Mycobacterial MazG Safeguards Genetic Stability via Housecleaning of 5-OH-dCTP

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.

吞噬细胞产生活性氧（reactive oxygen species）与活性氮（reactive nitrogen species）是清除病原微生物的重要固有免疫应答机制。已知脱氧核苷三磷酸（deoxynucleotides, dNTPs）作为DNA合成的前体核苷酸，是这类氧化剂的关键作用靶点之一；而氧化型dNTP掺入基因组DNA可引发突变甚至细胞死亡。本研究表明，分枝杆菌dNTP焦磷酸水解酶MazG可通过降解5-羟基脱氧胞苷三磷酸（5-OH-dCTP），阻止其掺入DNA，从而守护杆菌基因组的稳定性。在分枝杆菌中敲除编码（d）NTP焦磷酸水解酶的mazG基因后，无论是在氧化应激条件下还是生长静止期，菌株均会出现突变表型，表现为CG到TA的碱基突变频率升高。生化分析显示，分枝杆菌MazG可高效水解5-OH-dCTP——这是一种氧化型核苷酸，当其被聚合酶掺入DNA后可诱发CG到TA的突变。此外，化学遗传分析证实，向耻垢分枝杆菌（Mycobacterium smegmatis, Msm）的mazG缺失突变株中直接掺入5-OH-dCTP，会引发剂量依赖性的诱变表型，表明5-OH-dCTP是分枝杆菌MazG的天然底物。进一步研究发现，结核分枝杆菌（Mycobacterium tuberculosis, Mtb）的mazG基因缺失会导致其在活化巨噬细胞及感染小鼠脾脏中的存活能力显著下降。本研究不仅将分枝杆菌MazG鉴定为一种新型嘧啶特异性清洁酶，可通过降解5-OH-dCTP阻止CG到TA的突变，还揭示了结核分枝杆菌在宿主体内存活的基因组守护机制。