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

在所有细胞中，有一组非常庞大且保守的蛋白质致力于确保染色体的完整性。无法修复DNA损伤或无法重新启动被阻断的复制分叉对细胞存活是有害的，因此了解细胞如何组织不同修复途径的机制对理解生命是很重要的。最近，在单分子水平上实时研究蛋白质的相互作用已经成为可能。我们将采用单分子跟踪（SMT）的新技术来研究复制叉处理模型细菌枯草芽孢杆菌DNA损伤的方式。我们将研究两种可选择的翻译聚合酶对两种复制DNA聚合酶的交换，以及它们在复制叉外的假定功能。我们还将研究已知的修复蛋白在复制叉上的募集。使用可诱导和可逆的DNA路障，在单个停滞的复制叉上可视化蛋白质，将是向前迈出的重要一步。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体，以及大量突变体，并以最高的时空分辨率扩展我们在单个位点或复制叉上的复制重启酶和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.