Eisenstatt Jessica R, Boeckmann Lars, Au Wei-Chun, Garcia Valerie, Bursch Levi, Ocampo Josefina, Costanzo Michael, Weinreich Michael, Sclafani Robert A, Baryshnikova Anastasia, Myers Chad L, Boone Charles, Clark David J, Baker Richard, Basrai Munira A
Genetics Branch, Center for Cancer Research, National Cancer Institute.
Division of Developmental Biology, Eunice Kennedy Shriver National Institute for Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20894.
G3 (Bethesda). 2020 Jun 1;10(6):2057-2068. doi: 10.1534/g3.120.401131.
The evolutionarily conserved centromeric histone H3 variant (Cse4 in budding yeast, CENP-A in humans) is essential for faithful chromosome segregation. Mislocalization of CENP-A to non-centromeric chromatin contributes to chromosomal instability (CIN) in yeast, fly, and human cells and CENP-A is highly expressed and mislocalized in cancers. Defining mechanisms that prevent mislocalization of CENP-A is an area of active investigation. Ubiquitin-mediated proteolysis of overexpressed Cse4 () by E3 ubiquitin ligases such as Psh1 prevents mislocalization of Cse4, and strains display synthetic dosage lethality (SDL) with We previously performed a genome-wide screen and identified five alleles of and that encode the Dbf4-dependent kinase (DDK) complex, which regulates DNA replication initiation, among the top twelve hits that displayed SDL with We determined that strains exhibit defects in ubiquitin-mediated proteolysis of Cse4 and show mislocalization of Cse4 Mutation of () bypasses the requirement of Cdc7 for replication initiation and rescues replication defects in a strain. We determined that does not rescue the SDL and defects in proteolysis of in a strain, suggesting a DNA replication-independent role for Cdc7 in Cse4 proteolysis. The SDL phenotype, defects in ubiquitin-mediated proteolysis, and the mislocalization pattern of Cse4 in a strain were similar to that of and strains, suggesting that Cdc7 regulates Cse4 in a pathway that overlaps with Psh1 Our results define a DNA replication initiation-independent role of DDK as a regulator of Psh1-mediated proteolysis of Cse4 to prevent mislocalization of Cse4.
进化上保守的着丝粒组蛋白H3变体（芽殖酵母中的Cse4，人类中的CENP - A）对于准确的染色体分离至关重要。CENP - A在非着丝粒染色质上的错误定位会导致酵母、果蝇和人类细胞中的染色体不稳定（CIN），并且CENP - A在癌症中高度表达且定位错误。确定防止CENP - A错误定位的机制是一个活跃的研究领域。E3泛素连接酶如Psh1对过表达的Cse4进行泛素介导的蛋白水解可防止Cse4的错误定位，并且 菌株与 显示出合成剂量致死性（SDL）。我们之前进行了全基因组筛选，并在与 显示SDL的前十二个命中结果中鉴定出五个 的等位基因以及编码Dbf4依赖性激酶（DDK）复合物的基因，该复合物调节DNA复制起始。我们确定 菌株在Cse4的泛素介导的蛋白水解中表现出缺陷，并显示出Cse4的错误定位。 （ ）的突变绕过了Cdc7对复制起始的需求，并挽救了 菌株中的复制缺陷。我们确定 在 菌株中不能挽救SDL和 蛋白水解的缺陷，这表明Cdc7在Cse4蛋白水解中具有独立于DNA复制的作用。 菌株中的SDL表型、泛素介导的蛋白水解缺陷以及Cse4的错误定位模式与 和 菌株相似，表明Cdc7在与Psh1重叠的途径中调节Cse4。我们的结果确定了DDK作为Cse4的Psh1介导的蛋白水解的调节剂具有独立于DNA复制起始的作用，以防止Cse4的错误定位。
