Decoding nucleoside supplementation: how thymidine outperforms ribonucleosides in accelerating mammalian replication forks

TL;DR

This study shows that thymidine, not ribonucleosides, is more effective in boosting DNA replication speed and maintaining genomic stability in mammalian cells.

Contribution

The study clarifies how thymidine accelerates replication forks and counteracts replication stress compared to ribonucleosides.

Findings

Ribonucleoside supplementation mainly increases CTP and UTP levels but has minimal effect on replication fork speed.

Thymidine supplementation significantly raises dTTP and dGTP levels and accelerates replication fork progression.

Thymidine counteracts replication fork slowdown caused by elevated dUTP through its effect on DNA polymerase ϵ.

Abstract

Disruptions in deoxynucleoside triphosphate (dNTP) supply impair DNA replication and lead to genomic instability. While exogenous ribonucleosides (rNuc) have been suggested to alleviate replication stress by increasing dNTP levels, their precise metabolic effects remain unclear. Here, we show that rNuc supplementation primarily elevates CTP and UTP levels, with only modest increases in dCTP, and has minimal impact on replication fork speed across multiple mammalian cell lines. In contrast, thymidine (dThd), either alone or in combination with rNuc—as in EmbryoMax Nucleosides—significantly increases dTTP and dGTP levels, leading to accelerated replication fork progression. Notably, dThd, rather than rNuc, drives fork acceleration and counteracts fork slowdown caused by elevated dUTP, consistent with primer extension assays showing that dUTP transiently inhibits Pol ϵ-mediated DNA synthesis at template adenines. These results clarify the distinct roles of nucleosides in nucleotide metabolism, providing a mechanistic basis for how dThd promotes fork progression and preserves genomic stability.

Graphical Abstract

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