Role of Ddk Kinase in Regulation of DNA Replication

Ddk 激酶在 DNA 复制调节中的作用

• 批准号: 6910763
• 负责人: WEI JIANG
• 金额: $ 31.04万
• 依托单位: SANFORD BURNHAM PREBYS MEDICAL DISCOVERY INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6910763
• 关键词: DNA replication; DNA replication origin; HeLa cells; Xenopus; adenosinetriphosphatase; conformation; enzyme activity; enzyme substrate; eukaryote; gene duplication; genome; green fluorescent proteins; helicase; immunofluorescence technique; phosphorylation; protein kinase; protein structure function; western blottings

DESCRIPTION (provided by applicant): Our long-term goal is to understand the molecular mechanisms by which the initiation of DNA replication is regulated in higher eukaryotes. In particular, we will study the molecular mechanisms by which the S-phase-promoting kinase, Ddk (Dbf4-dependent kinase Cdc7), regulates and executes the initiation of DNA replication in mammalian cells. Previously, we identified human Ddk complex HsCdc7/HsDbf4 and showed that HsCdc7/HsDbf4 plays an essential role in DNA replication in mammalian cells. We demonstrated that HsCdc7/HsDbf4 selectively phosphorylates the MCM2 subunit of chromatin-associated MOM complex, a component of pre-replication complex (pre-RC) and the putative DNA replicative helicase that is required for the initiation of DNA replication. These results strongly suggest that HsCdc7/HsDbf4 may be directly involved in regulating the initiation of DNA replication by phosphorylating chromatin/replication origin-associated proteins, such as MCM2, that orchestrate the initiation of DNA replication in mammalian cells. In this proposal, we will determine how HsCdc7/HsDbf4 targets its downstream substrates to regulate the initiation of DNA replication. We will examine whether HsCdc7/HsDbf4 phosphorylation of MCM2 affects the formation or conformation of MCM heteromeric complexes and regulates the helicase activity of MCM complex(es). We will determine whether phosphorylation of MCM2 by HsCdc7/HsDbf4 regulates its chromatin association and controls the initiation of DNA replication. Since the initiation of DNA replication is highly regulated in eukaryotic cells and many proteins are involved in this complex process, it is unlikely that MCM2 protein is the only downstream target of I-lsCdc7/HsDbf4. To further understand how HsCdc7/HsDbf4 kinase controls DNA replication, we will identify novel HsCdc7/HsDbf4 substrates by an in vitro phosphorylation screen. These studies will shed more light on how this S-phase promoting kinase regulates DNA replication process. The studies will also lead to a better understanding of the fundamental biological processes of genome duplication and maintenance of its integrity, which are still enigmatic in higher eukaryotes, despite their importance for cell growth, cell cycle control and carcinogenesis.

描述(申请人提供)：我们的长期目标是了解分子 高等真核生物中DNA复制启动的调控机制。特别是，我们将研究S时相促进激酶DDK(DBF4依赖的激酶CDC7)调节和执行哺乳动物细胞中DNA复制启动的分子机制。此前，我们鉴定了人DDK复合体HsCDc7/HsDbf4，并证明HsCDc7/HsDbf4在哺乳动物细胞的DNA复制中起着重要作用。我们证明了HsCdc7/HsDbf4选择性地磷酸化染色质相关MOM复合体的MCM2亚单位，复制前复合体(Pre-RC)的一个组成部分，以及启动DNA复制所需的假定的DNA复制解旋酶。这些结果有力地表明，HsCDc7/HsDbf4可能通过磷酸化染色质/复制起源相关蛋白，如MCM2，直接参与调控DNA复制的启动。在这个提案中，我们将确定HsCDc7/HsDbf4如何针对其下游底物来调控DNA复制的启动。我们将研究MCM2的HsCdc7/HsDbf4磷酸化是否影响MCM异构体复合体的形成或构象，并调节MCM复合体(ES)的解旋酶活性。我们将确定HsCdc7/HsDbf4对MCM2的磷酸化 调节其染色质结合并控制DNA复制的启动。由于DNA复制的启动在真核细胞中受到高度调控，许多蛋白质参与了这一复杂的过程，因此MCM2蛋白不太可能是I-lsCDc7/HsDbf4的唯一下游靶点。为了进一步了解HsCDc7/HsDbf4激酶是如何控制DNA复制的，我们将通过体外磷酸化筛选来鉴定新的HsCDc7/HsDbf4底物。这些研究将进一步阐明这种S促进蛋白激酶是如何调控DNA复制过程的。这些研究还将有助于更好地理解基因组复制和维持其完整性的基本生物学过程，尽管它们对细胞生长、细胞周期控制和癌症发生具有重要意义，但在高等真核生物中仍是谜。