Subnuclear systems for synthesis of simian virus 40 DNA in vitro.

Abstract

Two subnuclear systems for DNA synthesis of SV-40 in vitro were developed. Chromatin is prepared from infected cells [African green monkey kidney BSC-1]. These chromatin bodies can be disrupted and large debris can be pelleted, leaving a supernatant (soluble system). Chromatin bodies and the soluble system incorporate deoxyribonucleoside triphosphates into nucleoprotein complexes that contain SV-40 DNA. The DNA labeled in short pulses sediments in neutral sucrose gradients slightly faster than mature SV-40 DNA, as expected for a replicating intermediate. When rebanded in alkaline sucrose gradients, about half of the radioactivity is found in short strands (200-300 nucleotides) and half in longer strands (up to full viral size). When these systems are supplemented with a cytoplasmic preparation from HeLa cells, synthesis is stimulated .apprx. 5-fold, and the short strands are converted into strands of up to full viral length and into covalently closed circles. These subnuclear DNA-replicating systems should be useful for biochemical fractionation and characterization of some of the proteins required for [eukaryotic] DNA replication.