Mobility of murine replication proteins in the nucleus

我们的长期目标是阐明在G1/S-相转变时DNA复制开始的机制。该项目将集中在组装和激活的前复制复合物的过程中的启动蛋白的动力学。我们将利用光漂白技术研究活细胞核中单个起始蛋白的动力学性质。编码绿色荧光蛋白（GFP）与起始蛋白（例如ORC 1 -6、MCM 2 -7、CDC 6、CDC 45、DBF 4、CDC 7、PLK、CDT 1和双生蛋白）的融合物的载体将用于测量复制蛋白的动力学。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）。将通过RNA干扰研究敲除单个蛋白质的表达对复制前复合物的组装和定位的影响。最后，翻译后修饰（磷酸化，乙酰化）的启动蛋白的复制机制的结构和功能动力学的影响将进行调查。

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.