通过校对活性去除错误掺入的核苷酸确保了DNA复制的保真度。而ε-外切核酸酶DnaQ是模型生物大肠杆菌中公认的校对者,已经表明,大多数细菌的校对依赖于复制聚合酶的聚合酶和组织糖醇磷酸酶(PHP)域,尽管存在结构和功能上不同于大肠杆菌DnaQ的DnaQ同系物。然而,这种非规范DnaQ的生物学功能尚不清楚。这里,我们提供了独立证据,证明非规范DnaQ可作为分枝杆菌的额外校对者.使用突变累积测定与全基因组测序相结合,我们发现在耻垢分枝杆菌中DnaQ的缺失导致突变率增加,导致在均聚物道中AT偏倚的诱变和增加的插入/缺失。我们的结果表明,分枝杆菌DnaQ与β钳结合,并与PHP域校对器协同作用以纠正复制错误。此外,dnaQ的丢失导致复制叉功能障碍,可能由于对叉子塌陷的脆弱性增加,导致亚抑制性氟喹诺酮类药物的生长减弱和诱变增加。通过分析结核分枝杆菌(Mtb)临床分离株dnaQ的序列多态性,我们证明了在Mtb谱系4.3中普遍存在的一种自然进化的DnaQ变异体可能具有超突变性,并且与耐药性相关.这些结果建立了一个共同校对模型,并建议在DnaQ和PHP领域校对器之间进行分工。这项研究还提供了现实世界的证据,表明在Mtb的适应过程中可能存在突变驱动的进化途径。
The removal of mis-incorporated nucleotides by proofreading activity ensures DNA replication fidelity. Whereas the ε-exonuclease
DnaQ is a well-established proofreader in the model organism Escherichia coli, it has been shown that proofreading in a majority of bacteria relies on the polymerase and histidinol phosphatase (PHP) domain of replicative polymerase, despite the presence of a DnaQ homolog that is structurally and functionally distinct from E. coli
DnaQ. However, the biological functions of this type of noncanonical
DnaQ remain unclear. Here, we provide independent evidence that noncanonical
DnaQ functions as an additional proofreader for mycobacteria. Using the mutation accumulation assay in combination with whole-genome sequencing, we showed that depletion of DnaQ in Mycolicibacterium smegmatis leads to an increased mutation rate, resulting in AT-biased mutagenesis and increased insertions/deletions in the homopolymer tract. Our results showed that mycobacterial
DnaQ binds to the β clamp and functions synergistically with the PHP domain proofreader to correct replication errors. Furthermore, the loss of dnaQ results in replication fork dysfunction, leading to attenuated growth and increased mutagenesis on subinhibitory fluoroquinolones potentially due to increased vulnerability to fork collapse. By analyzing the sequence polymorphism of dnaQ in clinical isolates of Mycobacterium tuberculosis (Mtb), we demonstrated that a naturally evolved DnaQ variant prevalent in Mtb lineage 4.3 may enable hypermutability and is associated with drug resistance. These results establish a coproofreading model and suggest a division of labor between DnaQ and PHP domain proofreader. This study also provides real-world evidence that a mutator-driven evolutionary pathway may exist during the adaptation of Mtb.