Studies on The Interaction Between DnaG Primase and ssDNA Template in Mycobacterium tuberculosis
10.16476/j.pibb.2023.0445
- VernacularTitle:结核分枝杆菌DnaG引物酶与ssDNA模板相互作用的研究
- Author:
Jiang CHEN
1
;
Hao LUO
1
;
Zhi-Ming ZHANG
1
;
Xu SONG
2
;
Gang-Gang WANG
1
Author Information
1. Key Laboratory of Environmental and Applied Microbiology, Key Laboratory of Environmental Microbiology of Sichuan Province, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China
2. College of Life Sciences, Sichuan University, Chengdu 610064, China
- Publication Type:Journal Article
- Keywords:
DnaG primase;
Mycobacterium tuberculosis;
specific recognition site;
domain rearrangement;
DNA replication
- From:
Progress in Biochemistry and Biophysics
2024;51(8):1920-1934
- CountryChina
- Language:English
-
Abstract:
ObjectiveDnaG 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. MethodsBy 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. ResultsThe 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. ConclusionThis 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.
