An investigation into the role of Ap4A and Ap4Ns in the inhibition of the initiation phase of DNA replication

Precise DNA replication is essential for the maintenance of a healthy genome. Although DNA replication has a very high fidelity, mutations can be introduced during this process, and the likelihood of mutation increases when a cell is under stress. Therefore, several signalling mechanisms exist, such as the ATM and ATR-dependent signalling in the DNA damage response, which stall DNA replication and promote the repair of damage. Diadenosine tetraphosphate (Ap4A) levels increase in response to cell stress, where it inhibits the initiation phase of DNA replication at micromolar levels. However, the mechanistic basis for its inhibitory activity has not yet been determined. In addition, there are other dinucleoside tetraphosphates (Ap4Ns) that have extracellular signalling roles, yet their synthesis and intracellular function have not been established. Here we present evidence suggesting that Ap4Ns can be synthesised by UBE1 in a biologically relevant manner. We have developed a technique for the extraction and detection of Ap4A, Ap4C, Ap4G and Ap4U by triple quadrupole mass spectrometry in extracts from a mammalian KBM7 cell line with a disrupted Nudt2 gene (NuKO). Furthermore, we have applied this technique to investigate the effect of DNA damaging agents and DNA stressing agents on Ap4N levels in NuKO cells. Finally, the inhibitory effect of each Ap4N in a cell-free DNA replication system containing late G1 nuclei and S-phase cytosolic extract demonstrates that Ap4Ns have the capacity to inhibit the initiation of DNA replication. Further analysis suggests that Ap4Ns reduce initiation by reducing the chromatin localisation of the key replication proteins Mcm2 and PCNA. Together, these data suggest that both Ap4A and Ap4Ns may regulate the DNA replication process and that they could represent an additional regulatory mechanism to prevent untimely entry into S-phase during genotoxic stress