During in vitro incubation, nuclei from polyoma-infected cells elongate the daughter strands of the replicative intermediate of pblyoma DNA. This process is now shown to involve the transient formation of short ft-agments (4-5 S), a process that is stimulated by the addition of ribonucleoside triphosphates. The prresence of stretches of RNA at the 5'-end of short DNA chains was determined from Cs2SO4 equilibrium centrifugation and from the finding that isotope from a-3"P-labeled deoxynucleoside triphosphates was recovered in 2'(3')-ribonucleotides after alkaline hydrolysis. Recent work with microbial systems by Kornberg and coworkers (4, 5) and Sugino et al. (6) has implicated RNA as a primer of DNA replication. We will present evidence for a similar participation of RNA during polyoma DNA replication in isolated nuclei. This process appears to involve the intermediate formation of short fragments, which sediment at about 4-5 S, consisting of mixed RNA-DNA polynucleotides. Short chains also accumulate in vivo during deoxynucleotide starvation after addition of hydroxyurea.
MATERIALS AND METHODSLabeled nucleoside triphosphates were purchased from New England Nuclear Corp. or were synthesized by the procedure of Symons (7). Other sources of materials, the infection of cells, the preparation of polyoma DNA, and the centrifugation procedures were as described (1-3). Nuclei were prepared from cells suspended in "isotonic Hepes" buffer (2) by Perry's procedure with 0.5% NP42 (8), rather than by homogenizaAbbreviation: Hepes, N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid. * Present address: Institut fur Genetik, Universitat zu Kln, D-5 Koln-Lindenthal, Germany. t Present address: Department of Biochemistry, Faculty of Arts and Sciences, University of Pittsburgh, Pittsburgh, Pa. 15213. 412 tion in "hypotonic Hepes" buffer (2). After centrifugation, the nuclei were resuspended in 1-4 volumes of isotonic buffer and incubated under standard conditions (2). In some experiments ribonucleoside triphosphates were added, as indicated in the legends.
RESULTS
Intermediate formation of short fragmentsInfected nuclei were incubated under standard conditions with ['H]dTTP, and the radioactive polyoma DNA formed was analyzed by centrifugation in alkaline sucrose gradients (Fig. 1). At short incubation times, radioactivity was distributed bimodally, with a sharp peak around 4-5 5 and a second, broader, peak at higher S-values. The relative amount of radioactivity present in this second peak increased with time. The different labeled species were shown to be polyoma specific by hybridization studies.In a pulse-chase experiment, the radioactivity from the short chains formed during the first 1.5 min was converted into longer chains on prolonged incubation (Fig. 2). These results suggest that the DNA sedimenting at 4-5 S represents an intermediate in the synthesis of longer chains.Involvement of RNA in the synthesis of short fragments ATP enhances the incorporation of ['H]dTTP into polyoma DNA (2). Additiori of a mixt...