In vitro replication of simian virus 40 DNA in a nucleoprotein complex

Abstract

A simian virus 40 (SV40) nucleoprotein complex, extracted from nuclei isolated from a monkey cell line infected with SV40, continued DNA replication in the presence of a nuclear extract, cytosol, ATP, and ATP-regenerating system, and the four deoxyribonucleoside triphosphates. The DNA products of replication were also found as nucleoprotein complexes. Forty percent of the replicating viral DNA, labeled in vivo, was converted into covalently closed, superhelical DNA during incubation in vitro. Although the remaining labeled DNA was not converted into mature viral DNA, it was elongated to its full genome length. Failure to terminate replication successfully was not caused by endonuclease activity, since covalently closed DNA, labeled in vivo, was not damaged during incubation in vitro. When [alpha-32P]dATP was present during the incubation, the label appeared first in replicating DNA and later in mature DNA; no unusual products were labeled in vitro. The covalently closed SV40 DNA made in vitro had the same superhelical density as viral DNA made in vivo. These data demonstrate that viral nucleoprotein complexes ("minichromosomes") are able to continue DNA replication outside of the nucleus.