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一组非常大但保存良好的蛋白质来确保的。由于无法修复DNA损伤或过度反应对细胞生存不利，因此了解DNA通路的详细机制对于理解生命很重要。最近，在单分子水平上实时研究蛋白质之间的相互作用已经成为可能。我们将利用新的单分子跟踪(SMT)技术来扩展和深化我们对模式细菌枯草芽孢杆菌DNA修复的知识。我们将使用可诱导的核酸内切酶系统，研究跨损伤聚合酶对两种复制DNA聚合酶的交换，修复蛋白在复制叉处的招募，以及修复蛋白在不同双链断裂时的动态。向前迈出的重要一步是，使用可诱导的DNA路障，在单个停滞不前的复制叉子上可视化蛋白质。我们将利用我们收集的参与同源重组修复的主要DNA修复蛋白的功能性荧光蛋白融合和大量突变体，并以尽可能高的时空分辨率扩展我们对单个位点或复制分叉或DNA断裂位点的复制重新启动酶和同源重组蛋白的研究。

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.