Replication of parvoviral DNA. I. Characterization of a nuclear lysate system

We have developed a nuclear lysate system from infected, synchronized cells capable of synthesizing unit-length parvoviral DNA in vitro. It was necessary to supplement the nuclear lysates with the polyamines, spermidine and spermine, to prevent degradation of template and product DNAs. In this system RF, RI, and single-stranded progeny DNAs were synthesized. Label incorporated in viral RF DNA in vivo appeared first in RI DNA and then in single-stranded DNA during incubation in vitro. By sedimentation the product DNAs were identical to those found in infected cells. Their viral identity was confirmed by hybridization. The addition of ribonucleotides, RNase, or alpha-amanitin did not affect parvoviral DNA synthesis in this system. The results with the specific inhibitors of mammalian DNA polymerases, aphidicolin, N-ethylmaleimide, and 2',3'-dideoxythymidine 5'-triphosphate indicated that DNA polymerase alpha was required for synthesis of parvoviral DNA in the nuclear lysates. This requirement was confirmed by experiments with antibody to bovine DNA polymerase alpha.