[Eukaryotic replication fork: three DNA polymerases on two DNA strands].
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Animals↗
Biomedical subjects
T Tsurimoto
Publications and source records attributed to T Tsurimoto.
Replication factors required for SV40 DNA replication in vitro. I. DNA structure-specific recognition of a primer-template junction by eukaryotic DNA polymerases and their accessory proteins.
Eukaryotic DNA polymerase delta and its accessory proteins are essential for SV40 DNA replication in vitro. A multi-subunit protein complex, replication factor C (RF-C), which is composed of subunits with apparent molecular weights of 140,000, 41,000, and 37,000, has primer/template binding and DNA-dependent ATPase activities. UV-cross-linking experiments demonstrated that the Mr = 140,000 subunit recognizes and binds to the primer-template DNA, whereas the Mr = 41,000 polypeptide binds ATP. Assembly of a replication complex at a primer-template junction has been studied in detail with synthetic, hairpin DNAs. Following glutaraldehyde fixation, a gel shift assay demonstrated that RF-C alone forms a weak binding complex with the hairpin DNA. Addition of ATP or its nonhydrolyzable analogue, ATP gamma S, increased specific binding to the DNA. Footprinting experiments revealed that RF-C recognizes the primer-template junction, covering 15 bases of the primer DNA from the 3'-end and 20 bases of the template DNA. Another replication factor, proliferating cell nuclear antigen (PCNA) binds to RF-C and the primer-template DNA forming a primer recognition complex and extends the protected region on the duplex DNA. This RF-C.PCNA complex has significant single-stranded DNA binding activity in addition to binding to a primer-template junction. However, addition of another replication factor, RF-A, completely blocked the nonspecific, single-stranded DNA binding by the RF-C.PCNA complex. RF-A therefore functions as a specificity factor for primer recognition. In the absence of RF-C, DNA polymerase delta (pol delta) and PCNA form a complex at the primer-template junction, protecting exactly the same site as the primer recognition complex. Addition of RF-C to this complex produced a higher order complex which is unstable unless its formation is coupled with translocation of pol delta. These results suggest that the sequential binding of RF-C, PCNA, and pol delta to a primer-template junction might directly account for the initiation of leading strand DNA synthesis at a replication origin. We demonstrate this directly in an accompanying paper (Tsurimoto, T., and Stillman, B. (1991) J. Biol. Chem. 266, 1961-1968).
Adenosine Triphosphate↗
Replication factors required for SV40 DNA replication in vitro. II. Switching of DNA polymerase alpha and delta during initiation of leading and lagging strand synthesis.
Replication factors A and C (RF-A and RF-C) and the proliferating cell nuclear antigen (PCNA) differentially augment the activities of DNA polymerases alpha and delta. The mechanism of stimulation by these replication factors was investigated using a limiting concentration of primed, single-stranded template DNA. RF-A stimulated polymerase alpha activity in a concentration-dependent manner, but also suppressed nonspecific initiation of DNA synthesis by both polymerases alpha and delta. The primer recognition complex, RF-C.PCNA.ATP, stimulated pol delta activity in cooperation with RF-A, but also functioned to prevent abnormal initiation of DNA synthesis by polymerase alpha. Reconstitution of DNA replication with purified factors and a plasmid containing the SV40 origin sequences directly demonstrated DNA polymerase alpha dependent synthesis of lagging strands and DNA polymerase delta/PCNA/RF-C dependent synthesis of leading strands. RF-A and the primer recognition complex both affected the relative levels of leading and lagging strands. These results, in addition to results in an accompanying paper (Tsurimoto, T., and Stillman, B. (1991) J. Biol. Chem. 266, 1950-1960), suggest that an exchange of DNA polymerase complexes occurs during initiation of bidirectional DNA replication at the SV40 origin.
Adenosine Triphosphate↗
Three envelope proteins of hepatitis B virus: large S, middle S, and major S proteins needed for the formation of Dane particles.
