Analysis of DNA repair in bacteria in real time and at the single molecule level

染色体的完整性是由所有细胞中非常大但保存良好的蛋白质组来保证的。由于无法修复DNA损伤或过度反应对细胞存活有害，因此了解DNA途径的详细机制对于了解生命非常重要。最近，在单分子水平上真实的时间内研究蛋白质的相互作用已经成为可能。我们将利用单分子追踪技术（SMT）来扩展和深化我们对枯草芽孢杆菌DNA修复的认识。我们将研究交换的两个复制的DNA聚合酶的translesion聚合酶，招聘的修复蛋白复制叉，和修复蛋白在不同的双链断裂的动力学，使用诱导型内切酶系统。一个重要的步骤将采取可视化的蛋白质在个别，停滞的复制叉，使用诱导型DNA路障。我们将利用我们收集的功能性荧光蛋白融合的主要DNA修复蛋白参与修复的同源重组，和一个大的突变体收集，并扩展我们的研究复制重启酶和同源重组蛋白在个别网站或复制叉或DNA断裂位点在最高可能的时空分辨率。

The integrity of chromosomes is ensured by a very large yet well conserved set of proteins in all cells. As inability to repair DNA damage or an overshooting response are detrimental to cell survival, knowledge on the detailed mechanisms of DNA pathways is important to understand life. Recently, it has become possible to study the interplay of proteins in real time, at a single molecule level. We will employ the new technology of single molecule tracking (SMT) to extend and deepen our knowledge on DNA repair in the model bacterium Bacillus subtilis. We will study the exchange of the two replicative DNA polymerases by translesion polymerases, the recruitment of repair proteins to replication forks, and the dynamics of repair proteins at distinct double strand breaks, using an inducible endonuclease system. An essential step forward will be taken by visualization of proteins at individual, stalled replication forks, using an inducible DNA roadblock. We will take advantage on our collection of functional fluorescent protein fusions of major DNA repair proteins involved in repair by homologous recombination, and a large mutant collection, and extend our study of replication restart enzymes and homologous recombination proteins at individual sites or replication forks or DNA breaks sites at highest possible spatio-temporal resolution.