Mobility of murine replication proteins in the nucleus

小鼠复制蛋白在细胞核中的迁移性

• 批准号: 5366286
• 负责人: Professor Dr. Friedrich Grummt (†)
• 金额: --
• 依托单位: Lehrstuhl für Biochemie I
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2002
• 资助国家: 德国
• 起止时间: 2001-12-31 至 2006-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/5366286?language=en
• 关键词: Mobility; murine; replication; proteins; nucleus

Our long-term aim is the elucidation of the mechanisms governing the onset of DNA-replication at the G1/S-phase transition. The project will be focused on the dynamics of the initiator proteins during the assembly and activation of the pre-replicative complex. We will study the kinetic properties of individual initiator proteins in the nuclei of living cells with photobleaching techniques. Vectors encoding fusions of green fluorescence protein (GFP) with initiator proteins, e.g. ORC1-6, MCM2-7, CDC6, CDC45, DBF4, CDC7, PLK, CDT1 and geminin proteins will be used to measure the dynamics of replication proteins. We will apply FRAP (Fluorescence Recovery After Photobleaching) and FLIP (Fluorescence Loss In Photobleaching). The effect of knocking-out the expression of individual proteins on the assembly and localization of the pre-replicative complex will be investigated by RNA-interference. Finally, the effect of posttranslational modifications (phosphorylation, acetylation) of initiator proteins on the structural and functional dynamics of the replication machinery will be investigated.

我们的长期目标是阐明在G1/S相变中控制DNA复制开始的机制。该项目将重点研究复制前复合体组装和激活过程中启动蛋白的动态变化。我们将用光漂白技术研究活细胞核中单个启动子蛋白的动力学特性。编码绿色荧光蛋白(GFP)与启动子蛋白ORC1-6、MCM2-7、CDC6、CDC45、DBF4、CDC7、PLK、CDT1和Gminin蛋白融合的载体将被用来测量复制蛋白的动态。我们将应用FRAP(光漂白后的荧光恢复)和FLIP(光漂白中的荧光损失)。通过RNA干扰研究敲除单个蛋白质表达对复制前复合体组装和定位的影响。最后，将研究启动蛋白的翻译后修饰(磷酸化、乙酰化)对复制机制结构和功能动力学的影响。