Polyoma virus minichromosomes: a soluble in vitro replication system

Exogenously added simian virus 40 (SV40) DNA can be replicated semiconservatively in vitro by a mixture of a soluble extract of HeLa cell nuclei and the cytoplasm from SV40-infected Cosl cells. When cloned DNA was used as a template, the clone containing the SV40 origin of DNA replication was active, but a clone lacking the SV40 origin was inactive. The major products of the in vitro reaction were form I and form II SV40 DNAs and a small amount of form III. DNA synthesis in extracts began at or near the in vivo origin of SV40 DNA synthesis and proceeded bidirectionally. The reaction was inhibited by the addition of anti-large T hamster serum, aphidicolin, or RNase but not by ddNTP. Furthermore, this system was partially reconstituted between HeLa nuclear extract and the semipurified SV40 T antigen instead of the CosI cytoplasm. It is clear from these two systems that the proteins containing SV40 T antigen change the nonspecific repair reaction performed by HeLa nuclear extract alone to the specific semiconservative DNA replication reaction. These results show that these in vitro systems closely resemble SV40 DNA replication in vivo and provide an assay that should be useful for the purification and subsequent characterization of viral and cellular proteins involved in DNA replication. MATERIALS AND METHODS Cells and virus. The SV40-transformed monkey cell CosI (10, 11) was obtained from Y. Gluzman. SV40 was propagated in the monkey cell line GC7, and SV40 DNA was extracted from purified virions by CsClethidium bromide equilibrium centrifugation as described previously (41).

Section: Discussionmentioning

confidence: 99%

Initiation of simian virus 40 DNA replication in vitro

Ariga¹,

Sugano²

1983

“…In this paper, we classify the DNA structures and quantitate RI DNA molecules found in fractions of the polyoma minichromosome gradients of Gourlie et al (14). To determine the absolute concentrations of replicating and nonreplicating polyoma DNA molecules, we used the technique of quantitative electron microscopy (3,5,19,21).…”

mentioning

confidence: 99%

Polyoma virus minichromosomes: associated DNA molecules

Krauss¹,

Benbow²

1981

Self Cite

Electron microscopy was used to identify and quantitate DNA molecules associated with 3H-labeled polyoma minichromosomes which had been fractionated on a sucrose gradient. The percentage ofreplicating DNA molecules observed in the fractions of the gradient normally designated the replicative intermediate region was up to ninefold higher than in fractions from the mature region. Nevertheless, because of the higher overall concentration of polyoma DNA molecules in the mature region, nearly as many replicating DNA molecules were computed to be in the mature region as in the replicative intermediate region. The replicating molecules in the mature region were predominantly early replicative intermediates. Almost all late replicative intermediates were found in the replicative intermediate region. Under aqueous spreading conditions, a substantial fraction of the replicating DNA structures appeared to be asymmetrical or otherwise unusual, suggesting that extensive single-stranded regions may exist in replicating polyoma minichromosomes.

“…1). The thymidine incorporation data coupled with data from earlier electron microscopic studies of similar gradients (11,19) indicated that fractions 5 to 10 represented the replicative intermediate (RI) region of the gradient, whereas mature minichromosomes sedimented in fractions 10 to 15. Fractions were subsequently assayed for poly(ADP-rib) polymerase activity by using endogenous acceptors.…”

Section: Resultsmentioning

confidence: 75%

Polyoma virus minichromosomes: poly ADP-ribosylation of associated chromatin proteins

Prieto-Soto¹,

Gourlie²,

Misawa³

et al. 1983

Self Cite

The host nuclear enzyme poly(ADP-ribose) polymerase has been shown to be associated with the replicative intermediate and mature forms of polyoma virus minichromosomes. Minichromosome-associated histones H2A and H2B as well as several nonhistone proteins were poly ADP-ribosylated by endogenous poly(ADP-ribose) polymerase. In addition, minichromosome fractions catalyzed the formation in vitro of dimers of endogenous histone Hi linked by poly(ADPribose). Poly ADP-ribosylated polyoma virus minichromosome chromatin labeled in vivo with [3H]thymidine could be retained and eluted from antipoly(ADP-ribose) immunoglobulin G-Sepharose. Pulse-labeled replicative intermediate minichromosomes were retained better on the antibody columns than were mature minichromosomes labeled for 2.5 h. The possible role of poly ADPribosylation of viral nucleosomes during polyoma replication or transcription is discussed.