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[1]刘文林,罗昊,罗爽,等.结核分枝杆菌DNA复制解旋酶与引物酶的相互作用[J].应用与环境生物学报,2019,25(06):1486-1491.[doi:10.19675/j.cnki.1006-687x.2019.03043]
 LIU Wenlin,,et al.Interactions between DnaB helicase with DnaG primase in Mycobacterium tuberculosis[J].Chinese Journal of Applied & Environmental Biology,2019,25(06):1486-1491.[doi:10.19675/j.cnki.1006-687x.2019.03043]
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结核分枝杆菌DNA复制解旋酶与引物酶的相互作用
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《应用与环境生物学报》[ISSN:1006-687X/CN:51-1482/Q]

卷:
25卷
期数:
2019年06期
页码:
1486-1491
栏目:
研究论文
出版日期:
2019-12-30

文章信息/Info

Title:
Interactions between DnaB helicase with DnaG primase in Mycobacterium tuberculosis
作者:
刘文林罗昊罗爽王刚刚
1中国科学院成都生物研究所中国科学院环境与应用微生物重点实验室 成都 610041 2中国科学院成都生物研究所环境微生物四川省重点实验室 成都 610041 3中国科学院大学 北京 100049
Author(s):
LIU Wenlin1 2 3 LUO Hao1 2 3 LUO Shuang1 2 3 & WANG Ganggang1 2**
1Key Laboratory of Environmental and Applied Microbiology of Chinese Academy of Sciences, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China 2Key Laboratory of Environmental Microbiology of Sichuan Province, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China 3University of Chinese Academy of Sciences, Beijing 100049, China
关键词:
DNA复制解旋酶引物酶ATP 酶活相互作用
Keywords:
DNA replication DnaB DnaG ATPase activity interaction
分类号:
Q936
DOI:
10.19675/j.cnki.1006-687x.2019.03043
摘要:
在细菌DNA复制过程中,DnaB解旋酶解开双链DNA为DNA聚合酶提供单链模板,DnaG引物酶合成引物支持后随链的合成,DnaB和DnaG紧密协调保证了DNA复制得以精确完成. 目前对结核分枝杆菌(Mycobacterium tuberculosis,Mtb)染色体复制机制仍然知之甚少,有关结核分枝杆菌DnaB和DnaG相互作用的研究还非常有限. 重组表达了Mtb解旋酶DnaB和引物酶C端结构域(MtbP16),经过亲和层析、离子交换层析和凝胶过滤层析制备得到高纯度目的蛋白. 孔雀石绿定磷法检测MtbDnaB ATP水解活性表明,MtbDnaB六聚体状态下ATP水解活性较单体时提高5.4倍;当加入MtbP16,可以使MtbDnaB的ATP水解活性提高15%. 利用凝胶迁移实验(Electrophoretic mobility shift assays,EMSA)分别检测MtbDnaB与ssDNA、MtbDnaB与MtbP1相互作用,发现可以形成稳定的MtbDnaB/ssDNA和MtbDnaB/MtbP16复合物. 进一步研究发现,当将MtbP16蛋白加入到MtbDnaB/ssDNA中,随着MtbP16的增加,并未检测到MtbDnaB/ssDNA/MtbP16三元复合条带,反而导致游离ssDNA逐渐增多. 上述结果表明,MtbDnaB可以与MtbP16形成稳定的二元复合物,MtbP16的结合可以增强MtbDnaB 的ATPase 活性,未来可进一步以MtbDnaB/MtbP16复合物为靶标,通过定磷法测定MtbDnaB/MtbP16 复合物ATPase酶活进行抑制剂筛选. (图6 表2 参23)
Abstract:
During 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. This coupling between helicase and primase ensures that DNA replication can be accurately accomplished. So far, the known DNA replication mechanism of Mycobacterium tuberculosis (Mtb) is still poorly understood. Only a few studies exist on the interaction between DnaB and DnaG in M. tuberculosis. The Mtb DnaB helicase and the primase C-terminal domain (P16) were expressed optimal light and proteins were purified by affinity chromatography, ion-exchange chromatography, and gel filtration chromatography. The ATPase activity of Mtb DnaB helicase was measured using the malachite green phosphorus method, and the binding ability of the Mtb DnaB to ssDNAA and MtbP16 was measured using electrophoretic mobility shift assay (EMSA). The ATP hydrolysis activity of the hexameric Mtb DnaB was 5.4 times higher than that of the monomer. When the Mtb P16 was added, the ATPase activity of Mtb DnaB increased by 15%. The results of the EMSA showed that stable Mtb DnaB/ssDNA and Mtb DnaB/Mtb P16 complexes were formed. However, when Mtb P16 was added to the Mtb DnaB/ssDNA complex sample, the Mtb DnaB/ssDNA/Mtb P16 ternary complex was not detected with the increase in the quantity of Mtb P16, but the quantity of free ssDNA gradually increased. These results show that it is possible to prepare a stable Mtb DnaB/Mtb P16 complex that could be prepared and the binding of Mtb P16 stimulates the ATPase activity of Mtb DnaB. In the future, the Mtb DnaB/Mtb P16 complex could be a promising target for screening inhibitors.

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更新日期/Last Update: 2019-12-25