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Related Experiment Video

Updated: Jan 15, 2026

Studying Ribonucleotide Incorporation: Strand-specific Detection of Ribonucleotides in the Yeast Genome and Measuring Ribonucleotide-induced Mutagenesis
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Decoding nucleoside supplementation: how thymidine outperforms ribonucleosides in accelerating mammalian replication

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Exogenous ribonucleosides minimally impact DNA replication, unlike thymidine which boosts deoxynucleoside triphosphate levels. Thymidine accelerates replication forks and enhances genomic stability, clarifying nucleoside roles in nucleotide metabolism.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • Deoxynucleoside triphosphate (dNTP) supply is crucial for DNA replication and genomic stability.
  • Exogenous ribonucleosides (rNuc) are proposed to mitigate replication stress by increasing dNTPs, but their exact metabolic effects are not fully understood.

Purpose of the Study:

  • To elucidate the distinct metabolic effects of exogenous ribonucleosides and thymidine on nucleotide pools and DNA replication dynamics.
  • To clarify the mechanisms by which these nucleosides influence replication fork progression and genomic stability.

Main Methods:

  • Supplementation with exogenous ribonucleosides (rNuc) and thymidine (dThd) in mammalian cell lines.
  • Quantification of intracellular nucleotide triphosphate levels (CTP, UTP, dCTP, dTTP, dGTP).
  • Measurement of DNA replication fork speed and primer extension assays using Pol ϵ.

Main Results:

  • rNuc supplementation primarily increased CTP and UTP, with minor increases in dCTP, and had minimal effect on replication fork speed.
  • Thymidine (dThd), alone or with rNuc, significantly elevated dTTP and dGTP levels, accelerating replication fork progression.
  • dThd, not rNuc, was responsible for fork acceleration and counteracting dUTP-induced fork slowdown, which was linked to Pol ϵ inhibition at template adenines.

Conclusions:

  • Exogenous ribonucleosides have limited impact on dNTP levels and replication fork speed.
  • Thymidine effectively increases key dNTPs (dTTP, dGTP), promoting replication fork progression and genomic stability.
  • This study clarifies the differential roles of nucleosides in nucleotide metabolism and DNA replication, providing mechanistic insights into maintaining genomic integrity.