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

目前的建议的目的是获得结构和功能的洞察COMA组件与Cse 4和/或着丝粒核小体的协会。由于Okp 1/Ame 1变体与Cse 4-N的结合增强，分离稳定的COMA-Cse 4复合物或亚复合物将可能使结构分析更加可行。通过X射线晶体学和/或冷冻EM分析获得的结构数据将提供对复合物内蛋白质的接触和化学计量的临界点的机械见解。鉴定Cse 4-甲基转移酶和分离新的抑制剂，拯救由于Cse 4-R37 Me缺失而导致的缺陷，有可能为我们提供染色质功能和动粒结构的全面图片。我一直着迷于染色质因子，DNA序列，DNA结构和DNA/蛋白质上的修饰具有调节基因表达并最终决定细胞命运的潜力。我博士研究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，但主要集中在研究染色体交易的监管方面。柏林洪堡大学Ann Escherhofer-Murray教授实验室的重点领域之一是通过分析组蛋白及其变体上的表观遗传修饰来了解染色质组装和功能的分子细节。主机实验室使用遗传，生物化学和结构的方法来研究翻译后修饰在染色质组装中的作用。Ann Escherhofer-Murray教授为我提供了一个探索酿酒酵母中动粒和着丝粒蛋白的功能和调节的机会，并有助于了解动粒调节及其对着丝粒生物学的影响。拟议的项目将生物化学/结构研究和遗传分析很好地结合在一起。这是一个很好的机会，让我把我的蛋白质生物化学知识，以使用。与此同时，它将使我发展我的技术技能和知识酵母遗传学。实验室良好的科学基础设施和充满活力的研究环境将帮助我培养良好的科学态度。我相信，这个项目有力量塑造我的学术生涯作为一个独立的研究人员。

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.