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The rate of DNA synthesis in cultures of human lymphoblasts decreased more than 80% within 30 min after the cells were exposed to methotrexate, a potent inhibitor of dihydrofolate reductase. Despite this rapid initial inhibition, DNA continued to be synthesized for at least an additional 6 h. The mode of this subsequent replication appeared to be semiconservative, as indicated by the buoyant density of 5-bromodeoxyuridine-substituted DNA in alkaline CsCl gradients. The growth rates of DNA chains in cells exposed to methotrexate were determined by sedimentation rate analysis in alkaline sucrose gradients. DNA synthesized during 2-min or 10-min pulses with labeled deoxycitidine in the presence of methotrexate had about the same sedimentation coefficient, 35 S, as controls. When methotrexate-treated cultures were pulse-labeled for 10 min and then chased for various times, DNA fragments of about 80 S accumulated. DNA synthesized in the presence of methotrexate was stable and elongated to bulk-size DNA after methotrexate inhibition of growth was removed by addition of thymidine and deoxycytidine. The data suggest that methotrexate reduces the rate of DNA replication by inhibiting chain initiation independently of chain elongation. [ABSTRACT FROM AUTHOR]

DNA replication was investigated in isolated HeLa cell nuclei from different phases of the cell cycle. The addition of S-phase cytoplasm to S-phase nuclei restored the ability of these nuclei to synthesize DNA. The S-phase cytoplasm requirement could be replaced by partially purified calfthymus DNA polymerases A and B. The activity of both the S-phase cytoplasm and purified DNA polymerases in the nuclear system was ATP-dependent. Using nuclei prelabelied with bromodeoxyuridine and CsCl equilibrium centrifugation, the DNA synthesis in vitro was shown to be of the replicative, not repair type when supported by either S-phase cytoplasm or DNA polymerase A. DNA polymerase B, but not DNA polymerase A, was capable of using G1 nuclei to synthesize DNA; however, this synthesis was not ATP-dependent. Gel-filtration chromatography of HeLa cell-free extracts have shown that DNA polymerases B and C are present in S-phase and missing in G1-phase cells. [ABSTRACT FROM AUTHOR]