Mobility of murine replication proteins in the nucleus

我们的长期目标是阐明G1/ s期dna复制开始的机制。该项目将重点关注在复制前复合体的组装和激活过程中启动蛋白的动力学。我们将利用光漂白技术研究活细胞核中单个引发蛋白的动力学性质。载体编码绿色荧光蛋白（GFP）与启动蛋白融合，如ORC1-6、MCM2-7、CDC6、CDC45、DBF4、CDC7、PLK、CDT1和gemini蛋白，用于测量复制蛋白的动态。我们将采用FRAP（荧光恢复后光漂白）和FLIP（荧光损失在光漂白）。敲除单个蛋白的表达对复制前复合体的组装和定位的影响将通过rna干扰进行研究。最后，将研究引发蛋白的翻译后修饰（磷酸化、乙酰化）对复制机制的结构和功能动力学的影响。

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.