NAT10-mediated histone β-hydroxybutyrylation facilitates DNA replication and maintains genomic stability

Lysine β-hydroxybutyrylation (Kbhb) is a histone posttranslational modification derived from the ketone body β-hydroxybutyrate (BHB), but its role in DNA replication has remained unexplored. N-acetyltransferase 10 (NAT10) is known for RNA acetylation; whether it functions as a β-hydroxybutyryltransferase to catalyze histone Kbhb and regulate replication dynamics is unknown. We demonstrate that NAT10 directly catalyzes histone Kbhb at multiple lysine residues via its β-hydroxybutyryltransferase activity, an activity abolished by the G641E and K989R mutants or by remodelin inhibition. NAT10-mediated Kbhb accelerates replication fork speed and shortens S-phase duration without affecting origin firing. BHB treatment via NAT10-mediated Kbhb reduces replication stress markers (γH2AX, ssDNA, RPA2, p -CHK1) and genomic instability (micronuclei, anaphase bridges, chromatid breaks)—effects abolished by NAT10 depletion or remodelin inhibition. Mechanistically, Kbhb reduces chromatin-bound NAT10 and enhances chromatin accessibility, as shown by ChIP-seq and ATAC-seq. These findings were validated in primary human fibroblasts (WI-38, 2BS). NAT10-mediated histone Kbhb constitutes a metabolic-epigenetic mechanism that promotes DNA replication fidelity while maintaining genome stability. This pathway offers a potential therapeutic target for replication stress-associated pathologies, including cancer and aging-related conditions.