The infectious particles of hepatitis B virus are called Dane particles and consist of viral nucleic acid encapsulated within a core particle that is enveloped by virus-coded surface proteins. The major S protein constitutes a significant fraction of these surface proteins. In addition, there are two other related proteins (large S and middle S), but their role in envelope formation has not yet been elucidated. We modified the translation initiation codon ATG of each of the envelope proteins by site-directed mutagenesis and found that mutant genomes that did not produce one or two of these proteins were unable to form Dane particles. The particles released into the culture medium by such mutants did not carry DNA. Synthesis of virus-coded RNA still occurred normally, and core particles carrying DNA accumulated intracellularly. The DNA in such core particles was mostly in the double-stranded open circular form, in contrast to the normal situation in which the particles contain mostly RNA and its complementary single-stranded DNA or else contain linear DNA that is partially single stranded and otherwise duplex. The role of the large S and middle S proteins in the formation of Dane particles is discussed.
Blotting, Northern↗
Sequential initiation of lagging and leading strand synthesis by two different polymerase complexes at the SV40 DNA replication origin.
Enzymatic synthesis of DNA from the simian virus 40 origin of DNA replication has been reconstituted in vitro with eight purified components. DNA polymerase alpha-primase complex first initiates DNA synthesis at the replication origin and continues as the lagging strand polymerase. Subsequently, the DNA polymerase delta complex initiates replication on the leading strand template. Some prokaryotic DNA polymerase complexes can replace the eukaryotic polymerase delta complex. A model for polymerase switching during initiation of DNA replication is presented.
Animals↗
Functions of replication factor C and proliferating-cell nuclear antigen: functional similarity of DNA polymerase accessory proteins from human cells and bacteriophage T4.
The proliferating-cell nuclear antigen (PCNA) and the replication factors A and C (RF-A and RF-C) are cellular proteins essential for complete elongation of DNA during synthesis from the simian virus 40 origin of DNA replication in vitro. All three cooperate to stimulate processive DNA synthesis by DNA polymerase delta on a primed single-stranded M13 template DNA and as such can be categorized as DNA polymerase accessory proteins. Biochemical analyses with highly purified RF-C and PCNA have demonstrated functions that are completely analogous to the functions of bacteriophage T4 DNA polymerase accessory proteins. A primer-template-specific DNA binding activity and a DNA-dependent ATPase activity copurified with the multisubunit protein RF-C and are similar to the functions of the phage T4 gene 44/62 protein complex. Furthermore, PCNA stimulated the RF-C ATPase activity and is, therefore, analogous to the phage T4 gene 45 protein, which stimulates the ATPase function of the gene 44/62 protein complex. Indeed, some primary sequence similarities between human PCNA and the phage T4 gene 45 protein could be detected. These results demonstrate a striking conservation of the DNA replication apparatus in human cells and bacteriophage T4.
Adenosine Triphosphatases↗
Multiple replication factors augment DNA synthesis by the two eukaryotic DNA polymerases, alpha and delta.
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.
Bacterial Proteins↗
An in vitro system for screening anti-hepatitis B virus drugs.
A human hepatoblastoma cell line (HB 611) that continuously synthesizes hepatitis B viral (HBV) DNA was grown in the presence of various inhibitors of DNA synthesis, and the DNA from the cells was analyzed by the Southern blotting method to examine selective inhibition of the viral DNA synthesis. Among those that showed selective inhibition, and interferons alpha and beta, acyclovir, and dideoxy cytidine were effective. This system should be useful for screening new antiviral agents against HBV.
Acyclovir↗
An in vitro system for infection with hepatitis B virus that uses primary human fetal hepatocytes.
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.
Carcinoma, Hepatocellular↗
Demethylation by 5-azacytidine results in the expression of hepatitis B virus surface antigen in transgenic mice.
In 14p3HB transgenic mice, which carry three tandem copies of hepatitis B virus (HBV) DNA, the HBV DNA was significantly methylated and no viral proteins were produced. To analyze the causal relationship between hypermethylation and gene inactivity, 5-azacytidine was injected into the mice to demethylate HBV DNA. When postnatal 14p3HB mice were treated with the drug, hepatitis virus surface antigen was produced in these mice by 3 weeks of age, and the integrated HBV DNA of the liver was less heavily methylated. Our results suggest that injection of 5-azacytidine can be used to efficiently activate a silent transgene such as HBV DNA in transgenic mice.
Animals↗
Purification of a cellular replication factor, RF-C, that is required for coordinated synthesis of leading and lagging strands during simian virus 40 DNA replication in vitro.
