During mitosis, all 23 human chromosomes must be faithfully transmitted to progeny cells. Errors in this process result in aneuploidy, a form of genetic instability associated with tumorigenesis. Thus, mechanisms controlling chromosome segregation are increasingly viewed as significant to cell homeostasis and human health. Centromeric regions of chromosomes are essential for chromosome segregation because they mediate attachment to the mitotic spindle and act as cohesion sites linking sister chromatids. The central hypothesis underlying this proposal is that the ubiquitin-related Smt3/SUMO-1 protein modifier is a key regulator of these two aspects of centromere function. We have found the yeast Smt3/SUMO-1 deconjugase Smt4 interacts with the Mif2 kinetochore protein and smt4 mutants display a defect in centromere chromatin organization. Based on these data, our first aim tests the hypothesis that Smt3/SUMO-1 modification regulates a pathway connecting Mif2 and centromere/kinetochore integrity. We will analyze this pathway by isolating mif2 mutants defective for Smt4 interaction and suppressors of the smt4 defect in CEN structure. Our data also indicate that smt4 mutants fail to maintain cohesion at centromeric regions. Thus, our second aim tests the hypothesis that Smt3/SUMO-1 regulates a mechanism that specifically controls cohesion at centromeres. It has been proposed that centromeric cohesion is controlled by a tensiometer, a chromatin domain that modulates cohesion in response to tension exerted by the mitotic spindle. To examine whether Smt3/SUMO-1 modification regulates a tensiometer-like activity we will visualize centromere cohesion dynamics in Smt3/SUMO-1 conjugation mutants and develop a chromatin affinity purification technique to identify Smt3/SUMO-1 substrates that may be tensiometer components. Finally, we have isolated DNA topoisomerase II (Top2) as a Smt3/SUMO-1 substrate that can suppress the Smt4 cohesion defect.
Our third aim tests the hypothesis that Top2 Smt3/SUMO-1 modification coordinates chromatid topology with centromere cohesion dynamics. We will determine whether chromatid intertwining is associated with centromeric cohesion and examine the function of Top2 Smt3/SUMO-1 modification by isolating factors requiring this modification for survival. These studies will elucidate potentially conserved mechanisms controlling chromosome segregation and identify additional Smt3/SUMO-1 substrates regulating centromere function.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM066190-03
Application #
6892818
Study Section
Cell Development and Function Integrated Review Group (CDF)
Program Officer
Carter, Anthony D
Project Start
2003-06-01
Project End
2008-05-31
Budget Start
2005-06-01
Budget End
2006-05-31
Support Year
3
Fiscal Year
2005
Total Cost
$255,052
Indirect Cost
Name
University of California Riverside
Department
Anatomy/Cell Biology
Type
Schools of Earth Sciences/Natur
DUNS #
627797426
City
Riverside
State
CA
Country
United States
Zip Code
92521
Gasparyan, Hovik J; Xu, Ling; Petreaca, Ruben C et al. (2009) Yeast telomere capping protein Stn1 overrides DNA replication control through the S phase checkpoint. Proc Natl Acad Sci U S A 106:2206-11
Lee, Ming-Ta; Bachant, Jeff (2009) SUMO modification of DNA topoisomerase II: trying to get a CENse of it all. DNA Repair (Amst) 8:557-68
Baldwin, Melissa L; Julius, Jeffrey A; Tang, Xianying et al. (2009) The yeast SUMO isopeptidase Smt4/Ulp2 and the polo kinase Cdc5 act in an opposing fashion to regulate sumoylation in mitosis and cohesion at centromeres. Cell Cycle 8:3406-19
Baldwin, Melissa; Bachant, Jeff (2009) Top2 SUMO conjugation in yeast cell lysates. Methods Mol Biol 582:209-19
Baldwin, Melissa; Warsi, Tariq; Bachant, Jeff (2009) Analyzing Top2 distribution on yeast chromosomes by chromatin immunoprecipitation. Methods Mol Biol 582:119-30
Warsi, Tariq H; Navarro, Michelle S; Bachant, Jeff (2008) DNA topoisomerase II is a determinant of the tensile properties of yeast centromeric chromatin and the tension checkpoint. Mol Biol Cell 19:4421-33
Clarke, Duncan J; Bachant, Jeff (2008) Kinetochore structure and spindle assembly checkpoint signaling in the budding yeast, Saccharomyces cerevisiae. Front Biosci 13:6787-819
Khalil, Al-Muataz; Julius, Jeffrey A; Bachant, Jeff (2007) One step construction of PCR mutagenized libraries for genetic analysis by recombination cloning. Nucleic Acids Res 35:e104
Bachant, Jeff; Jessen, Shannon R; Kavanaugh, Sarah E et al. (2005) The yeast S phase checkpoint enables replicating chromosomes to bi-orient and restrain spindle extension during S phase distress. J Cell Biol 168:999-1012