Mycobacterial MazG safeguards genetic stability via housecleaning of 5-OH-dCTP.
Mycobacterial MazG safeguards genetic stability via housecleaning of 5-OH-dCTP.
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DOI：
10.1371/journal.ppat.1003814
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发表时间：
2013
期刊：
影响因子：
6.7
通讯作者：
Zhao GP
中科院分区：
文献类型：
--
作者：
Lyu LD;Tang BK;Fan XY;Ma H;Zhao GP
在吞噬细胞内产生活性氧和活性氮是消灭微生物病原体的重要先天免疫反应机制。众所周知，脱氧核苷酸（dNTPs）是DNA合成的前体核苷酸，是这些氧化剂的重要靶标之一，将氧化的dNTPs纳入基因组DNA可能导致突变甚至细胞死亡。在这里，我们表明分枝杆菌dNTP焦磷酸水解酶MazG通过降解5-OH-dCTP来保护杆菌基因组，从而阻止其并入DNA。分枝杆菌中编码(d)NTP焦磷酸水解酶的mazG的缺失导致在氧化应激和生长固定阶段的突变表型，导致CG到TA突变增加。生化分析表明，分枝杆菌MazG可以有效水解5-OH-dCTP，这是一种氧化核苷酸，在聚合酶结合后诱导CG向TA突变。此外，化学遗传学分析表明，将5-OH-dCTP直接掺入到MazG缺失突变株（Msm）中会导致剂量依赖性突变表型，这表明5-OH-dCTP是分枝杆菌MazG的天然底物。此外，结核分枝杆菌（Mtb）中mazG的缺失导致活化巨噬细胞和感染小鼠脾脏的存活率降低。本研究不仅将分枝杆菌MazG描述为一种新型嘧啶特异性内室清洁酶，通过降解5-OH-dCTP阻止CG向TA突变，而且揭示了Mtb在体内存活的基因组保护机制。细胞核苷酸库是活性氧和活性氮氧化的重要靶点。已知将这些氧化的非规范核苷酸错误地掺入DNA会引起突变，并可能与致癌、衰老和神经变性有关。细胞已经进化出一组生物降解的内室清洁酶，这些酶可以特异性地从核苷酸库中清除某些非规范核苷酸，从而防止它们与DNA结合。最具特征的内室清洁酶是特异性水解嘌呤核苷酸的mutt样蛋白，如8-oxo-dGTP和2-OH-dATP。细胞中缺乏MutT活性导致AT-CG突变显著增加和遗传不稳定。然而，对氧化嘧啶核苷酸特异性的室内清洁酶尚未被确定。在这里，我们发现来自分枝杆菌的dNTP焦磷酸水解酶MazG是一种5- oh - dctp特异性的内室清洁酶。分枝杆菌中mazG的缺失导致氧化应激和生长平稳期CG到TA突变增加。生化和化学遗传分析表明，5-OH-dCTP是分枝杆菌MazG的天然底物。此外，Mtb中mazG的缺失导致活化巨噬细胞和感染小鼠脾脏的存活率降低。这些结果揭示了分枝杆菌维持遗传稳定性和在体内存活的一种新的清洁途径。
Generation of reactive oxygen species and reactive nitrogen species in phagocytes is an important innate immune response mechanism to eliminate microbial pathogens. It is known that deoxynucleotides (dNTPs), the precursor nucleotides to DNA synthesis, are one group of the significant targets for these oxidants and incorporation of oxidized dNTPs into genomic DNA may cause mutations and even cell death. Here we show that the mycobacterial dNTP pyrophosphohydrolase MazG safeguards the bacilli genome by degrading 5-OH-dCTP, thereby, preventing it from incorporation into DNA. Deletion of the (d)NTP pyrophosphohydrolase-encoding mazG in mycobacteria leads to a mutator phenotype both under oxidative stress and in the stationary phase of growth, resulting in increased CG to TA mutations. Biochemical analyses demonstrate that mycobacterial MazG can efficiently hydrolyze 5-OH-dCTP, an oxidized nucleotide that induces CG to TA mutation upon incorporation by polymerase. Moreover, chemical genetic analyses show that direct incorporation of 5-OH-dCTP into mazG-null mutant strain of Mycobacterium smegmatis (Msm) leads to a dose-dependent mutagenesis phenotype, indicating that 5-OH-dCTP is a natural substrate of mycobacterial MazG. Furthermore, deletion of mazG in Mycobacterium tuberculosis (Mtb) leads to reduced survival in activated macrophages and in the spleen of infected mice. This study not only characterizes the mycobacterial MazG as a novel pyrimidine-specific housecleaning enzyme that prevents CG to TA mutation by degrading 5-OH-dCTP but also reveals a genome-safeguarding mechanism for survival of Mtb in vivo. The cellular nucleotide pool is a significant target for oxidation by reactive oxygen species and reactive nitrogen species. Misincorporation of these oxidized non-canonical nucleotides into DNA is known to cause mutations, and may be related to carcinogenesis, aging and neurodegeneration. Cells have evolved a group of bio-degradation housecleaning enzymes that may specifically eliminate certain non-canonical nucleotide from the nucleotide pool and thus prevent their incorporation into DNA. The most well-characterized housecleaning enzymes are the MutT-like proteins which specifically hydrolyze the oxidized purine nucleotides, such as 8-oxo-dGTP and 2-OH-dATP. Lack of MutT activity in cells leads to significant increase of AT-CG mutation and genetic instability. However, housecleaning enzymes specific for oxidized pyrimidine nucleotides are yet to be identified. Here we show that the dNTP pyrophosphohydrolase MazG from mycobacteria is a 5-OH-dCTP-specific housecleaning enzyme. Deletion of mazG in mycobacteria results in increased CG to TA mutation under oxidative stress and in the stationary phase of growth. Both biochemical and chemical genetic analyses demonstrate that 5-OH-dCTP is a natural substrate of mycobacterial MazG. Furthermore, deletion of mazG in Mtb leads to reduced survival in activated macrophages and in the spleen of infected mice. These results reveal a novel housecleaning pathway for mycobacteria to maintain genetic stability and survival in vivo.
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DOI：
10.1084/jem.175.4.1111
发表时间：
1992-04-01
期刊：
The Journal of experimental medicine
影响因子：
--
作者：
Chan J;Xing Y;Magliozzo RS;Bloom BR
通讯作者：
Bloom BR
DOI：
10.1073/pnas.1003219107
发表时间：
2010-07-06
影响因子：
11.1
作者：
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通讯作者：
McKinney, John D.
影响因子：
4.8
作者：
Fujikawa, K;Kamiya, H;Kasai, H
通讯作者：
Kasai, H
影响因子：
64.5
作者：
Boshoff, HIM;Reed, MB;Mizrahi, V
通讯作者：
Mizrahi, V
影响因子：
56.9
作者：
Darwin, KH;Ehrt, S;Nathan, CF
通讯作者：
Nathan, CF