Imbalance in the level of the pyrimidine degradation products dihydrouracil and dihydrothymine is associated with cellular transformation and cancer progression. Dihydropyrimidines are degraded by dihydropyrimidinase (DHP), a zinc metalloenzyme that is upregulated in solid tumors but not in the corresponding normal tissues. How dihydropyrimidine metabolites affect cellular phenotypes remains elusive. Here we show that the accumulation of dihydropyrimidines induces the formation of DNA–protein crosslinks (DPCs) and causes DNA replication and transcriptional stress. We used Xenopus egg extracts to recapitulate DNA replication invitro. We found that dihydropyrimidines interfere directly with the replication of both plasmid and chromosomal DNA. Furthermore, we show that the plant flavonoid dihydromyricetin inhibits human DHP activity. Cellular exposure to dihydromyricetin triggered DPCs-dependent DNA replication stress in cancer cells. This study defines dihydropyrimidines as potentially cytotoxic metabolites that may offer an opportunity for therapeutic-targeting of DHP activity in solid tumors.

Eukaryotic genomes are duplicated from thousands of replication origins that fire sequentially forming a defined spatiotemporal pattern of replication clusters. The temporal order of DNA replication is determined by chromatin architecture and, more specifically, by chromatin contacts that are stabilized by RIF1. Here, we show that RIF1 localizes near newly synthesized DNA. In cells exposed to the DNA replication inhibitor aphidicolin, suppression of RIF1 markedly decreased the efficacy of isolation of proteins on nascent DNA, suggesting that the isolation of proteins on nascent DNA procedure is biased by chromatin topology. RIF1 was required to limit the accumulation of DNA lesions induced by aphidicolin treatment and promoted the recruitment of cohesins in the vicinity of nascent DNA. Collectively, the data suggest that the stabilization of chromatin topology by RIF1 limits replication-associated genomic instability.

Eukaryotic genomes are duplicated from thousands of replication origins that fire sequentially forming a defined spatiotemporal pattern of replication clusters. The importance of the organization of replisomes into functional clusters, also called replication factories, is still poorly understood. Here we identified the multifunctional protein RIF1 as a structural component of replication factories. RIF1 depletion did not impair the velocity of replication forks, neither in basic conditions nor in presence of a molecule that interferes with replication fork progression but increased the frequency of DNA lesions induced in S phase. Isolation of replication-associated proteins from RIF1-depleted cells revealed a major defect in the clustering of replication factors on nascent DNA, without any noticeable impact on DNA synthesis or replisome stability. We found that the changes in replication patterns commonly observed upon RIF1 depletion are induced by DNA replication stress. The data suggest that RIF1 encases replication factories to ensure the organization of replication clusters against chromatin rearrangements induced by DNA replication stress.