DNA聚合酶ε亚基在结核分枝杆菌耐药中的作用机制

The main shortage of TB chemotherapy is the requirement of lengthy treatment which is mainly attributed to the drug tolerance of Mycobacterium tuberculosis (Mtb). Recently, we found that protecting DNA from damage and the maintenance of genome integrity are essential for the survival of Mtb under antibiotics treatment (PNAS, Cover Paper). dCTP can be oxidized by reactive oxygen species which are induced by antibiotics. The oxidized dCTP is incorporated into DNA via error-prone DNA polymerase DnaE2 and then double strand breaks (DSB) are produced which are lethal to bacteria. It was suggested in that study that error-prone DNA polymerase is the key factor causing DNA damage and affecting drug tolerance phenotype. We found that the mutation in ε subunit of DNA polymerase Ⅲ leads to reduced antibiotics and oxidizing agent killing by 100 times compared with WT; this ε subunit has DNA exonuclease activity and excises ssDNA but not dsDNA. Therefore, a hypothesis was proposed: this ε subunit is involved in excision repair of oxidized DNA, DSB is produced in this repair process and results in cell death. In order to prove this hypothesis, the biochemical function of this ε subunit, the role of this ε subunit in maintaining genome integrity and drug tolerance in Mtb will be studied in this project. The results of this study will provide new insights about the mechanism that maintaining genome integrity under antibiotics treatment in Mtb.

结核分枝杆菌（Mtb）的药物耐受是结核病治疗过程漫长的重要原因。我们近期研究揭示保护DNA免受损伤、维护基因组稳定是Mtb耐受药物杀菌的关键机制（PNAS，封面文章）。被抗生素诱导产生的活性氧氧化的dCTP，在易错DNA聚合酶DnaE2作用下掺入DNA，进而产生对细菌致命的DNA双链断裂（DSB）。该研究揭示了易错DNA聚合酶是造成DNA损伤、影响药物耐受表型的主要因素。近期我们发现DNA聚合酶Ⅲ的ε亚基突变导致分枝杆菌对抗生素及氧化剂杀菌的敏感性降低100倍；该亚基具有核酸外切酶活性，能降解ssDNA，不能降解dsDNA。由此我们提出以下假说：该亚基参与氧化损伤DNA的切除修复，修复过程中产生的DSB是导致细菌死亡的原因。本项目拟研究ε亚基的生化功能、及其在Mtb基因组稳定性维护及药物耐受中的作用，以增进对药物作用下Mtb基因组稳定性维护机制的认知。

结核分枝杆菌 （Mycobacterium tuberculosis，Mtb）的药物耐受可产生抗药突变，是结核病疗程漫长的重要原因。DNA 聚合酶Ⅲ是高保真 DNA 聚合酶，具有错配校正功能。我们前期研究发现DNA 聚合酶Ⅲ的 ε 亚基可能与分枝杆菌的药物耐受有关。本项目以耻垢分枝杆菌 (Mycobacterium smegmatis) 为模式菌研究其DNA 聚合酶Ⅲ ε 亚基的功能。我们分别构建了DNA 聚合酶Ⅲ ε 亚基的敲除菌株（ΔdnaQ， ΔMs4259和ΔMs4259ΔdnaQ），通过检测ε亚基敲除菌株在自然条件及氧化压力条件下的突变频率、SOS应答相关基因（recA、dnaE2）的表达，阐明了DNA 聚合酶Ⅲ的ε亚基在维护分枝杆菌基因组稳定性中起着重要作用。通过检测抗结核药物对ε亚基突变株的MIC、杀菌曲线及平板药敏实验，阐明了ε亚基突变株对抗生素的耐受性降低。ε亚基缺失后导致了菌株突变频率增加，基因组稳定性降低，从而更易被抗生素杀死。ε亚基有望作为抗结核药物的靶点，以期缩短结核病治疗疗程。