In vitro polyoma DNA synthesis: characterization of a system from infected 3T3 cells

Abstract

A lysate from hypotonically swollen polyoma-infected BALB/3T3 cells incorporated labeled deoxynucleotide triphosphates into both viral and cellular DNAs. The incorporation was stimulated by the presence of ATP, deoxynucleotide triphosphates, thiols, and magnesium ions. Strong inhibition of incorporation was observed with thiol reagents and arabinosyl nucleotide triphosphates. The rate of in vitro synthesis increased with the temperature of incubation as expected. Incorporation into cellular DNA for up to 2 h was observed in lysates from virus-infected and serum-stimulated cells but not from resting cells. Synthesis in the system, therefore, appeared to reflect the physiological state of the cells before preparation of the lysate. Incorporation into viral DNA stopped far sooner than that into cellular DNA. During the initial phase of the in vitro incubation, incorporation occurred into viral replicative intermediates (RI). These RIs had identical properties to those isolated after in vivo pulse labeling and a substantial proportion of them was matured to form I DNA at later times in the incubation through all the stages known to occur in vivo. Density labeling of the in vitro product showed that practically all of the RIs pre-existing in the infected cell took part in the in vitro reaction. Analysis of DNA labeled in vitro in the presence of 5-bromodeoxyuridine triphosphate showed that synthesis occurred on RIs at all stages of replication and that the progeny strands were elongated by up to 80% of unit viral DNA length. Pre-existing RIs, pulse labeled in vivo, showed evidence of a pool at a late stage of replication which required elongation of their progeny strands by approximately 25% during conversion to form I molecules. From density-labeling experiments, we were also able to show that viral DNA synthesis in vitro was semiconservative. The major reason for cessation of viral DNA synthesis in vitro was the very limited ability of the lysate to initiate new rounds of viral DNA synthesis.