Characterization of the role of serine phosphorylation on CenH3 function in Saccharomyces cerevisae

着丝粒是染色体上确定的区域，指导着丝粒的组装，确保在细胞分裂期间姐妹染色单体的正确分离。酿酒酵母的着丝粒DNA被包装在含有着丝粒组蛋白H3变体Cse4代替通常的组蛋白H3的单个核小体中。在此提议之前的工作中，我们已经确定了Cse4的一种新的翻译后修饰（PTM），即丝氨酸33的磷酸化。在这里，我们将确定这种修改的功能意义。为此，将对该位点突变为丙氨酸进行遗传分析，并确定其对细胞周期进程、动粒组装和染色体分离的影响。此外，将产生丝氨酸33磷酸化特异性抗体，并将确定修饰的游离和染色质结合的Cse4的存在。最后，这种抗体将用于寻找负责这种修饰的酶。这些对着丝粒组蛋白变体的新修饰的研究将允许重要的新见解通过PTM调节着丝粒功能，从而将开辟一条迄今为止尚未探索的新的研究途径。

Centromeres are defined regions of the chromosomes that direct the assembly of the kinetochores, which ensure proper segregation of sister chromatids during cell division. The centromeric DNA of Saccharomyces cerevisiae is packaged in a single nucleosome containing the centromeric histone H3 variant Cse4 in place of the usual histone H3. In work preceding this proposal, we have identified a novel posttranslational modification (PTM) of Cse4, the phosphorylation of serine 33. Here, we will determine the functional significance of this modification. To this end, genetic analysis of mutation of this site to alanine will be performed, and its effect on cell cycle progression, kinetochore assembly and chromosome segregation will be determined. Furthermore, a serine 33 phosphorylation-specific antibody will be generated, and the presence of modified free and chromatin-bound Cse4 will be determined. Finally, this antibody will be used to search for the enzyme responsible for this modification. These studies of a novel modification on the centromeric histone variant will allow important new insights into the regulation of centromere function by PTMs and thus will open up a new avenue of investigation that so far has not been explored.