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

当前提案的目的是获得对Coma组分与Cse4和/或着丝粒核小体的关联的结构和功能的洞察。由于Okp1/Ame1变异体与Cse4-N结合增强，分离出稳定的ComA-Cse4复合体或亚复合体将有可能使结构分析变得更加可行。通过X射线结晶学和/或低温电子显微镜分析获得的结构数据将提供对复合体中蛋白质的接触点和化学计量比的机械洞察。Cse4-甲基转移酶的鉴定和修复Cse4-R37Me缺陷的新型抑制子的分离为我们提供了染色质功能和动粒结构的全面图景。我一直对染色质因子、DNA序列、DNA结构和DNA/蛋白质的修饰具有调节基因表达并最终决定细胞命运的想法很感兴趣。我博士工作的一个重要部分是通过分析体外DNA-蛋白质和蛋白质-蛋白质相互作用来理解减数分裂染色体配对的机制。随着时间的推移，我对酵母遗传学、蛋白质生物化学和成像产生了兴趣。作为一名博士后研究员，我希望继续在染色体生物学领域工作，但主要专注于研究染色体交易的调控方面。我想精通酵母遗传学，并进一步加强我在蛋白质生物化学方面的技能。柏林洪堡大学Ann Ehrenhofer-Murray教授的实验室的重点领域之一是通过分析组蛋白及其变体的表观遗传修饰来了解染色质组装和功能的分子细节。主办实验室使用遗传、生化和结构方法来研究染色质组装中翻译后修饰的作用。Ann Ehrenhofer-Murray教授为我提供了一个探索酿酒酵母着丝粒和着丝粒蛋白的功能和调控的机会，有助于理解着丝粒调控及其对着丝粒生物学的影响。拟议的项目很好地结合了生化/结构研究和遗传分析。这对我来说是一个很好的机会，可以把我在蛋白质生物化学方面的知识用上。同时，它将使我发展我的技术技能和酵母遗传学的知识。实验室里优良的科学基础设施和充满活力的研究环境将帮助我养成良好的科学态度。我相信这个项目有力量塑造我作为一名独立研究员的学术生涯。

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.