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Proofreading01:31

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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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Translesion (TLS) polymerases rescue stalled DNA polymerases at sites of damaged bases by replacing the replicative polymerase and installing a nucleotide across the damaged site. Doing so, TLS allows additional time for the cell to repair the damage before resuming regular DNA replication.
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The DNA replication, transcription, and translation processes are intricately coupled in bacteria, allowing efficient gene expression and rapid protein synthesis. While this physical and functional coordination is advantageous, it introduces challenges that bacteria overcome through specific regulatory mechanisms.Coupling of Replication, Transcription, and TranslationThe coupling of replication, transcription, and translation is a hallmark of bacterial gene expression. As the replisome unwinds...
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Direct Restart of a Replication Fork Stalled by a Head-On RNA Polymerase
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Structural dynamics as a contributor to error-prone replication by an RNA-dependent RNA polymerase.

Ibrahim M Moustafa1, Victoria K Korboukh1, Jamie J Arnold1

  • 1From the Department of Biochemistry and Molecular Biology.

The Journal of Biological Chemistry
|November 8, 2014
PubMed
Summary

Altering RNA-dependent RNA polymerase (RdRp) fidelity impacts viral attenuation. This study reveals how nucleotide-binding site dynamics in poliovirus RdRp influence nucleotide incorporation accuracy, crucial for designing new vaccines.

Keywords:
Lethal MutagenesisPlus-stranded RNA VirusPoliovirusPolymerase FidelityPopulation GeneticsRNA PolymeraseRNA VirusViral Replication

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Area of Science:

  • Virology
  • Molecular Biology
  • Biochemistry

Background:

  • RNA viruses rely on RNA-dependent RNA polymerase (RdRp) for replication.
  • RdRp fidelity, the accuracy of nucleotide incorporation, is critical for viral genome stability and can be a target for antiviral strategies.
  • Perturbing RdRp fidelity is a potential avenue for rational vaccine design.

Purpose of the Study:

  • To investigate the molecular mechanisms underlying nucleotide incorporation fidelity in poliovirus RdRp.
  • To compare the conformational dynamics of wild-type (WT) RdRp with a high-fidelity mutant (H273R).
  • To elucidate how enzyme dynamics relate to the fidelity checkpoint during nucleotide selection.

Main Methods:

  • X-ray crystallography to determine enzyme structures.
  • Molecular dynamics simulations to model enzyme behavior.
  • NMR spectroscopy to probe enzyme dynamics and nucleotide interactions.
  • Pre-steady-state kinetics to measure enzyme reaction rates.

Main Results:

  • The nucleotide-binding site of RdRp exists in two states: occluded and competent.
  • Primed template RNA binding enhances conformational dynamics between these states.
  • Wild-type RdRp favors the occluded state, promoting fidelity; the H273R mutant favors the competent state, increasing errors.
  • NMR data indicated Met-187 resonance reflects the enzyme's ability to verify nucleotide correctness.
  • Kinetic data support the role of conformational dynamics in the fidelity checkpoint.

Conclusions:

  • Faithful nucleotide incorporation is achieved by linking the equilibrium of nucleotide-binding pocket conformations and active site dynamics to the correctness of the bound nucleotide.
  • Understanding these dynamics is essential for the rational design of viral vaccine candidates by manipulating RdRp fidelity.
  • Multiple biophysical and biochemical approaches are necessary to fully understand polymerase fidelity mechanisms.