Adenovirus DNA replication in vitro: identification of a host factor that stimulates synthesis of the preterminal protein-dCMP complex.

A protein factor that participates in the formation of a covalent complex between the 80,000-dalton precursor of the adenovirus (Ad) terminal protein (pTP) and 5'-dCMP has been isolated and characterized. This 47,000-dalton protein, isolated from nuclear extracts of uninfected HeLa cells, has been designated nuclear factor I. It is free of detectable DNA polymerase alpha, beta, and gamma activities. In the presence of Ad DNA-prot, the Ad-protein fraction (containing the pTP and the Ad-associated DNA polymerase), ATP, Mg2+, and dCTP, nuclear factor I stimulates formation of the pTP-dCMP complex. Addition of the Ad DNA binding protein (Ad DBP) renders the formation of the pTP-dCMP complex completely dependent on the addition of nuclear factor I. When Ad DNA-prot is replaced with phi X174 single-stranded circular DNA, pTP-dCMP complex formation requires only the Ad-protein fraction; Ad DBP and ATP are inhibitory and nuclear factor I has no effect on this reaction. This suggests that the initiation reaction observed with Ad DNA-prot in the absence of Ad DBP occurs at single-stranded DNA sites. In the presence of Ad DBP, these sites are blocked thus creating a requirement for nuclear factor I in pTP-dCMP complex formation.     

Template requirements for the initiation of adenovirus DNA replication.

The first step in the replication of the adenovirus genome is the covalent attachment of the 5'-terminal nucleotide, dCMP, to the virus-encoded terminal protein precursor (pTP). This reaction can be observed in vitro and has been previously shown to be dependent upon either viral DNA or linearized plasmid DNA containing viral terminal sequences. Plasmids containing deletions or point mutations within the viral terminal sequence were constructed by site-directed mutagenesis. In the case of linear double-stranded templates, pTP-dCMP formation required sequences located within the first 18 base pairs of the viral genome. This sequence contains a segment of 10 base pairs that is conserved in all human adenovirus serotypes. Point mutations within the conserved segment greatly reduced the efficiency of initiation, while a point mutation at a nonconserved position within the first 18 base pairs had little effect. Single-stranded DNAs can also support pTP-dCMP formation in vitro. In contrast to the results obtained with duplex templates, experiments with a variety of single-stranded templates, including phage M13-adenovirus recombinants, denatured plasmids, and synthetic oligodeoxynucleotides, failed to reveal any requirements for specific nucleotide sequences. With single-stranded templates containing no dG residues, the specific deoxynucleoside triphosphate requirements of the initiation reaction were altered.     

Separation of the adenovirus terminal protein precursor from its associated DNA polymerase: role of both proteins in the initiation of adenovirus DNA replication.

A complex containing the 80,000-dalton precursor to the adenovirus (Ad)-encoded terminal protein (pTP) and a 140,000-dalton protein is required for Ad DNA replication in vitro. This complex has been separated into subunits by glycerol gradient centrifugation in the presence of urea. The isolated 140,000-dalton subunit contains a DNA polymerase activity which can be differentiated from all host DNA polymerases. No enzyme activity was detected with the isolated pTP. The requirements for reactions involved in the initiation of Ad DNA replication were determined by using the isolated subunits. The covalent addition of dCMP, the first nucleotide in the DNA chain, to the pTP, which serves as the primer for replication, required the DNA polymerase subunit as well as the pTP. Synthesis of viral DNA in vitro also required both subunits. The properties of the DNA polymerase suggest that it may be a viral gene product.     

Adenovirus DNA replication in vitro: purification of the terminal protein in a functional form.

The 80,000-dalton form of the adenovirus (Ad) terminal protein (pTP) has been purified from Ad-infected HeLa cells. pTP was assayed by its ability to form a covalent complex with dCMP. The protein copurified with an activity that is essential for in vitro Ad DNA replication (Ad protein activity) as well as with a DNA polymerase activity that was distinguished from those of HeLa cell DNA polymerases alpha, beta, and gamma. The Ad protein-associated DNA polymerase activity was detected with activated DNA but not with poly(rA).oligo(dT) as template and was insensitive to aphidicolin and sensitive to N-ethylmaleimide. The Ad protein, DNA polymerase, and pTP-dCMP complex-forming activities sedimented in a glycerol gradient as a single peak with an apparent molecular size of 180,000 daltons. NaDodSO4/polyacrylamide gel analysis of the glycerol gradient fraction showed major bands of 80,000 and 140,000 daltons. The 80,000-dalton band was identified as pTP by comparison of its tryptic peptide map with that of the 55,000-dalton form of the terminal protein, which was purified from Ad virions.     