Cell extracts (S100) derived from human 293 cells were separated into five fractions by phosphocellulose chromatography and monitored for their ability to support simian virus 40 (SV40) DNA replication in vitro in the presence of purified SV40 T antigen. Three fractions, designated I, IIA, and IIC, were essential. Fraction IIC contained the known replication factors topoisomerases I and II, but in addition contained a novel replication factor called RF-C. The RF-C activity, assayed in the presence of I, IIA, and excess amounts of purified topoisomerases, was detected in both cytosol and nuclear fractions, but was more abundant in the latter fraction. RF-C was purified from the 293 cell nuclear fraction to near homogeneity by conventional column chromatography. The reconstituted reaction mix containing purified RF-C could replicate SV40 origin-containing plasmid DNA more efficiently than could the S100 extract, and the products were predominantly completely replicated, monomer molecules. Interestingly, in the absence of RF-C, early replicative intermediates accumulated and subsequent elongation was aberrant. Hybridization studies with strand-specific, single-stranded M13-SV40 DNAs showed that in the absence of RF-C, abnormal DNA synthesis occurred preferentially on the lagging strand, and leading-strand replication was inefficient. These products closely resembled those previously observed for SV40 DNA replication in vitro in the absence of proliferating-cell nuclear antigen. These results suggest that an elongation complex containing RF-C and proliferating-cell nuclear antigen is assembled after formation of the first nascent strands at the replication origin. Subsequent synthesis of leading and lagging strands at a eucaryotic DNA replication fork can be distinguished by different requirements for multiple replication components, but we suggest that even though the two polymerases function asymmetrically, they normally progress coordinately.
Cell-Free System↗
Simian virus 40 DNA replication in vitro: identification of multiple stages of initiation.
A cell-free DNA replication system dependent upon five purified cellular proteins, one crude cellular fraction, and the simian virus 40 (SV40)-encoded large tumor antigen (T antigen) initiated and completed replication of plasmids containing the SV40 origin sequence. DNA synthesis initiated at or near the origin sequence after a time lag of approximately 10 min and then proceeded bidirectionally from the origin to yield covalently closed, monomer daughter molecules. The time lag could be completely eliminated by a preincubation of SV40 ori DNA in the presence of T antigen, a eucaryotic single-stranded DNA-binding protein (replication factor A [RF-A]), and topoisomerases I and II. In contrast, if T antigen and the template DNA were incubated alone, the time lag was only partially decreased. Kinetic analyses of origin recognition by T antigen, origin unwinding, and DNA synthesis suggest that the time lag in replication was due to the formation of a complex between T antigen and DNA called the T complex, followed by formation of a second complex called the unwound complex. Formation of the unwound complex required RF-A. When origin unwinding was coupled to DNA replication by the addition of a partially purified cellular fraction (IIA), DNA synthesis initiated at the ori sequence, but the template DNA was not completely replicated. Complete DNA replication in this system required the proliferating-cell nuclear antigen and another cellular replication factor, RF-C, during the elongation stage. In a less fractionated system, another cellular fraction, SSI, was previously shown to be necessary for reconstitution of DNA replication. The SSI fraction was required in the less purified system to antagonize the inhibitory action of another cellular protein(s). This inhibitor specifically blocked the earliest stage of DNA replication, but not the later stages. The implications of these results for the mechanisms of initiation and elongation of DNA replication are discussed.
Antigens, Viral, Tumor↗
Replication of SV40 in vitro using proteins derived from a human cell extract.
In the presence of large T antigen and plasmids containing a functional origin of replication, extracts from a human cell line will support multiple rounds of simian virus 40 (SV40) replication in vitro. Fractionation of this extract has led to the identification of several factors, some of which have been purified to homogeneity. The characterisation of these proteins has led to the separation of SV40 replication in vitro into multiple stages. Two proteins, the cell cycle-regulated proliferating cell nuclear antigen and replication factor-C, have been shown to be essential for coordinating leading and lagging strand synthesis in this system. Another protein, replication factor-A, is a multi-subunit protein of 70, 34 and 11K (K = 10(3) Mr) polypeptides which, because of its high affinity for DNA, is thought to function as a eukaryotic single-stranded DNA binding protein. Interactions between other cellular factors are also described that effect the initiation of DNA replication, but are not required in a more purified system. In addition a model for a hypothetical replication fork is described, which suggests a role for both alpha- and delta-polymerases in this system, and may be applicable to higher eukaryotes.
Antigens, Viral↗
Anti-hepatitis B virus activities of purine derivatives of oxetanocin A.
