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.

In order to pass on an intact copy of the genome during cell division, complete and faithful DNA replication is crucial. Yet, certain areas of the genome are intrinsically challenging to replicate, which manifests as high local mutation propensity. Such regions include trinucleotide repeat sequences, common chromosomal fragile sites (CFSs), and early replicating fragile sites (ERFSs). Despite their genomic instability CFSs are conserved, suggesting that they have a biological function. To shed light on the potential function of CFSs, this review summarizes the similarities and differences of the regions that challenge DNA replication with main focus on CFSs. Moreover, we review the mechanisms that operate when CFSs fail to complete replication before entry into mitosis. Finally, evolutionary perspectives and potential physiological roles of CFSs are discussed with emphasis on their potential role in neurogenesis.