Studies on the initiation of simian virus 40 replication in vitro: RNA primer synthesis and its elongation (simian virus 40 large tumor antigen/DNA synthesis/DNA polymerase a/DNA primase)

DNA primase-dependent synthesis of oligo- ribonucleotides 10-15 nucleotides long was observed in the presence of ATP, UTP, GTP, and CTP by using the purified components of the simian virus 40 (SV40) DNA replication system. The DNA primase-catalyzed reaction required the SV40 large tumor antigen (T antigen), DNA polymerase a (pol-a), the three-subunit human single-stranded DNA binding protein (HSSB), and topoisomerase I. The synthesis of small RNAs was unaffected by the addition of activator 1, prolifer- ating cell nuclear antigen, and DNA polymerase 8, proteins that can support extensive leading-strand synthesis. The RNA primers were derived predominantly from transcription of the lagging-strand template, even after prolonged incubation, in- dicating that the leading strand did not serve as a template. When the four dNTPs were added after oligoribonudeotide synthesis, pol-a extended the RNA primers hybridized to SV40 DNA. Pulse-chase experiments revealed that the small RNA chains were elongated to Okazaki-sized products. T7 DNA polymerase was also shown to rapidly extend oligoribonucle- otide primers in the presence of aphidicolin or antibodies against pol-a, conditions under which pol-a was markedly inhibited. These findings suggest that interactions between T antigen, pol-a-primase, and HSSB position the pol-a-primase complex on the lagging-strand template for RNA primer syn- thesis.