The centromeric nucleosome in yeast: Interaction with the inner kinetochore and assembly by the chaperone Yta7/ ATAD2

在有丝分裂过程中，遗传信息从母体细胞到子细胞的忠实传递要求每条染色体的姐妹染色单体被正确地分离到子细胞。为此，动粒在一侧附着于着丝粒染色质，在另一侧附着于微管，从而提供物理连接，允许染色单体在染色体分离期间被拉向细胞的相反两极。在上一个研究期间，我们研究了酿酒酵母中着丝粒核小体与内部动粒的连接。具体来说，我们确定了异二聚体Okp 1 CENP Q/Ame 1 CENP-U和酵母CENP-A同源物Cse 4的延伸氨基末端之间的物理相互作用，我们发现这种相互作用受到Cse 4 CENP-A N-末端上的翻译后修饰的调节。Okp 1 CENP Q/Ame 1 CENP-U是内动粒的CTF 19复合物的一部分，内动粒是人类CCAN的酵母等同物。此外，我们已经鉴定了组蛋白伴侣Yta 7 ATAD 2作为酵母中着丝粒Cse 4 CENP-A水平的新型调节剂。我们假设Yta 7是一种与解折叠酶具有同源性的含溴结构域的AAA+ ATP酶，其功能是解折叠Cse 4/ H4并将其交给CENP-A伴侣Scm 3 HJURP以掺入着丝粒核小体。目前的项目建议建立在这项工作的基础上，并获得Okp 1 CENP Q/Ame 1 CENP-U如何与Cse 4 N-末端相互作用的分子见解。具体而言，我们将使用接近的信息，使用双正交氨基酸和交联/质谱以及结构分析，以确定酵母的着丝粒核小体和Okp 1/Ame 1的内部动粒之间的相互作用的分子细节。这对于理解动粒与染色质的物理附着是如何实现的是很重要的。此外，我们将详细阐明Yta 7 ATAD 2如何调节着丝粒染色质的机制。我们将描述其与Cse 4/ H4和Cse 4伴侣Scm 3的相互作用及其在染色质组装中的作用，使用生化和结构方法。我们将联合收割机与体内分析相结合，以确定Yta 7的功能相关残基和结构域。总而言之，这个项目将提供在着丝粒的调节机制的见解，这是很重要的，以了解在这个过程中的缺陷如何有助于染色体的错误分离。

The faithful transmission of genetic information from mother to daughter cells during mitosis requires that the sister chromatids of each chromosome are properly segregated to the daughter cells. For this, the kinetochore attaches on one side to the centromeric chromatin and on the other side to the microtubule and thus provides a physical link that allows the chromatids to be pulled towards opposite poles of the cell during chromosome segregation. In the last grant period, we have characterized the attachment of the inner kinetochore to the centromeric nucleosome in the yeast Saccharomyces cerevisiae. Specifically, we identified a physical interaction between the heterodimer Okp1 CENP Q/ Ame1 CENP-U and the extended amino-terminus of the yeast CENP-A homolog Cse4, and we found this interaction to be modulated by posttranslational modifications on the Cse4 CENP-A N-terminus. Okp1 CENP Q/ Ame1 CENP-U is part of the CTF19 complex of the inner kinetochore, the yeast equivalent of human CCAN. Furthermore, we have identified the histone chaperone Yta7 ATAD2 as a novel regulator of centromeric Cse4 CENP-A levels in yeast. We postulate that Yta7, a bromodomain-containing AAA+ ATPase with homology to unfoldases, functions to unfold Cse4/ H4 and to hand it to the CENP-A chaperone Scm3 HJURP for incorporation into the centromeric nucleosome. The current project proposes to build on this work and to obtain molecular insights into how Okp1 CENP Q/ Ame1 CENP-U interacts with the Cse4 N-terminus. Specifically, we will use proximity information obtained using biorthogonal amino acids and cross-linking/ mass spectrometry as well as structural analysis to determine the molecular details of the interaction between the centromeric nucleosome of yeast and Okp1/ Ame1 of the inner kinetochore. This is important in order to understand how physical attachment of the kinetochore to chromatin is achieved. Furthermore, we will elucidate in mechanistic detail how Yta7 ATAD2 regulates centromeric chromatin. We will characterize its interaction with Cse4/ H4 and the Cse4 chaperone Scm3 and its role in chromatin assembly using biochemical and structural approaches. We will combine this with an in vivo analysis to determine functionally relevant residues and domains of Yta7. Altogether, this project will provide mechanistic insights into regulation at the centromere that are important to understand how defects in this process contribute to chromosome missegregation.