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[1]刘文林 罗昊 罗爽 王刚刚**.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):1-13.[doi:10.19675/j.cnki.1006-687x.2019.03043]
 LIU Wenlin,,et al.Interactions between DnaB helicase with DnaG primase from Mycobacterium tuberculosis*[J].Chinese Journal of Applied & Environmental Biology,2019,25(06):1-13.[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:
1-13
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 from Mycobacterium tuberculosis*
作者:
刘文林1;?2;?3 罗昊1;?2;?3 罗爽1;?2;?3 王刚刚1;?2**
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 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
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
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8 表2 参23)
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.

备注/Memo

备注/Memo:
收稿日期 Received: 2019-03-20 接受日期 Accepted: 2019-04-11
*国家自然科学基金项目(31470742, U1432102, 31700664, 31270783)和中国科学院百人计划项目资助
**通讯作者 Corresponding author (E-mail: wanggg@cib.ac.cn)
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更新日期/Last Update: 2019-04-12