DNA synthesis by two eukaryotic DNA polymerases, alpha and delta, was studied using a single‐strand M13 DNA template primed at a unique site. In the presence of low amounts of either DNA polymerase alpha or delta, DNA synthesis was limited and short DNA strands of approximately 100 bases were produced. Addition of replication factors RF‐A, PCNA and RF‐C, which were previously shown to be required for SV40 DNA replication in vitro, differentially stimulated the activity of both DNA polymerases. RF‐A and RF‐C independently stimulated DNA polymerase alpha activity 4‐ to 6‐fold, yielding relatively short DNA strands (less than 1 kb) and PCNA had no effect. In contrast, polymerase delta activity was stimulated co‐operatively by PCNA, RF‐A and RF‐C approximately 25‐ to 30‐fold, yielding relatively long DNA strands (up to 4 kb). Neither RF‐C nor RF‐A appear to correspond to known polymerase stimulatory factors. RF‐A was previously shown to be required for initiation of DNA replication at the SV40 origin. Results presented here suggest that it also functions during elongation. The differential effects of these three replication factors on DNA polymerases alpha and delta is consistent with the model that the polymerases function at the replication fork on the lagging and leading strand templates respectively. We further suggest that co‐ordinated synthesis of these strands requires dynamic protein‐protein interactions between these replication factors and the two DNA polymerases.

PCNA (proliferating cell nuclear antigen), originally characterized as a DNA polymerase accessory protein, functions as a DNA sliding clamp for DNA polymerase delta and is an essential component for eukaryotic chromosomal DNA replication. Recent studies have revealed a striking feature of PCNA in its ability to interact with multiple partners, involved, for example, in Okazaki fragment joining, DNA repair, DNA methylation and chromatin assembly. Since these reactions take place mainly on replicating DNA, PCNA has applications as a marker for DNA synthesis. It is of interest that proteins involved in cell cycle regulation may also exhibit PCNA binding activity. For example, the CDK inhibitor, p21 (Cip1/Waf1) interacts with PCNA blocking its activity necessary for DNA replication and also affecting interactions with other PCNA binding proteins. The available data indicate that DNA sliding clamps have generated additional functions with evolution of eukaryotes from simple prokaryotes. In mammalian cells, they play key roles in controlling DNA synthesis reactions and the reorganization of replicated DNA at replication forks. Several cell cycle regulation proteins target these processes by affecting PCNA actions

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An in vitro culture of human fetal hepatocytes has been employed for infection by hepatitis B virus (HBV) virions that are produced by an established human hepatoma cell line, HB 611. HBV surface antigen and e antigen were released into the medium 3-4 days after infection, and production continued thereafter. RNA synthesis with similar kinetics was observed. Viral DNA replication started 2 days after infection, and replicative HBV DNA that included relaxed circles, single-stranded minus strands, and closed circles accumulated during 16 days of incubation. Immunofluorescent study using fluorescein isothiocyanate-labeled rabbit antisera directed against HBV core antigen revealed that this antigen is present in the nuclei in 12% of the infected cells. Particles containing HBV DNA were detected in the culture medium and were infectious. Thus, this in vitro infection system closely mimics infection in vivo and it allows detailed studies on early events associated with human HBV entry into cells and subsequent replication and integration.Hepatitis B virus (HBV) is a world-wide pathogen that causes hepatitis. Infection with the virus may be chronic and is associated with subsequent development of hepatocellular carcinoma. Although chimpanzees can be infected in vivo with HBV (1) and woodchucks, ground squirrels, and Peking ducks can be infected with their respective HBV-like viruses (2-4), use of whole animals poses certain limitations for detailed molecular studies. The most vexing technical problem in the biology of HBV has been lack of a practical in vitro system that allows infection and replication of this virus under controlled experimental conditions. Several investigators have attempted to infect human hepatic cells by using human adult and fetal hepatocytes (5, 6), fetal hepatocyte organ culture (7), oval cells originating from human liver (8), and HeLa cells (6). However, no signs of viral penetration, replication, or particle production have been observed except for a transient expression of some viral markers.We have established a cell line (HB 611) derived from a human hepatocellular carcinoma cell line (Huh6c15) by transfecting with a plasmid containing tandemly arranged HBV DNA. The HB 611 cells carry chromosomally integrated HBV genomes, and they allow expression of viral RNA, DNA, and proteins and produce Dane-particle-like virions (9). These particles have all the properties characteristic of the Dane particles from the patient's blood (10 MATERIALS AND METHODS Virus Sources and Cell Culture. The cell line HB 611, which produces Dane-particle-like double-shelled particles from integrated HBV DNA, has been described (9). A monolayer cell culture of HB 611 in 10% fetal calf serum/Dulbecco's modified Eagle's medium (10% FCS/DMEM) kept at 37°C was used as the source of infection.Hepatocytes were isolated from human fetal liver by a modification of the methods of Leffert and Paul (13). The liver tissuie was placed immediately in cold 10% FCS/DMEM supplemented with penicillin (100 units/ml) and s...