Structural topography of simian virus 40 DNA replication

Abstract

Applying an in situ cell fractionation procedure, we analyzed structural systems of the cell nucleus for the presence of mature and replicating simian virus 40 (SV40) DNA. Replicating SV40 DNA intermediates were tightly and quantitatively associated with the nuclear matrix, indicating that elongation processes of SV40 DNA replication proceed at this structure. Isolated nuclei as well as nuclear matrices were able to continue SV40 DNA elongation under replication conditions in situ, arguing for a coordinated and functional association of SV40 DNA and large T molecules at nuclear structures. SV40 DNA replication also was terminated at the nuclear matrix. While the bulk of newly synthesized, mature SV40 DNA molecules then remained at this structure, some left the nuclear matrix and accumulated at the chromatin.

Aphidicolin-resistant polyomavirus and subgenomic cellular DNA synthesis occur early in the differentiation of cultured myoblasts to myotubes

“…Such a pattern of Py DNA synthesis is distinct from that seen with permissive 3T6 cells and is a characteristic of aphidicolin-treated (but not untreated) differentiating C2C12 and Aza-ClB myoblast cells (4a). This replication pattern is also very similar to the punctate replication centers seen with bromodeoxyuridine pulse-labeling on en-try into S phase of cultured rat fibroblasts (38) and somewhat similar to patterns reported for simian virus 40, herpesvirus, and adenovirus (17,60,73,76).…”

DNA polymerase epsilon may be dispensable for SV40- but not cellular-DNA replication.

“…The nuclei were prepared from SV40-infected cells by a method that leaves them attached to the culture plates. Under these conditions, SV40 chromatin remains associated with the nuclear matrix and its replication may resemble more closely the in vivo process, compared with that of isolated SV40 chromatin (Schirmbeck and Deppert, 1991). Moreover, the crosslinking signals obtained with nuclear monolayers were stronger than those seen with nuclei in suspension (not shown), probably due to the lower self-absorption of UV-light by the monolayer.…”

“…The conditions of cell growth and SV40 infection were described previously (Nethanel et al, 1988). Nuclei were isolated by the in situ fractionation method of Schirmbeck and Deppert (1991) from SV40 infected CV-1 cells, near-confluent monolayers of uninfected CV-1 cells or CV-1 cells stimulated to proliferate by deprivation and replenishment of serum. In the latter case, the cells were grown in medium supplemented with 5% calf serum to 70% confluency and brought to quiescence by incubation for 72 h in a medium containing 0.25% calf serum.…”

What the papers say: The organization of replication centres in higher eukaryotes

“…This implies that the organization which appears to exist in vivo is not essential to the mechanics of the replication process and undermines the concept of fixed replication. However, despite the fact that soluble systems will reproduce SV40 DNA with high fidelity there is evidence to suggest that once inside a cell the viral genome is forced to use nuclear matrix-associated DNA polymerase complexes (27). These facts may emphasise the organizational, rather than operational, importance of nuclear structure.…”