Structural Biology Study On Mycobacterium Smegmatis Primase DnaG And The Synthesis Of C-di-GMP In Vitro

DnaG plays a significant role in the regulation of DNA replication in prokaryotes. Studies have shown that that there is an interaction between c-di-GMP and M. smegmatis primase DnaG. However, the specific binding sites,the interaction mechanism and how they involve in DNA replication remain unclear. Our study used M. smegmatis DnaG as material. This study started from the terms of the characterstics and structure of the protein to figure out the interaction mechanism of DnaG and c-di-GMP. The main results were listed as follows:1. Full-length and different structural domains were cloned. We constructed 19 prokaryotic expression plasimids, while only DnaG116-630, 121-460, 116-440, 461-636, 477-630, 121-630 these six fragments were successfully expressed and purified. Then we used these purified proteins to screen crystals.2. By the means of bioinformatics, limited protease digestion and mass spectrometry, we found out that 460-477 is disordered, connecting TOPRIM domain and a C-terminal helicase binding domain.3. After adding additives, we successfully improved 121-460 crystal’s diffraction resolution from 7 ? to 3.8 ?. However, the data is still not qualified, the structure could not be solved. The optimization needs to be continued.4. Expressed and purified Yde H-p ET-28 a. Synthesised c-di-GMP in vitro. Currently, with the optimization of the process, 1 mg c-di-GMP can be synthesized with 12 mg Yde H in 3 h, when GTP can all be turned into c-di-GMP5. Co-crystallization of DnaG121-460/c-di-GMP complex, crystals are gained. After optimization,the resolution was still poor, crystal optimization needs to be carried on.In this study, a large number of protein expression and crystallization had been done. We worked out a method for DnaG protein purification and crystallization, and identified the disorder which affect the stability of the crystals, laying a foundation for future work to solve DnaG three-dimensional structure. Comparing with extraction of c-di-GMP by HPLC, the application of ion exchange chromatography is an easy and rapid way to gain c-di-GMP with large amount. However, in order to obtain high-resolution crystals, the optimization of crystal conditions require further exploration. The three-dimensional structures of DnaG and DnaG/c-di-GMP complex need to be further investigated.