Is Ap4A an activator of eukaryotic DNA replication?

The most well established fact concerning Ap4A metabolism is that the concentration of this compound is cell cycle and cell proliferation dependent. An additional intriguing fact is that Ap4A can stimulate DNA synthesis in cell extracts, and when injected into living cells. In view of these facts, it is not surprising that Ap4A has been postulated to regulate the initiation of DNA replication. However, in our opinion, experimental efforts designed to test this hypothesis do not conclusively link Ap4A to DNA replication. Work on the mechanism of stimulation of DNA synthesis in vitro indicates that Ap4A and a variety of adenylated nucleotides increase DNA synthetic rates by acting as primers. Thus far there is no evidence that this primer function plays a role in the initiation of normal DNA replication in vivo, or that Ap4A is unique in this capacity to stimulate initiation processes. Additional experiments have shown an association of partially purified DNA alpha polymerase with both tryptophanyl-tRNA synthetase and a protein capable of binding Ap4A. The Ap4A-binding protein appears to be necessary for Ap4A to assume the correct conformation for priming, since physiological levels of Ap4A are not stimulatory for highly purified DNA alpha polymerase. The relevance of tRNA synthetases to the regulation hypothesis is their ability to produce Ap4A. Ironically, mammalian tryptophanyl-tRNA synthetase does not appear to have this capacity. Furthermore, the association of alpha polymerase with either Ap4A-binding protein or tryptophanyl-tRNA synthetase in vivo has not been conclusively demonstrated. Although Ap4A has been postulated to regulate many phenomena in eukaryotes and bacteria, such as entry into S phase and the response to oxygen deprivation, the links between Ap4A and these processes are still only circumstantial. It is tempting to extrapolate from the alarmone and stringent responses of bacteria to other systems, but these phenomena are not known to occur in eukaryotic cells. Similar deprivation and inhibition experiments in mammalian cells have been shown to stop growth at a synchronous position in cell cycle, and the Ap4A concentration has been found simply to vary accordingly. The addition or depletion of Ap4A from intracellular pools has not been shown to alter cell cycle. Therefore, while the speculation concerning the role of Ap4A in vivo is a good source of future experiments, at this point its role as an important regulatory compound is far from demonstrated.