In vitro synthesis of simian virus 40 DNA. II. Evidence for a repair mechanism

Abstract

The technique of density labeling of DNA by BrdU was used to characterize the material synthesized in vitro by cytoplasmic extracts of SV40 infected cells incubated in the presence of simian virus 40 (SV40) DNA component I molecules (Girard et al, Biochimie, this volume). In a first experiment, the template was labeled beforehand in vivo using [14C]-BrdU, and the in vitro incubation was carried out in the presence of [3H]-dGTP and [3H]-dTTP. In a second experiment, the template was labeled in vivo with 32P, and the in vitro incubation was in the presence of [3H]-dGTP and BrdUTP. After digestion with the restriction endonuclease Hind II + III, the fragments from the end products of the reaction were analyzed by density gradient centrifugation, at pH 7 and pH 13. In both experiments the DNA product molecules had the same density as the resepctive DNA templates. Cellular enzymes seem to be responsible for this in vitro synthesis of DNA, since cytoplasmic extracts from uninfected cells were almost as active as those from SV40 infected cells. The system was proved efficient in the conversion of "open circular" molecules (component II DNA molecules) to covalently closed circular DNA molecules (relaxed component I molecules). The use of DNA complexed with histones did not impart viral specificity to the system. It is concluded that the cytoplasmic extract is only capable of supporting the repair synthesis of added viral DNA.