细菌基因组上DNA磷硫酰化修饰的丰度和序列特异性

DNA phosphorothioations are an epigenetic modification firstly discovered on the DNA backbone, which are widespread in many species of bacteria with stereo-specificity and diverse sequence contexts. To detect the distribution of phosphorothioate modifications across the Escherichia coli B7A genome, two new complementary technologies: single molecule, real-time (SMRT) sequencing and deep sequencing of iodine-induced cleavage at phosphorothioation (ICDS) have been developed, and showed that phosphorothioation was detected mainly in GpsAAC/GpsTTC sequences on both strands in B7A. The sequence diversity, abundance and the distribution of DNA phosphorothioate modification in several different bacterial genomes will be investigated by using both SMRT and ICDS sequencing. The molecular mechanism of partial modification of consensus sequence GAAC/GTTC in E.coli B7A, and the sequence specificity of phosphorothioation consensus sites will be elucidated. The goal of this project is to develop and apply novel sequencing technologies to localize phosphorothioate modifications in bacterial genomes as part of a multifaceted effort to define the biochemical mechanism and the biological function of phosphorothioate modifications.

DNA磷硫酰化修饰是首次在DNA大分子骨架上发现的表观遗传修饰。该修饰广泛存在于细菌中并具有序列和手性特异性。为了检测大肠杆菌Escherichia coli B7A基因组水平上DNA磷硫酰化修饰位点，发展了单分子实时测序（single molecule real-time sequencing, SMRT）和碘切割依赖的深度测序（deep sequencing of iodine-induced cleavage,ICDS)两种技术。B7A基因组磷硫酰化修饰主要发生在部分的双链的GpsAAC/GpsTTC序列上。本项目在此基础上，拟测定多个细菌基因组磷硫酰化修饰位点，深入开展磷硫酰化修饰的序列多样性和在基因组中的分布研究，解析B7A基因组中GAAC/GTTC位点部分修饰的分子机制，阐明DNA磷硫酰化修饰的序列特征。为揭示DNA磷硫酰化修饰的生物化学机制和生物学功能奠定基础。

DNA磷硫酰化一种DNA骨架非桥联氧原子被硫原子取代的一种新型DNA修饰，该修饰广泛存在于细菌中并且具有序列选择性和R-型手性特异性，但这种新型表观遗传学修饰在细菌基因组上的分布规律以及修饰的丰度并不清楚。本研究发展了定量检测基因组上DNA磷硫酰化修饰位点的PT-IC-seq检测方法，这种方法可以用于全基因组水平上定量研究磷硫酰化修饰位点的修饰频率。PT-IC-seq分析结果表明，大肠杆菌B7A中大多数磷硫酰化修饰位点都是保持着较低（<5%）的修饰频率，并且它们都表现出类似于甲基化修饰的异质性特征。该研究清楚地证明了磷硫酰化修饰在细菌群体中的异质性的特征，为磷硫酰化修饰在整个基因组水平的精确定位和鉴定提供了技术方法。在沙门氏菌Cerro 87中，DNA磷硫酰化修饰是由dptBCDE的4个基因介导的。本研究发现在沙门氏菌Cerro 87中DNA磷硫酰化基因dptB-E作为一个操作子共转录，敲除了dptB基因导致细胞内磷硫酰化修饰水平增强，转录分析显示DptB抑制了dptBCDE的表达，表明dptB在转录水平上负调控DNA磷硫酰化修饰基因的转录。同时，通过定位DptB在DNA磷硫酰化修饰基因上游调控区的2个DNA结合区域，发现每个靶序列中都含有1对保守的正向重复序列。本研究也利用构建的沙门氏菌全细胞裂解液催化体系，以及dptCDE基因敲除突变株细胞裂解液添加纯化的DptCDE反应体系，在体外再现了DNA磷硫酰化修饰反应。发现DNA磷硫酰化修饰蛋白形成复合体而发挥作用，为阐明DNA磷硫酰化修饰的生物化学过程建立了重要基础。研究还发现来自鸭疫里默士杆菌的DNA磷硫酰化修饰蛋白DndEi具有ATP依赖的解旋酶活性，并且是磷硫酰化修饰是必需的。同时，发现DndEi具有一种ATP酶活性，这种活性被具有GAAC/GTTC基序的DNA底物激活。这些研究不仅拓展了对DNA磷硫酰化修饰分子机制的理解，也为阐明DNA磷硫酰化修饰的生理功能建立了基础。