Adenovirus DNA replication in vitro: synthesis of full-length DNA with purified proteins.

A protein required for the elongation of replicating intermediates of adenovirus (Ad) DNA to full length has been isolated and characterized. This factor, isolated from nuclear extracts of uninfected HeLa cells, has been designated nuclear factor II. In the presence of Ad DNA with proteins at each 5' end (Ad DNA-protein) and three proteins coded for by the Ad genome [the preterminal protein (pTP), the DNA polymerase (Ad Pol), and the DNA binding protein (Ad DBP)], nuclear factor II complementing activity is detected only in the presence of host nuclear factor I. Highly purified preparations of nuclear factor II that are free of detectable DNA polymerase alpha, beta, and gamma activities contain a DNA topoisomerase activity. Furthermore, type I DNA topoisomerases purified from HeLa cells and calf thymus substitute for nuclear factor II complementing activity in the in vitro Ad DNA replication system. These results indicate that a protein that is involved in higher order DNA structure is required for Ad replication. This protein plus the purified proteins described above carry out the initiation and synthesis of full-length 36,000-base-pair Ad DNA.     

Specific binding of a cellular DNA replication protein to the origin of replication of adenovirus DNA.

Nuclear factor I, a 47-kilodalton protein, purified from nuclear extracts of uninfected HeLa cells, is involved in the initiation and possibly the elongation of replicating adenovirus (Ad) DNA in vitro. The binding of nuclear factor I to DNA has been monitored by a filter binding assay of nuclear factor I to DNA has been monitored by a filter binding assay using plasmid pLA1 DNA, which contains a 3,290 base-pair fragment derived from the left-hand terminus (coordinates, 0-9.4 map units) of Ad serotype 5 DNA. Nuclear factor I binds selectively to a double-stranded fragment spanning nucleotides 0-451 to the Ad genome. The retention of the 451-base-pair DNA fragment-nuclear factor I complex on nitrocellulose filters does not require Mg2+ or ATP and is resistant to high ionic strength. DNase I protection experiments revealed that nuclear factor I binds to a nucleotide sequence located at position 17-48, close to the terminus of Ad DNA. This 32-nucleotide sequence contains four "consensus" sequences present in various serotypes of Ad DNA and is capable of forming higher ordered structures. The role of nuclear factor I and this DNA sequence in the generation of Ad preterminal protein-dCMP initiation complex is discussed.     

Function of adenovirus terminal protein in the initiation of DNA replication.

An early event in the initiation of adenovirus DNA replication is the formation of a covalent complex between the 87,000-dalton adenovirus terminal protein precursor and 5'- dCMP (pTP-dCMP complex). Nuclear extracts prepared from adenovirus-infected HeLa cells catalyzed complex formation in the presence of ATP, Mg2+, and adenovirus DNA-protein complex but were not active when Pronase-treated DNA was used as template. The activity has been partially purified by chromatography on denatured DNA-cellulose and used to examine whether the 55,000-dalton terminal protein on adenovirus DNA is required for pTP-dCMP complex formation. Results obtained with either DNA-protein complex or Pronase-treated DNA were identical to those obtained using crude nuclear extracts. However, after treatment with piperidine to remove residual peptides. Pronase-treated DNA supported complex formation with the partially purified activity but not with the crude extracts. In addition, when a plasmid containing an origin of adenovirus DNA replication was used as template, the pTP-dCMP complex was formed provided the plasmid was linearized in such a way that the origin was located at the end of the molecule. Neither linearized plasmid DNA with an internal origin nor supercoiled plasmid DNA supported complex formation. Furthermore, after heat denaturation, the linear plasmid DNA still supported complex formation, again provided that the origin was located at the end of the molecule. The partially purified protein fraction supported a limited amount of DNA chain elongation, which permitted exact positioning of the initiation site. These results suggest that enzymes responsible for complex formation recognize a DNA sequence at the origin and that the terminal protein on the template DNA plays a subordinate role.     

The 140-kDa adenovirus DNA polymerase is recognized by antibodies to Escherichia coli-synthesized determinants predicted from an open reading frame on the adenovirus genome.

