细菌DNA复制叉解链和引物合成的偶联机制研究

In DNA replication, unwinding of dsDNA by hexameric helicase advances the motion of replication fork, periodically, primers are synthesized by primase on the lagging strand. So far, it is still a mystery how the replication machinery couples dsDNA unwinding to synthesis of the primer in bacteria. The Bacillus subtilis bacteriophage SSP1 helicase, G40P, is a homology of bacterial replicative helicase, it shares 35% and 45% sequence identity with the counterparts from E. coli and B. subtilis, respectively. The G40P helicase functions by interplaying with DnaG primase. Here, we will investigate the crystal structures of G40P/DnaG and G40P/DnaG/ssDNA complexes. Furthermore, biochemical assays will be performed to testify the results that are revealed by the structures. We believe the structural and functional studies here will show light on the mechanism of the coupling of the motion of replication fork to primer synthesis. Potentially, these data could be applied into the pharmaceutical industry for the development of novel anti-bacteria medicine.

在DNA复制叉形成过程中，解旋酶六聚体在复制起点附近装配，将双链模板解开，引物酶以解旋酶提供的单链为模板合成引物, 支持后随链的复制。解旋酶与引物酶的协同作用保证了复制叉解链和引物合成协调衔接，先导链和后随链复制同步完成。然而, 复制叉解链和引物合成的偶联机制仍然不清楚。在本研究项目中，将研究引物酶DnaG全长蛋白、解旋酶G40P/引物酶DnaG全长蛋白复合物、G40P/DnaG/ssDNA三元复合物晶体结构，并与G40P的结构进行比较，揭示G40P/DnaG/ssDNA相互作用诱导发生的构象变化以及G40P/DnaG/ssDNA相互作用的结构基础；基于晶体结构研究的结果，设计关键残基突变体，开展功能研究,进一步探讨晶体结构研究揭示的结果，综合分析结构与功能研究结果，阐明复制叉解链和引物合成的偶联机制。