We have used chromosome transmission fidelity <I>(ctf)</I>mutants and the deletion strain collections of <I>S. cerevisiae</I>to identify and characterize genes required for kinetochore function and checkpoint function. Studies with the <I>ctf </I>mutants led to the identification and characterization for a role of <I>SPT4</I>and <I>NUP170</I>in chromosome segregation and spindle assembly checkpoint (SAC) function. We established a novel role for Spt4p in heterochromatic silencing. Using cross-species approach we showed that the yeast <I>spt4</I>strains are complemented by human <I>SPT4</I>. Most importantly, we showed that <I>S. cerevisiae SPT4</I>contributes to the proper localization of histone H3 variant Cse4p. We investigated the mechanism of Cse4p localization and have recently established that mislocalization of Cse4p and altered histone stoichiometry lead to defects in chromosome transmission. We wish to examine if chromatin modifiers and post-translational modification of kinetochore proteins affect the assembly/function of CenH3 chromatin. Our recent results with Cse4p localization and histone dosage in <I>S. cerevisiae</I>are consistent with those in <I>S. pombe</I>suggesting conservation of the underlying mechanisms. Thus, studies in <I>S. cerevisiae</I>that elucidate a mechanism for Cse4p localization and the role of chromatin modifications in centromere function may help us understand analogous pathways in humans and other systems. We also wish to establish the molecular role of Spt4p and its interacting partners Spt5p and Spt6p as well as histones in chromatin structure, chromosome segregation and gene silencing in both yeast and humans. To demonstrate the functional relevance of our findings in <I>S. cerevisiae</I>, we plan to extend our research to higher eukaryotes. To this end we are collaborating with Drs. Caplen and Roschke in RNAi studies to investigate the role of human Spt4p/Spt5p/Spt6p in chromosome segregation and function of CENP-A. Our studies with the nuclear pore complex (NPC) gene <I>NUP170</I>allowed us to establish a novel relationship between SAC proteins Mad1p and Mad2p and the NPC in <I>S. cerevisiae</I>. Subsequent to our work, several other studies including ones with human cell lines, have reported roles for NPC components in kinetochore function. Our studies have led to the first report of Mad1p, Mad2p and Bub3p localization to the kinetochore upon SAC activation in <I>S. cerevisiae</I>. We recently defined a domain of Mad1p that is required for chromosome transmission and checkpoint function. Further relevance for a role of NPC in mitosis is based on our collaboration with Dr. Belanger that show genetic interactions between spindle pole body (SPB) and mitotic exit network mutants. In addition to chromosome segregation, the DNA damage and replication checkpoint pathways ensure genome stability by halting the cell cycle in response to genotoxic stress. We have recently established a functional relationship between oxidative stress genes <I>SOD1</I>and <I>CCS1</I>and the <I>MEC1</I>mediated checkpoint pathway for DNA damage and replication arrest. Recent results from genetic analysis have shown that Sod1p and Ccs1p have a role in DNA repair, genome stability and telomere maintenance. Our studies with Sod1p and Ccs1p will unravel molecular mechanisms that correlate oxidative stress, redox state and checkpoint pathways in <I>S. cerevisiae</I> that may be applicable to other systems. Our research on the molecular determinants of faithful chromosome transmission in <I>S. cerevisiae</I>will help us understand analogous processes in humans and their implications in human disease. Our laboratory is uniquely poised to utilize the conventional genetic, biochemical, and cell biology approaches, as well as high-throughput genomic analysis for our research projects. We use an array of gene-deletion strains and a colony picking robot for the identification of possible cancer drug targets and also for genetic screens by Synthetic Genome (SGA) analysis, developed in the laboratory of Charlie Boone (Univ. of Toronto).

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC010822-03
Application #
7965724
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
3
Fiscal Year
2009
Total Cost
$1,142,454
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Mishra, Prashant K; Thapa, Kriti S; Chen, Panyue et al. (2018) Budding yeast CENP-ACse4 interacts with the N-terminus of Sgo1 and regulates its association with centromeric chromatin. Cell Cycle 17:11-23
Ohkuni, Kentaro; Levy-Myers, Reuben; Warren, Jack et al. (2018) N-terminal Sumoylation of Centromeric Histone H3 Variant Cse4 Regulates Its Proteolysis To Prevent Mislocalization to Non-centromeric Chromatin. G3 (Bethesda) 8:1215-1223
Shrestha, Roshan L; Ahn, Grace S; Staples, Mae I et al. (2017) Mislocalization of centromeric histone H3 variant CENP-A contributes to chromosomal instability (CIN) in human cells. Oncotarget 8:46781-46800
Mishra, Prashant K; Ciftci-Yilmaz, Sultan; Reynolds, David et al. (2016) Polo kinase Cdc5 associates with centromeres to facilitate the removal of centromeric cohesin during mitosis. Mol Biol Cell 27:2286-300
Ohkuni, Kentaro; Takahashi, Yoshimitsu; Fulp, Alyona et al. (2016) SUMO-Targeted Ubiquitin Ligase (STUbL) Slx5 regulates proteolysis of centromeric histone H3 variant Cse4 and prevents its mislocalization to euchromatin. Mol Biol Cell :
Mishra, Prashant K; Guo, Jiasheng; Dittman, Lauren E et al. (2015) Pat1 protects centromere-specific histone H3 variant Cse4 from Psh1-mediated ubiquitination. Mol Biol Cell 26:2067-79
Ohkuni, Kentaro; Takahashi, Yoshimitsu; Basrai, Munira A (2015) Protein purification technique that allows detection of sumoylation and ubiquitination of budding yeast kinetochore proteins Ndc10 and Ndc80. J Vis Exp :e52482
Lai, Xianning; Beilharz, Traude; Au, Wei-Chun et al. (2013) Yeast hEST1A/B (SMG5/6)-Like Proteins Contribute to Environment-Sensing Adaptive Gene Expression Responses. G3 (Bethesda) 3:1649-59
Haase, Julian; Mishra, Prashant K; Stephens, Andrew et al. (2013) A 3D map of the yeast kinetochore reveals the presence of core and accessory centromere-specific histone. Curr Biol 23:1939-44
Mishra, Prashant K; Ottmann, Alicia R; Basrai, Munira A (2013) Structural integrity of centromeric chromatin and faithful chromosome segregation requires pat1. Genetics 195:369-79

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