细菌DNA复制解旋酶水解ATP和解开dsDNA的偶联机制研究

The replicative helicase is essential enzyme for DNA replication in bacteria, which hydrolyzes ATP and unwinds duplex DNA at the replication fork. To date, extensive studies have been performed on a few replicative helicases, including the helicase domains of SV40 large T antigen, papillomavirus E1 helicase and T7 gp4 helicase. All these efforts lead to variable models in term of ATP hydrolysis, which implies the diversity in the mechanism for the replicative helicase. Evidence from previous studies indicates that the eukaryotic and prokaryotic helicases may use distinct DNA unwinding mechanisms for replication, which may be a reflection of the differences in the replication machineries in the two systems. .So far, it is still a mystery how the replicative helicase couples ATP hydrolysis and DNA unwinding 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. Here, we will investigate the crystal structure on the complexes of G40P/ATP、G40P/ADP and G40P/ssDNA. 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 that G40P helicase unwinds duplex DNA by consuming the ATP. Potentially, these results could be applied into the pharmaceutical industry for the development of novel anti-bacteria medicine.

细菌解旋酶在DNA复制过程中发挥重要作用，尽管有关细菌DNA复制机理研究的文献已经很多，但细菌解旋酶水解ATP和解开dsDNA的偶联机制仍然不清楚。枯草杆菌噬菌体SPP1解旋酶G40P蛋白具有复制相关解旋酶的典型特征和性质，与细菌解旋酶氨基酸序列同源性（Sequence Identity）较高，其与大肠杆菌和枯草杆菌解旋酶的氨基酸序列同源性分别为35%和45%。 本项目将研究解旋酶G40P/ATP、G40P/ADP和G40P/ssDNA系列复合物的晶体结构，并与解旋酶G40P的结构进行比较，揭示ATP结合、水解诱导G40P发生的构象变化以及G40P/DNA相互作用的分子机理；在结构研究的基础上，将设计G40P突变体，通过生物化学实验进一步验证晶体结构研究揭示的结果，进而综合结构与功能研究数据，阐明G40P水解ATP和解开dsDNA的偶联机制。

细菌DnaB解旋酶和引物酶在DNA复制过中发挥重要作用，在DNA复制起始过程中, DnaB解旋酶与引物酶DnaG协同作用, 完成后随链的复制，但到目前为止，引物酶DnaG在复制叉处如何识别特异起始位点还不清楚。 我们表达了枯草芽胞杆菌引物酶并进行结晶，解析了引物酶RNA聚合酶结构域(RNAP)的结构，结构比较表明，在缺失ZBD结构域时，Loop1反时针旋转约25度，而Loop1与DNA结合相关, 凝胶阻滞试验证明了ZBD结构域对引物酶识别特异序列模板具有重要作用. 生化实验结果表明引物酶突变体体W167A, R202A，R204A， R148A, R224A, K232A，N235A结合含特异识别位点ssDNA的能力不同程度降低, 根据以上结果，我们基本可以确定特异ssDNA序列的结合表面。基于本研究的晶体结构数据和生化实验结果，结合文献报道结果, 我们提出了引物酶RPD/ssDNA/ATP复合物结构模型。这些研究结果对于我们认识细菌DNA复制机制有重要意义