DNA磷硫酰化修饰的的基因组分布及修饰蛋白复合物的结构与功能

DNA phosphorothioation (PT) is a epigenetic modification for decades in which a non-bridging oxygen in the DNA phosphate backbone is replaced with sulfur. However, the biochemistry mechanism, physiological function and genomic distribution of this novel epigenetic modification remains poorly defined and needs futher studies. We demonstrated that PT participated a restriction-modification system of which the restriction mechanism was distict with the traditional ones and that dnd genes confer the host strain the resistance to hydrogen peroxide. Further insights into PT biology is our recent observation of genomic distribution of PT sites in Escherichia coli B7A and Vibrio cyclitrophicus FF75 that only a small fraction of unusually short consensus sequences are modified, with no further strict consensus sequences beyond the modification motifs in both cases. These observations raise many questions about the novel epigentic modification of PT. The phenomenon of partial PT modification in short consensus sequences, especially in the face of restriction system, suggests a regulation of the frequency of PT modification and site selection by PT-modifying enzymes. We have developed and applied iodine-induced cleavage deep sequencing (ICDS) for genomic mapping of PT sites in bacterial. More bacterial genomic PT sites will be demonstrated by using this method to find the law of distribution which could provide insights of its physiolgical function of PT. Our recent study reavealed that three of the five PT-modifying enzymes formed a large protein complex, with all of subunits in tetrameric conformations, suggesting a complex pathway of PT biosynthesis. Our efforts are made on the construction of in vitro DNA PT modification by the PT-modifying protein complex to investigate the mechanism of PT biosythesis and regulation. DNA methylation is one of the most well established epigenetic modification, we also investigate its effects on DNA PT modification. By using combination of small angle X-ray, neutron scattering, cryo-EM and X-ray crystallography, we will determine the three-dimensional structure of phosphorothioate modifying protein complex. Based on our recent observation of high abundance of PT modification in microbiome that play an important role in colitis, we are going to investigate the physiological function of PT modifications in microbiome. These above studies will lead to further understanding of PT biology.

DNA磷硫酰化修饰是近年发现的硫原子取代DNA骨架磷原子上非桥联氧原子形成一种新型表观遗传修饰。该修饰广泛存在于细菌中并具有序列和手性特异性，但这种新型修饰的过程和分布规律及生理功能等基本科学问题仍然不清楚。本项目在发现细菌基因组磷硫酰化修饰位点具有部分修饰特征基础上，对DNA磷硫酰化修饰的生物学进行深入研究。解析修饰蛋白复合物的三维空间结构，研究生物体内部分修饰的机制。揭示去磷硫酰化和磷硫酰化控制基因组整体修饰水平的调控机制。以建立的ICDS测序技术，解析大肠杆菌B7A基因组DNA磷硫酰化修饰位点的时空分布。以构建的体外催化体系，揭示甲基化修饰和DNA磷硫酰化修饰的相互关系。通过分析DNA磷硫酰化修饰在肠道菌群中的分布，研究磷硫酰化修饰肠道菌的致病作用。预期将对DNA磷硫酰化修饰的过程做出系统阐述，并获得对其生物学功能更深入的理解，丰富对这一新型表观遗传学修饰领域重要科学问题的基本认识。

DNA磷硫酰化(PT)修饰是近年发现的硫原子取代DNA骨架磷原子上非桥联氧原子形成一种新型修饰。该修饰广泛存在于细菌中并具有序列和手性特异性，但这种新型修饰的生化过程、基因组分布规律及生理功能等基本科学问题仍然不清楚。本研究中，我们建立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，PT-IC-seq分析结果表明细菌中大多数PT修饰位点具有较低的修饰频率，并且它们都表现出类似于甲基化修饰的异质性特征。基于微滴式数字PCR技术，我们也开发了PT-IC-ddPCR方法，进一步印证了这一重要发现。该研究对揭示修饰的过程和修饰的生理功能具有重要的指导意义。利用磷硫酰化修饰DNA抵抗核酸酶剪切的特点，发展了普适性的检测DNA修饰和损伤的单碱基分辨率的Nick-seq技术，实现了基因组DNA上的多种化学修饰全覆盖、高准确率、单碱基分辨率的定位和定量分析，为相关表观遗传调控机理、DNA损伤修复、癌症发生机理、人工干预表观遗传修饰等多方面的研究提供了通用技术平台。运用同位素标记和LC-MS分析, 我们发现在无氧化压力胁迫的大肠杆菌和沙门氏菌中PT上硫原子的置换速率明显低于次氯酸处理下PT置换速率，而在无氧化压力胁迫以及过氧化氢或次氯酸氧化压力下PT修饰的总量未发生改变。这些研究结果表明PT是一种动态且不稳定的DNA修饰，导致在氧化胁迫下基因组的不稳定。我们调查了PT修饰基因在人体微生物组中的分布，发现超过2000株含PT修饰基因的细菌分布于包括肠道的身体不同部位。在肠道微生物DNA中发现了6种新型PT修饰二核苷，证明了PT修饰基因进化的多样性与修饰类型紧密相关。这些研究表明PT修饰广泛分布于人体微生物组中，为调查人体微生物组中PT修饰的生物学功能奠定了基础。通过体内遗传和体外生化分析，证明了假单胞菌中转录调控因子SpfB负调控PT修饰，获得了高纯度的修饰蛋白复合物，利用小角X射线和冷冻电镜研究了修饰蛋白复合物的三维结构，为全面解析PT修饰的生化过程和生物学功能奠定了重要基础。