NAD⁺ regulates nucleotide metabolism and genomic DNA replication

The intricate orchestration of enzymatic activities involving nicotinamide adenine dinucleotide (NAD⁺) is essential for maintaining metabolic homeostasis and preserving genomic integrity. As a co-enzyme, NAD⁺ plays a key role in regulating metabolic pathways, such as glycolysis and Kreb's cycle. ADP-ribosyltransferases (PARPs) and sirtuins rely on NAD⁺ to mediate post-translational modifications of target proteins. The activation of PARP1 in response to DNA breaks leads to rapid depletion of cellular NAD⁺ compromising cell viability. Therefore, the levels of NAD⁺ must be tightly regulated. Here we show that exogenous NAD⁺, but not its precursors, has a direct effect on mitochondrial activity. Short-term incubation with NAD⁺ boosts Kreb's cycle and the electron transport chain and enhances pyrimidine biosynthesis. Extended incubation with NAD⁺ results in depletion of pyrimidines, accumulation of purines, activation of the replication stress response and cell cycle arrest. Moreover, a combination of NAD⁺ and 5-fluorouridine selectively kills cancer cells that rely on de novo pyrimidine synthesis. We propose an integrated model of how NAD⁺ regulates nucleotide metabolism, with relevance to healthspan, ageing and cancer therapy.