Proliferating-cell nuclear antigen (PCNA) mediates the replication of simian virus 40 (SV40) DNA by reversing the effects of a protein that inhibits the elongation reaction. Two other protein fractions, activator I and activator II, were also shown to play important roles in this process. We report that activator II isolated from HeLa cell extracts is a PCNA-dependent DNA polymerase 8 that is required for efficient replication of DNA containing the SV40 origin of replication. PCNA-dependent DNA polymerase 8 on a DNA singly primed 4X174 single-stranded circular DNA template required PCNA, a complex of the elongation inhibitor and activator I, and the single-stranded DNA-binding protein essential for SV40 DNA replication. DNA polymerase 6, in contrast to DNA polymerase a, hardly used RNA-primed DNA templates. These results indicate that both DNA polymerase a and 8 are involved in SV40 DNA replication in vitro and their activity depends on PCNA, the elongation inhibitor, and activator I.The in vitro origin of replication-dependent simian virus 40 (SV40) DNA replication has been established by using cytosolic extracts of human or monkey cells supplemented with SV40 large tumor antigen (Tag) (1-3). SV40 Tag is a multifunctional protein possessing origin-specific DNA-binding, ATPase, and DNA helicase activities; this antigen is required for initiating DNA replication (4-7).We have described a system capable of catalyzing a Tag and SV40 origin-dependent replication reaction using purified proteins. In addition to Tag and DNA containing the SV40 origin of replication (ori'DNA), immunopurified DNA polymerase a-DNA primase complex, topoisomerase (topo) I and/or II and a multisubunit single-stranded DNA-binding protein (SSB), all isolated from HeLa cells, are required (8,9). Replication reactions containing these proteins will hereafter be identified as the monopolymerase system.Proliferating-cell nuclear antigen (PCNA), a known accessory factor for DNA polymerase 6 (Pol-6) (10-12), was shown essential for leading-strand DNA synthesis in crude systems; without PCNA only small-sized DNA products arising from the lagging strand were synthesized (13). In contrast, PCNA had no effect on the monopolymerase system. The reasons for this difference were traced to the effect of multiple protein factors [an elongation inhibitor (El), activators I (Al) and II (All)], which were present in crude fractions but not in the monopolymerase system (14, 15). The El has been characterized as a 120-kDa protein that binds to ends of DNA chains, thus inhibiting enzymes that act at such sites (such as exonuclease III, 5' --3' exonuclease, and DNA ligase).Pol-6 was initially isolated and distinguished from other DNA polymerases by its tightly associated 3' -k 5' exonuclease activity and its PCNA dependency (16,17). Recently another polymerase, also called Pol-6, which does not require PCNA, has been isolated from several mammalian sources (17-20). These different Pol-8 preparations share some common properties, including 3' -+ 5' exonuclea...