An additional proofreader contributes to DNA replication fidelity in mycobacteria

• 成果类型: Article
• 署名作者: Deng, Ming-Zhi; Liu, Qingyun; Cui, Shu-Jun; Wang, Yi-Xin; Zhu, Guoliang; Fu, Han; Gan, Mingyu; Xu, Yuan - Yuan; Cai, Xia; Wang, Sheng; Sha, Wei; Zhao, Guo-Ping; Fortune, Sarah M.; Lyu, Liang-Dong
• 署名单位: Fudan University; Harvard University; Harvard T.H. Chan School of Public Health; Fudan University; Chinese Academy of Sciences; Center for Excellence in Molecular Plant Sciences, CAS; Chinese Academy of Sciences; University of Chinese Academy of Sciences, CAS; Fudan University; University of North Carolina; University of North Carolina Chapel Hill; University of North Carolina School of Medicine
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-8590
• DOI: 10.1073/pnas.2322938121
• 发表日期: 2024-08-20
• 关键词: escherichia-coli; polymerase-iii; epsilon-subunit; spontaneous mutations; fluctuation analysis; exonuclease subunit; efficient method; tuberculosis; repair; roles

摘要

The removal of mis- incorporated nucleotides by proofreading activity ensures DNA replication fidelity. Whereas the 8- 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 DnaQhomolog 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 beta 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.