Characterization of physical interaction between replication initiator protein DnaA and replicative helicase from Mycobacterium tuberculosis H37Rv.
In the pathogenic Mycobacterium tuberculosis H37Rv, the causative agent of tuberculosis, the genetic and biochemical mechanisms for initiation of DNA replication are largely unknown. In the present study, we have characterized the physical interactions between M. tuberculosis DnaA and DnaB using both in vivo methods, such as bacterial two-hybrid assays, and in vitro techniques, such as surface plasmon resonance (SPR) and Pull-down/Western blotting. The full-length N-terminus (1-206 residues) of DnaB was found to interact with DnaA, while the shorter N-terminal domain of DnaB (1-125 residues), which lacked the linker region, did not. Further SPR and electrophoretic mobility shift assays indicated that the N-terminus (1-206 residues) of DnaB also had a critical role in regulating DnaA complex formation at the origin of replication (OriC). This regulatory effect was not obviously observed for DNA substrates containing only two DnaA-boxes. This is the first report showing a physical interaction between DnaA and replicative helicase DnaB from M. tuberculosis and the role in subsequent DnaA-OriC interactions. The findings reported here further the understanding of the regulatory mechanisms for initiation of DNA replication in this important human pathogen.
Xie
Yunchang
Y
National Key Laboratory of Agricultural Microbiology, Center for Proteomics Research, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, 430070, China.
He
Zheng-Guo
ZG
D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism*; D053488:DnaB Helicases / Q000378:metabolism*; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D018741:Replication Origin / Q000235:genetics; D020349:Surface Plasmon Resonance; D020798:Two-Hybrid System Techniques
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D011994:Recombinant Proteins; D053488:DnaB Helicases