结核分枝杆菌DnaG引物酶与ssDNA模板相互作用的研究

Studies on The Interaction Between DnaG Primase and ssDNA Template in Mycobacterium tuberculosis

目的结核分枝杆菌(Mycobacterium tuberculosis)引物酶DnaG(MtuDnaG)在其基因组DNA复制中起着至关重要的作用,因而被认为是抗结核药物研发的新靶点。然而MtuDnaG起始引物合成的机制尚不清楚,这阻碍了MtuDnaG抑制剂的筛选。本研究将鉴定MtuDnaG结合模板的特异性识别位点,探讨MtuDnaG与ssDNA模板之间的相互作用。方法本研究以MtuDnaG的双结构域蛋白MtuP49(包含了锌指结合结构域和RNA聚合酶结构域)为研究对象,利用生物化学和生物物理学方法,研究MtuDnaG与含不同三联体的ssDNA模板之间的相互作用,鉴定MtuDnaG的特异性识别位点。结果5'-GCG/C-3'三联体可能是MtuDnaG结合模板ssDNA的特异性识别位点。此外,在结核分枝杆菌基因组的复制起始点附近也存在可以与MtuP49特异结合的5'-GCG/C-3'位点,其3'端侧翼序列显著影响ssDNA与MtuP49的亲和力。突变实验表明,位于锌指结合结构域中的Arg31对MtuP49结合ssDNA的活性具有重要贡献。基于预测的MtuP49结构,推测在MtuP49结合模板ssDNA过程中,锌指结合结构域会发生分子内重排。结论本研究首次鉴定了MtuDnaG结合模板ssDNA的特异性识别位点,揭示了影响MtuDnaG与模板ssDNA相互作用的主要因素。本文的研究结果不但有助于阐明MtuDnaG起始引物合成的机制,也为靶向DnaG的新型抗结核药物开发提供了新的信息。

Objective DnaG primase in Mycobacterium tuberculosis(MtuDnaG)plays a vital role in DNA replication,making it a target for novel antituberculosis drug discovery.However,the mechanism of MtuDnaG priming is not fully understood,which hinders the screening of MtuDnaG inhibitors.In this work,the specific recognition sites(SRS)in ssDNA for MtuDnaG binding was investigated and the interactions between MtuDnaG and ssDNA template was discussed.Methods By biochemical and biophysical methods,the binding of the didomain of MtuDnaG(MtuP49,containing the zinc-binding domain and RNA polymerase domain)to ssDNA template with various trinucleotide sites was evaluated,the affinity of MtuP49 to ssDNA template was measured.Results The present study suggested the 5'-GCG/C-3'as the potential SRS in ssDNA for specific binding to MtuDnaG.Besides,5'-GCG/C-3'sites were further identified within the oriC region of M.tuberculosis genome.Importantly,the 3'sequence flanking the 5'-GCG/C-3'site markedly affected the binding affinity of ssDNA to MtuP49.Mutagenesis studies showed that substitution of residue Arg31 in the zinc-binding domain affected the binding activity of MtuP49 to template ssDNA.Combined with the predicted structure of MtuP49,an intramolecular rearrangement of zinc-binding domain relative to the RNA polymerase domain was implied to be essential in the binding of MtuP49 to template ssDNA.Conclusion This study firstly identified the SRS in ssDNA for MtuDnaG binding,the key factors affecting MtuDnaG binding to ssDNA was revealed.The above results provide evidence to shed light on the mechanism of MtuDnaG priming,and pave the way for development of novel DnaG-targeted antituberculosis drugs.