Semiconservative DNA replication in vitro. I. Properties of two systems derived from mouse P-815 cells by permeabilization or lysis with Brij-58

Abstract

To study DNA replication in vitro, mouse P-815 cells were either permeabilized by hypotonic treatment or gently lysed with the detergent Brij-58. In the presence of KCl, EGTA, creatine phosphokinase, creatine phosphate, sucrose, dithiothreitol, CTP, GTP, UTP, and HEPES at pH 7.8, both in vitro systems required similar concentrations of all four deoxyribonucleoside triphosphates, ATP, Mg2+, and dextran. Incorporation of [3H] dTTP was due to semiconservative DNA replication and was restricted to S-phase nuclei. No repair replication was detectable. In the first 20 min, the rate of DNA replication in vitro was 30--40% of the in vivo rate, and after 60 min, about 3% of the genome were replicated. Preexisting bulk DNA was not fragmented during permeabilization or lysis with Brij-58 and during replication in vitro. Size distributions of growing strands in vitro were similar to those found in vivo. Neither cycloheximide (100 mug/ml) nor hydroxyurea (3 mM) inhibited DNA replication in vitro.