Structural and functional analysis of the kinetochore – chromatin interface in Saccharomyces cerevisiae

• Applicant: Dr. Rucha Kshirsagar, Ph.D.
• Subject Area: General Genetics and Functional Genome Biology ; Cell Biology
• Term: from 2018 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 411741701

The aim of the current proposal is to gain structural and functional insights into the association of the COMA components with Cse4 and/or the centromeric nucleosome. Isolation of stable COMA-Cse4 complex or sub complexes due to the enhanced binding of Okp1/ Ame1 variants to Cse4-N will potentially make structural analysis more feasible. The structural data obtained by X-ray crystallography and/or cryo-EM analysis will provide mechanistic insights into the critical points of contact and stoichiometry of proteins within the complex. Identification of the Cse4- methyltransferase and isolation of novel suppressors that rescue the defects due to the loss of Cse4-R37Me have the potential to provide us with a comprehensive picture of chromatin function and kinetochore architecture.I have always been fascinated by the idea that chromatin factors, DNA sequence, DNA structure and modifications on DNA/proteins have the potential to modulate gene expression and ultimately determine cell fate. A significant part of my doctoral work was based on understanding the mechanisms of meiotic chromosome pairing by analyzing in vitro DNA-protein and protein-protein interactions. Over the time, I have developed an interest in yeast genetics, protein biochemistry, and imaging. As a post-doctoral researcher, I wish to continue working in the field of chromosome biology, but with a major focus on studying regulatory aspects ofchromosome transactions. I want to gain proficiency in yeast genetics and further strengthen my skills in protein biochemistry.One of the focus areas of Prof. Ann Ehrenhofer-Murray’s laboratory at Humboldt University of Berlin is to understand the molecular details of chromatin assembly and function by analysing epigenetic modifications on histones and its variants. The host laboratory uses genetic, biochemical, and structural approaches to study the role of post-translational modifications in chromatin assembly. Prof. Ann Ehrenhofer-Murray has offered me an opportunity to explore the function and regulation of kinetochore and centromere proteins in Saccharomyces cerevisiae and contribute to the understanding of kinetochore regulation and its impact on centromerebiology. The proposed project has a good mix of biochemical/structural studies and genetic analyses. It is an excellent opportunity for me to put my knowledge on protein biochemistry to use. At the same time, it will allow me to develop my technical skill setand knowledge on yeast genetics. The excellent scientific infrastructure and the dynamic research environment in the lab will help me develop good scientific attitude. I believe that this project has the strength to shape my academic career as an independent researcher.

• DFG Programme: Research Grants