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Adenine↗
Identification of cellular components required for SV40 DNA replication in vitro.
To investigate the cellular proteins involved in simian virus 40 (SV40) replication, extracts derived from human 293 cells have been fractionated into multiple components. When such fractions are combined with the virus-encoded T antigen (TAg) and SV40 origin containing plasmid DNA, efficient and complete replication is achieved, while each fraction alone is inactive. At present, a minimum of eight such cellular components have been identified. Previous experiments have demonstrated one of these to be the cell-cycle-regulated proliferating-cell nuclear antigen (PCNA). As PCNA has been identified as a processivity factor for DNA polymerase delta, we suggest that both polymerases alpha and delta are involved in this system. Three further fractions have been identified. One is a partially purified fraction which, under certain conditions, is required with TAg for the formation of a pre-synthesis complex of proteins at the replication origin. The second of these factors, RF-A, is a complex of three polypeptides which may function as a eucaryotic SSB. The third, RF-C, is a factor which is required, with PCNA, for coordinated leading- and lagging-strand synthesis at the replication fork. Complete synthesis and segregation of the daughter molecules also requires the presence of topoisomerases I and II. These results suggest a model for DNA synthesis which involves multiple stages prior to and during replicative DNA synthesis.
Adenosine Triphosphate↗
Direction of transcription affects the replication mode of lambda in an in vitro system.
A set of artificial circular plasmids, named plasmoids, was constructed. They are about 1 kb in size and consist of a 178 bp lambda dv minimal DNA replication origin (ori) which has four direct repeats and the A + T-rich region conferring polarity to the ori fragment, a 775 bp DNA segment that codes for the CAT amino acid sequence and the 99 bp lac promoter (plac). They carry no other functional genes or genetic sites. The constructions involved various combinations of relative orientations of these components. These molecules do not replicate in vivo because they lack genes coding for initiation proteins, but they do replicate in an in vitro system (Fuller et al. 1981), and can be used for studies of interactions between transcription and replication. In these plasmids, major transcription starts from the strong plac, and some weak unscheduled transcription starts from several other initiation sites. The major RNA synthesis was found to interfere with the unscheduled RNA synthesis, which was occurring on the opposite strand. The most active replication took place when the major RNA synthesis went through the lambda origin region in the direction which occurs naturally in the lambda genome. Under these conditions, DNA synthesis going against such transcription was less than that going along with the major transcription. When RNA synthesis through the lambda origin region was in the opposite direction, DNA synthesis in the same direction was about half of that observed in the above case, whereas that going against transcription was very weak.
Bacteriophage lambda↗
Purification of hepatitis B virus gene X product synthesized in Escherichia coli and its detection in a human hepatoblastoma cell line producing hepatitis B virus.
A fused gene containing 94% of the hepatitis B virus (HBV) open reading frame X was expressed in Escherichia coli, and its 17-kDa product was purified by ion-exchange chromatography. Antibody elicited against the X-gene product reacted with materials proximal to the nuclear membrane of a human hepatoblastoma cell line producing HBV particles. No such reaction was observed with the same cell line that did not produce HBV particles.
Carcinoma, Hepatocellular↗
Stable expression and replication of hepatitis B virus genome in an integrated state in a human hepatoma cell line transfected with the cloned viral DNA.
A human hepatocellular carcinoma cell line (Huh6-c15) was transfected with a recombinant DNA molecule that consists of tandemly arranged hepatitis B virus (HBV) genome and a neomycin-resistant gene. One clone resistant to G-418 produces and releases surface antigen and e antigen into medium at a high level and accumulates core particles intracellularly. This clone has a chromosomally integrated set of the original recombinant DNA and produces a 3.5-kilobase transcript corresponding to the pregenome RNA as well as HBV DNAs in an extrachromosomal form. Most of these DNAs were in single-stranded or partially double-stranded form and were packaged in the intracellular core particles. In the medium, particles were detected that contained HBV DNA and were morphologically indistinguishable from Dane particles. These results demonstrate that the HBV genome in an integrated state acted as a template for viral gene expression and replication. The cells were maintained for more than 6 months without losing the ability to produce the extrachromosomal HBV DNA and Dane-like particles. Thus, the cells can be used as a model system for analyses of gene expression and DNA replication of HBV in human hepatocytes.
Carcinoma, Hepatocellular↗