Analysis of simian virus 40 chromosome-T-antigen complexes: T-antigen is preferentially associated with early replicating DNA intermediates.

Abstract

The fraction and DNA composition of simian virus 40 chromosomes that were complexed with large T-antigens (T-Ag) were determined at the peak of viral DNA replication. Simian virus 40 chromatin containing radiolabeled DNA was extracted by the hypotonic method of Su and DePamphilis (Proc. Natl. Acad. Sci. U.S.A. 73:3466-3470, 1976) and then fractionated by sucrose gradient sedimentation into replicating (90S) and mature (70S) chromosomes. Viral chromosomes containing T-Ag were isolated by immunoprecipitation with saturating amounts of either an anti-T-Ag monoclonal antibody or an anti-T-Ag hamster serum under conditions that specifically precipitated T-Ag protein from cytosol extracts. An average of 10% of the uniformly labeled DNA in the 90S pool and 7.5% in the 70S pool was specifically precipitated, demonstrating that under these conditions immunologically reactive T-Ag was tightly bound to only 8% of the total viral chromosomes. In contrast, simian virus 40 replicating intermediates (RI) represented only 1.2% of the viral DNA, but most of these molecules were associated with T-Ag. At the shortest pulse-labeling periods, an average of 72 +/- 18% of the radiolabeled DNA in 90S chromosomes could be immunoprecipitated, and this value rapidly decreased as the labeling period was increased. Electron microscopic analysis of the DNA before and after precipitation revealed that about 55% of the 90S chromosomal RI and 72% of the total RI from both pools were specifically bound to T-Ag. Comparison of the extent of replication with the fraction of RI precipitated revealed a strong selection for early replicating DNA intermediates. Essentially all of the RI in the 70S chromosomes were less than 30% replicated and were precipitated with anti-T-Ag monoclonal antibody or hamster antiserum. An average of 88% of the 90S chromosomal RI which were from 5 to 75% replicated were immunoprecipitated, but the proportion of RI associated with T-Ag rapidly decreased as replication proceeded beyond 70% completion. By the time sibling chromosomes had separated, only 3% of the newly replicated catenated dimers in the 90S pool (<1% of the dimers in both pools) were associated with T-Ag. Measurements of the fraction of radiolabeled DNA in each quarter of the genome confirmed that T-Ag was preferentially associated with newly initiated molecules in which the nascent DNA was nearest the origin of replication. These results are consistent with a specific requirement for the binding of T-Ag to viral chromosomes to initiate DNA replication, and they also demonstrate that T-Ag does not immediately dissociate from chromosomes once replication begins. The biphasic relationship between the fraction of T-Ag-containing RI and the extent of DNA replication suggests either that 1 or 2 molecules of T-Ag remain stably bound until replication is about 70% completed or that 4 to 6 molecules of T-Ag are randomly released from each RI at a uniform rate throughout replication.