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Proofreading01:31

Proofreading

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Synthesis of new DNA molecules is carried out by the enzyme DNA polymerase, which adds nucleotides on the daughter strand complementary to the template DNA strand. DNA polymerase has a higher affinity to add the correct base and ensures fidelity during DNA replication. Furthermore,  it exhibits proofreading activity during replication, using an exonuclease domain that cuts off incorrect nucleotides from the nascent DNA strand.
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Organisms are capable of detecting and fixing nucleotide mismatches that occur during DNA replication. This sophisticated process requires identifying the new strand and replacing the erroneous bases with correct nucleotides. Mismatch repair is coordinated by many proteins in both prokaryotes and eukaryotes.
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Base complementarity between the three base pairs of mRNA codon and the tRNA anticodon is not a failsafe mechanism. Inaccuracies can range from a single mismatch to no correct base pairing at all. The free energy difference between the correct and nearly correct base pairs can be as small as 3 kcal/ mol. With complementarity being the only proofreading step, the estimated error frequency would be one wrong amino acid in every 100 amino acids incorporated. However, error frequencies observed in...
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DNA replication has three main steps: initiation, elongation, and termination. Replication in prokaryotes begins when initiator proteins bind to the single origin of replication (ori) on the cell's circular chromosome. Replication then proceeds around the entire circle of the chromosome in each direction from the two replication forks, resulting in two DNA molecules.
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DNA replication is carried out by a large complex of proteins that act in a coordinated matter to achieve high-fidelity DNA replication. Together this complex is known as the DNA replication machinery or the replisome.
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Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis
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An additional proofreader contributes to DNA replication fidelity in mycobacteria.

Ming-Zhi Deng1, Qingyun Liu2, Shu-Jun Cui1,3

  • 1Key Laboratory of Medical Molecular Virology of the Ministry of Education/Ministry of Health, Department of Medical Microbiology and Parasitology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China.

Proceedings of the National Academy of Sciences of the United States of America
|August 14, 2024
PubMed
Summary

Noncanonical DnaQ acts as a second proofreader in mycobacteria, working with the PHP domain to ensure DNA replication fidelity. Its loss causes increased mutations and replication stress, potentially aiding adaptation in Mycobacterium tuberculosis.

Keywords:
DNA replication fidelityDnaQMycobacteriumdrug resistanceproofreading

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Genetics

Background:

  • DNA replication fidelity is crucial for genomic stability, maintained by proofreading mechanisms.
  • While Escherichia coli uses ε-exonuclease DnaQ, many bacteria employ the polymerase and histidinol phosphatase (PHP) domain for proofreading.
  • The role of distinct, noncanonical DnaQ homologs in bacterial proofreading remains largely uncharacterized.

Purpose of the Study:

  • To investigate the function of noncanonical DnaQ as a proofreader in mycobacteria.
  • To elucidate the interaction and synergistic action of DnaQ with the PHP domain during DNA replication.
  • To explore the implications of DnaQ function and variation in Mycobacterium tuberculosis (Mtb) adaptation and drug resistance.

Main Methods:

  • Mutation accumulation assays coupled with whole-genome sequencing in Mycolicibacterium smegmatis.
  • Analysis of DnaQ binding to the β clamp and its interaction with the PHP domain.
  • Sequence polymorphism analysis of dnaQ in clinical isolates of Mycobacterium tuberculosis.

Main Results:

  • Depletion of DnaQ in M. smegmatis significantly increased mutation rates, exhibiting AT-biased mutagenesis and elevated insertions/deletions in homopolymer tracts.
  • Mycobacterial DnaQ was shown to bind the β clamp and function synergistically with the PHP domain proofreader.
  • Loss of DnaQ function led to replication fork dysfunction, attenuated growth, and increased mutagenesis under fluoroquinolone stress.
  • A prevalent Mtb DnaQ variant in lineage 4.3 was associated with hypermutability and drug resistance.

Conclusions:

  • Noncanonical DnaQ functions as an additional, essential proofreader in mycobacteria, complementing the PHP domain's activity.
  • A coproofreading model with a division of labor between DnaQ and the PHP domain is proposed.
  • DnaQ variation in Mtb can drive hypermutability and contribute to drug resistance, suggesting a mutator-driven evolutionary pathway for adaptation.