FIG. 3.

Delayed-pulse and pulse-chase in vitro DNA replication assays. (A) Schematic diagram of the order of addition of the components in the procedures used throughout this study. CP, creatine phosphate; CPK, creatine phosphokinase. (B) In the delayed pulse experiment, replication reactions were begun with ATP but not the other nucleotides. At 30 min, 1 μCi of [α-³²]dATP, along with cold dTTP, dCTP, dGTP, CTP, GTP, and UTP, was added, and DNA synthesis proceeded for 30 min at 37°C. In the pulse-chase assays, all ribonucleotides and deoxyribonucleotides including 1 μCi of [α-³²]dATP were added from the beginning. After 30 min, the label was chased with an excess of cold dATP for 30 min. Human topo I (100 ng) was added either from the beginning (topo I at 0′) or at 30 min (topo I at 30′). After the reactions were stopped, the labeled replicative products were purified and analyzed as described for Fig. 1A.