The Interactions Between DNA Replication Initiators In Mycobacterium Tuberculosis

In the pathogenic Mycobacterium tuberculosis H37Rv, the specific regulation mechanisms for DNA replication are largely unknown. Therefore, the studies on the interactions between its replication initiators will facilitate understanding the pathogenic DNA replication mechanisms and uncovering potential drug targets.In this study, based on the screening a library and the characterization of the interactive proteins with two critical replicative proteins MtbDnaB and MtbDnaE2, we further studied the interactions in detail among the MtbDnaA, MtbDnaB and DNA replication origin (OriC). Four major results were obtained as follows: (1) By bacterial two-hybird technique, we primarily constructed a protein-protein interaction network involved in MtbDnaB and MtbDnaE2. (2) We have characterized carefully the interactive domains between MtbDnaA and MtbDnaB by bacterial two-hybird assay in combination with Pull-down and SPR techniques. (3) Further surface plasmon resonance (SPR) and electrophoretic mobility shift assays (EMSA), indicated that the N-terminus of DnaB could inhibit the binding activity for MtbDnaA on entire MtbOriC, but could not directly influence MtbDnaA binding local DnaA boxes. (4) Our result showed that there exists a physical interaction between MtbDnaE2 and MtbDnaN.Thus, here we for the first time studied the interactions between DNA replication initiators in M. tuberculosis, and preliminarily analyzed the protein interaction between MtbDnaN and MtbDnaE2. All these results will greatly contribute to a further understanding on the DNA replication and infection mechanisms of M. tuberculosis, and will also provide important clues for searching new durg targets.