NAT10-mediated β-hydroxybutyrylation Affects DNA Replication

Accurate DNA replication is essential for genome integrity, with dysregulated replication dynamics, replication stress and genomic instability-hallmarks of cancer and aging. Here, we observe NAT10 is a β-hydroxybutyryl-transerase and NAT10-mediated β-hydroxybutyrylation (Kbhb) of histones that appears to affect replication fork progression without significantly impacting origin firing, potentially to reduce replication stress and to help maintain genomic stability. DNA fiber analyses show β-hydroxybutyrate (BHB) treatment enhances replication efficiency while maintaining fork symmetry, effects abolished by NAT10 depletion or inhibition. BrdU/EdU labeling, and EdU-FACS analyses reveal that NAT10-mediated Kbhb accelerates replication fork velocity and shortens S-phase duration. LC-MS/MS profiling shows no significant changes in origin firing following BHB treatment. Assessment of replication stress markers, including γH2AX foci, non-denaturing BrdU incorporation, RPA2 foci, S317-CHK1 phosphorylation, and levels of γH2AX and RPA2 on chromatin, suggests that NAT10-mediated Kbhb reduces replication stress. Evaluation of genomic instability, measured by micronuclei formation, sister chromatid bridges, and chromatid breaks/gaps during mitosis, indicates that NAT10-mediated Kbhb also reduces genomic instability. Mechanistically, NAT10-mediated Kbhb modulates chromatin association, thereby modulating chromatin accessibility to establish a replication-permissive environment. This epigenetic remodeling serves to moderate replication stress markers and genomic instability. Conserved effects in transformed and primary cell models position NAT10 as a metabolic-epigenetic nexus linking nutrient signaling to replication fidelity. Our findings suggest targeting Kbhb signaling as a potential therapeutic strategy against replication stress-associated pathologies.