Untersuchung der Funktion von SMC Proteinen bei der Chromosomensegregation und DNA Reparatur

SMC（染色体结构维持）蛋白在从细菌到人类的所有细胞的各种基本染色体动力学中起着关键作用。我们正在研究与ScpA和ScpB蛋白复合物中起作用的原核SMC蛋白。SMC复合体对染色体的压实和分离至关重要，它定位于类核（包含染色体）上的两个不同的位置，每个细胞一半一个。我们的数据表明，SMC复合体在亚细胞中心局部凝聚新复制的DNA，在那里它似乎形成了玫瑰状的超结构。纯化的SMC以环状结构与DNA结合，但尚不清楚它是如何凝聚DNA的。我们还发现SMC复合物在遗传水平上与拓扑异构酶IV相互作用，并对全局蛋白质合成产生强烈影响。我们将使用DNA阵列来研究这种效应是否基于SMC对转录的影响。我们将使用各种生化技术来阐明SMC蛋白的确切DNA结合模式和功能。蛋白质结构域将与与ssDNA而不是与dsDNA结合的RecN SMC蛋白交换，以阐明不同的结合模式。先进的荧光显微镜技术将被用来发现亚细胞中心是如何形成的，细胞SMC是如何和在哪里装载到DNA上的，以及它何时与ScpA和ScpB相互作用。

SMC (structural maintenance of chromosomes) proteins are key players in a variety of essential chromosome dynamics in all cells from bacteria to man. We are studying the prokaryotic SMC protein that acts in a complex with ScpA and ScpB proteins. The SMC complex is essential for chromosome compaction and segregation, and localizes to two distinct sites on the nucleoid (containing the chromosome), one in each cell half. Our data suggest that the SMC complex locally condenses newly replicated DNA in the subcellular centres, where it appears to form rosette-like super structures. Purified SMC binds to DNA as a ring like structure, but it is unclear how it condenses DNA. We have also found that the SMC complex interacts with topoisomerase IV at a genetic level, and has a strong influence on global protein synthesis. We will use DNA arrays to investigate if this effect is based on an effect of SMC on transcription. We will use various biochemical techniques to elucidate the exact mode of DNA binding and function for SMC protein. Protein domains will be swapped with the RecN SMC protein that binds to ssDNA rather than to dsDNA, to elucidate the different binding modes. Advanced fluorescence microscopy techniques will be employed to find out how the subcellular centres are formed, how and where in the cell SMC is loaded onto DNA, and when it interacts with ScpA and ScpB.