Analysis of DNA replication restart in bacteria at the single molecule level

一组非常大且保存良好的蛋白质致力于确保所有细胞中染色体的完整性。无法修复DNA损伤或无法重新启动受阻的复制叉会损害细胞的生存，因此了解细胞如何组织不同修复途径的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。我们将使用单分子跟踪(SMT)的新技术来研究复制叉处理模式细菌枯草杆菌DNA损伤的方式。我们将研究两种可供选择的跨损伤聚合酶对这两种复制DNA聚合酶的交换，以及它们在复制分叉外的可能功能。我们还将调查已知修复蛋白在复制分叉上的招募情况。向前迈出的重要一步是，使用可诱导和可逆的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主要DNA修复蛋白作为功能性荧光蛋白融合蛋白和大量突变蛋白，并以尽可能高的时空分辨率扩展我们对复制重新启动酶和单个位置或复制叉处的同源重组蛋白的研究。

A very large and well conserved set of proteins is dedicated to ensure chromosomes integrity in all cells. Inability to repair DNA damage or fail to restart blocked replication forks are detrimental to cell survival, so understanding the mechanisms of how cells organize different repair 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 study the way replication forks deal with DNA damage in the model bacterium Bacillus subtilis. We will study the exchange of the two replicative DNA polymerases by the two alternative translesion polymerases, as well as their putative function outside of replication forks. We will also investigate the recruitment of known repair proteins to replication forks. An essential step forward will be taken by visualization of proteins at individual, stalled replication forks, using an inducible and reversible DNA roadblock. We will take advantage of our collection of major DNA repair proteins as functional fluorescent protein fusions, and a large mutant collection, and extend our study of replication restart enzymes and homologous recombination proteins at individual sites or replication forks at highest possible spatio-temporal resolution.