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Interactions between DnaB helicase with DnaG primase from Mycobacterium tuberculosis*(PDF)

Chinese Journal of Applied & Environmental Biology[ISSN:1006-687X/CN:51-1482/Q]

Issue:
2019 06
Page:
1-13
Research Field:
Articles
Publishing date:

Info

Title:
Interactions between DnaB helicase with DnaG primase from Mycobacterium tuberculosis*
Author(s):
LIU Wenlin1 2 3 LUO Hao1 2 3 LUO Shuang1 2 3 & WANG Ganggang 1 2**
1 Key Laboratory of Environmental and Applied Microbiology of Chinese Academy of Sciences, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China
2 Key Laboratory of Environmental Microbiology of Sichuan Province, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China
3 University of Chinese Academy of Sciences, Beijing 100049, China
Keywords:
DNA replication DnaB DnaG ATPase activity interaction
CLC:
-
PACS:
DOI:
10.19675/j.cnki.1006-687x.2019.03043
DocumentCode:

Abstract:
In bacterial DNA replication, the replicative ring helicase ( DnaB) unwinds duplex DNA into single-stranded DNA (ssDNA), while DnaG primase produces short RNA primers on the lagging strand that form the initiation site for the replicative DNA polymerase. The coupling between helicase and primase ensures that DNA replication can be accurately accomplished. So far, the DNA replication mechanism of Mycobacterium tuberculosis ( Mtb) is still poorly understood. Only few studies on the interaction between DnaB and DnaG havebeen performed in M. tuberculosis. The Mtb DnaB helicase and the primase C-terminal domain (P16) were expressed, the proteins were purified by affinity chromatography, ion exchange chromatography and gel filtration chromatography. The ATPase activity of MtbDnaB helicase was measured by the malachite green phosphorus method, and the binding ability of the MtbDnaB to ssDNAA and MtbP16 were detected by EMSA assay. The ATP hydrolysis activity of hexamer MtbDnaB was 5.4 times higher than that of monomer; when the MtbP16 was added, the ATPase activity of MtbDnaB was increased by 15%. The results of EMSA showed that the stable MtbDnaB/ssDNA and MtbDnaB/MtbP16 complexes were formed; however, when MtbP16 was added into MtbDnaB/ssDNA complex sample, the MtbDnaB/ssDNA/MtbP16 ternary complex was not detected with the increase of MtbP16 amount, but the free ssDNA was gradually increased. These results show that stable MtbDnaB/MtbP16 complex could be prepared and the binding of MtbP16 stimulates the ATPase activity of MtbDnaB. In the future, the MtbDnaB/MtbP16 complex could be promising target for inhibitor screening.

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Last Update: 2019-04-12