细菌引物酶识别特异序列DNA模板的机制研究

Primase is an essential enzyme that synthesizes RNA primers by DNA polymerases during DNA replication. The synthesis of primer by DNA primase occurs continuously on the lagging strand to provide primers for DNA polymerase to initiate the synthesis of Okazaki fragments. Primase requires sequence-specific DNA template to initiate synthesis of RNA primers rather than a random site, however, it is still a mystery how the enzyme recognizing sequence-specific DNA template to initiate synthesis of primers. In our previous work, the structure of the RNA polymerase domain from Bacillus subtilis has been determined. Based on the structural and biochemical data, a model for the sequence-specific DNA binding to primase was proposed. In this study, the structures of primase from Bacillus subtilis (BsuDnaG) will be investigated by crystallography and small angle X-ray scattering, including the BsuDnaG/sequence-specific DNA template and BsuDnaG/sequence-specific DNA template/NTPs complexs. Furthermore, structure-based function will be studied by biochemical assay. The results will show light on the mechanism of sequence-specific DNA template recognizing by DnaG primase in Bacteria, and provide theoretical basis for the development of novel anti-bacteria medicine.

引物酶是DNA复制的关键酶，在DNA复制过程中为DNA聚合酶合成RNA引物。DNA复制中后随链的合成是分段进行，需要引物酶不断合成RNA引物，为DNA聚合酶合成冈崎片段提供引发末端。引物酶在特异序列模板处启动引物合成而不是随机的，但是引物酶识别特异序列DNA模板的机制还不甚清楚。在前期的工作中，我们解析了枯草芽孢杆菌（Bacillus subtilis）引物酶（BsuDnaG）RNA聚合酶结构域的晶体结构，根据结构与功能性质实验，我们提出了特异序列DNA模板结合引物酶的模型。在本研究中，我们将综合X射线晶体衍射和小角散射法研究引物酶BsuDnaG/特异序列DNA模板，BsuDnaG/特异序列DNA模板/三磷酸核苷（NTPs）的晶体及溶液结构，结合功能性质实验，揭示细菌引物酶识别特异序列DNA模板的机制，为筛选引物酶抑制剂及新型抗菌药物的开发奠定基础。

引物酶是DNA复制的关键酶，在DNA复制过程中为DNA聚合酶合成RNA引物。DNA复制中后随链的合成是分段进行，需要引物酶不断合成RNA引物，为DNA聚合酶合成冈崎片段提供引发末端。引物酶在特异序列模板处启动引物合成而不是随机的，但是引物酶识别特异序列DNA模板的机制还不甚清楚。在本项目研究中，我们系统研究了结核分枝杆菌引物酶RBD/ZBD（P49）结构域与含不同识别位点的ssDNA结合能力以及HBD（P16）结构域与解旋酶DnaB及ssDNA的相互作用。本项目的研究结果加深了我们对结核分枝杆菌DNA复制系统的认识，为筛选引物酶抑制剂及新型抗菌药物的开发奠定基础。