Regulation of DNA replication in S phase nuclei by ATP and ADP pools

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Abstract
Synchronized 3T6 (mouse fibroblast) ghost monolayers (isolated nuclei) were utilized to study the effects of ATP and ADP levels on DNA replication in vitro. A system yielding discontinuous semiconservative DNA replication (without any detectable repair) in synchronized S phase nuclei was developed. Lack of initiation of new sites was observed in isolated S phase 3T6 nuclei without the presence of cytoplasmic material; DNA synthesis is comprised only of elongation at sites where initiation had previously taken place. DNA synthesis in S phase nuclei proceeded optimally at an ATP concentration of 4-5 mM. High ATP levels and high ATP/ADP ratio (produced by an ATP-regenerating system at a variety of ATP concentrations) yielded marked inhibition of [3H]dTTP incorporation. The cellular and nuclear pools of ATP and ADP in intact synchronized 3T6 cells were accurately determined by high-pressure liquid chromatography. A good correlation with the studies on isolated nuclei was observed. Whereas total cellular ATP pools increase during the progression of 3T6 cells from G1 to S phase of the cell cycle, nuclear ATP pools do not increase and the nuclear ATP/ADP ratios decrease once the cells enter the S phase of their cycle. Apparently nuclear ATP pools and ATP/ADP ratios act as S phase controls, regulating DNA elongation at sites where its synthesis has previously been initiated by cytoplasmic factors.

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