Dbf4-Dependent Kinase (DDK)-Mediated Proteolysis of CENP-A Prevents Mislocalization of CENP-A in Saccharomyces cerevisiae. Dbf4-Dependent Kinase (DDK)-Mediated Proteolysis of CENP-A Prevents Mislocalization of CENP-A in Saccharomyces cerevisiae

Restricting the localization of the centromeric histone H3 variant CENP-A to centromeres is essential to prevent chromosomal instability (CIN). Mislocalization of overexpressed CENP-A contributes to CIN in yeast, fly, and human cells. CENP-A is overexpressed in many cancers. Therefore, defining mechanisms that prevent CENP-A mislocalization will help us understand how CENP-A overexpression contributes to CIN in cancer. A genome-wide screen to characterize essential genes required for growth when CENP-A is overexpressed identified the replication initiation Dbf4-Dependent Kinase (DDK) complex. We show that DDK regulates ubiquitin-mediated proteolysis of Cse4 and prevents mislocalization of Cse4 independently of its role in DNA replication. Overall design: Cse4 ChIP-seq was carried out in wild-type and cdc7-7 yeast strains overexpressing epitope-tagged Cse4 or tagged Cse4 expressed from the endogenous promoter. Samples of input and immunoprecipitated DNA were analyzed.

将着丝粒组蛋白H3变体CENP-A（centromeric histone H3 variant CENP-A）的定位限定于着丝粒区域，是防止染色体不稳定性（chromosomal instability, CIN）的必要前提。过表达的CENP-A发生错误定位，会在酵母、果蝇与人类细胞中诱发染色体不稳定性。CENP-A在多种癌症中呈现过表达状态。因此，阐明抑制CENP-A错误定位的分子机制，将有助于我们理解CENP-A过表达如何在癌症中促成染色体不稳定性。本研究通过全基因组筛选，在CENP-A过表达的条件下鉴定生长必需的功能基因，最终发现了复制起始Dbf4依赖性激酶（replication initiation Dbf4-dependent kinase, DDK）复合体。我们的研究证实，DDK可通过调控Cse4的泛素介导蛋白水解过程，且不依赖其在DNA复制中的功能，来阻止Cse4的错误定位。实验整体设计：针对过表达表位标记Cse4，或由内源启动子表达的标签化Cse4的野生型与cdc7-7酵母菌株，开展了Cse4染色质免疫共沉淀测序（ChIP-seq）实验，并对输入样本与免疫沉淀得到的DNA进行了分析。