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Related Experiment Video

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Studying Ribonucleotide Incorporation: Strand-specific Detection of Ribonucleotides in the Yeast Genome and Measuring Ribonucleotide-induced Mutagenesis
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Ribonucleotide incorporation by yeast DNA polymerase ζ.

Alena V Makarova1, Stephanie A Nick McElhinny2, Brian E Watts2

  • 1Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA.

DNA Repair
|March 29, 2014
PubMed
Summary

DNA polymerases can incorporate ribonucleotides (rNMPs) into DNA, impacting genome stability. Pol ζ, crucial for mutagenesis, incorporates rNMPs at a low frequency, suggesting rare incorporation during DNA repair.

Keywords:
DNA polymeraseMutagenesisRibonucleotidesTranslesion synthesis

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • Ribonucleotide (rNMP) incorporation into DNA by replicative polymerases can compromise genome stability.
  • Pol ζ is a key eukaryotic enzyme involved in translesion DNA synthesis and damage-induced mutagenesis.

Purpose of the Study:

  • To investigate the capacity of Pol ζ to incorporate ribonucleotides (rNMPs) into DNA.
  • To quantify rNMP incorporation by Pol ζ under various cellular conditions and cofactor interactions.

Main Methods:

  • In vitro assays measuring rNMP incorporation by purified Pol ζ.
  • Enzyme kinetics studies under varying dNTP/rNTP concentrations and in the presence of cofactors (RPA, RFC, PCNA) and Rev1.

Main Results:

  • Pol ζ incorporates rNMPs into DNA and can extend primers with 3'-ribonucleotides.
  • Under unstressed conditions, Pol ζ incorporates rNMPs at a frequency of 1 per 200-300 dNMPs.
  • Under damage-induced conditions (elevated dNTPs), rNMP incorporation by Pol ζ decreases to 1 per 1300 dNMPs.
  • Interaction with Rev1 did not significantly alter rNMP incorporation frequencies.

Conclusions:

  • Pol ζ exhibits ribonucleotide incorporation activity, though at a frequency comparable to or slightly higher than high-fidelity replicative polymerases under normal conditions.
  • The frequency of rNMP incorporation by Pol ζ decreases significantly under conditions mimicking DNA damage.
  • These findings suggest that ribonucleotide incorporation into DNA during Pol ζ-mediated mutagenesis in vivo is likely a rare event.