:e1002288