Sequence studies of the adenovirus 2 genome have revealed the presence of a large open reading frame (ORF) from 22.9 to 14.2 map units that is believed to encode most of the adenovirus DNA polymerase (Ad Pol). An 838-base-pair fragment (19.6-17.3 map units) containing approximately 25% of this ORF has been cloned and expressed in a beta-galactosidase-chloramphenicol acetyltransferase (lacZ-CAT) expression vector under the control of the trp-lac hybrid promoter. This recombinant vector directed the synthesis of a 58-kDa lacZ-Ad Pol-CAT fusion protein that has CAT activity. This fusion protein was easily purified by affinity chromatography in which chloramphenicol, the substrate for CAT, was covalently bound to a matrix. Antisera were prepared against the purified 58-kDa lacZ-Ad Pol-CAT fusion protein and were found to react specifically with the 140-kDa Ad Pol by ELISA and immunoblot analysis. In addition, these antisera recognized 120- and 29-kDa polypeptides in immunoblot analysis of partially purified terminal protein precursor (pTP)-Ad Pol complex. The exact nature of the 120- and 29-kDa polypeptides is not known, but they may be breakdown products of Ad Pol. Although the lacZ-Ad Pol-CAT fusion protein is not active in any of the Ad Pol enzymatic reactions, antibody against the prokaryotic fusion protein should be useful for screening bacteria harboring plasmids that have been constructed to express the entire Ad Pol ORF.     

Reconstitution of simian virus 40 DNA replication with purified proteins.

Replication of plasmid DNA molecules containing the simian virus 40 (SV40) origin of DNA replication has been reconstituted with seven highly purified cellular proteins plus the SV40 large tumor (T) antigen. Initiation of DNA synthesis is absolutely dependent upon T antigen, replication protein A, and the DNA polymerase alpha-primase complex and is stimulated by the catalytic subunit of protein phosphatase 2A. Efficient elongation of nascent chains additionally requires proliferating cell nuclear antigen, replication factor C, DNA topoisomerase I, and DNA polymerase delta. Electron microscopic studies indicate that DNA replication begins at the viral origin and proceeds via intermediates containing two forks that move in opposite directions. These findings indicate that the reconstituted replication reaction has many of the characteristics expected of authentic viral DNA replication.     

Rolling circle DNA replication in vitro by a complex of herpes simplex virus type 1-encoded enzymes.

Extracts of insect cells infected with baculoviruses recombinant for the herpes simplex virus 1 (HSV-1)-encoded enzymes that are required for its replication can promote the rolling circle replication of circular plasmid templates. Replication is independent of a HSV-1 origin of replication (oris) or the HSV-1 origin binding protein and is inhibited by the origin binding protein when the plasmid contains oris. Replication is dependent on a complex composed of the HSV-1-encoded DNA polymerase and its processivity enhancing factor (the UL42 protein), ICP8 (the HSV-1-encoded single-strand DNA binding protein), and the HSV-1-encoded helicase-primase. The complex can be purified by size-exclusion and anion-exchange chromatography.     

Escherichia coli Tus protein acts to arrest the progression of DNA replication forks in vitro.

A polar DNA replication barrier is formed when the DNA-binding protein Tus forms a complex with any of the four 23-base-pair terminator (ter) sites found in the terminus region of the Escherichia coli chromosome. We have used a plasmid DNA replication system reconstituted with purified proteins in vitro to investigate the interaction of the Tus protein with the replication fork. Purified Tus protein alone is necessary and sufficient to arrest DNA replication on ColE1-type plasmid templates containing ter sites. Tus protein-catalyzed termination depends upon the orientation of the ter site in the plasmid DNA. Nucleotide resolution mapping of the terminated nascent DNA shows that leading-strand DNA synthesis arrests at the point of contact with the Tus protein, while the final lagging-strand primer sites are 50-70 nucleotides upstream. In addition, the distribution of leading-strand arrest sites changes when the composition of the proteins on the lagging-strand side of the replication fork is altered.     

Evidence for an altered adenovirus DNA polymerase in cells infected with the mutant H5ts149

The N complementation group of adenovirus (Ad) serotype 5 mutants, which are temperature sensitive for viral DNA synthesis in vivo, has been used to study a 140,000-dalton DNA polymerase (Pol) that copurified with the 80,000-dalton terminal protein precursor (pTP). Extracts prepared from HeLa cells infected with the N group mutant H5ts149 at nonpermissive temperature were unable to synthesize viral DNA. The defect in these extracts was specifically reversed by addition of the Pol purified from wild-type Ad-infected cytosol. Addition of the pTP, free of the Pol, did not restore replicative activity to H5ts149 extracts. The reactions studied depend on the presence of the DNA template and include the initiation reaction (the covalent attachment of dCMP to the pTP) and the selective replication of Ad DNA restriction endonuclease fragments containing the origin sequences. Glycerol gradient sedimentation showed that a replicative activity representing the pTP-Pol complex was greatly reduced in H5ts149 extracts as compared with wild-type extracts, suggesting some alteration in the mutant. A pool of pTP free of Pol was detected on these gradients in extracts from both wild-type and H5ts149-infected cells. In addition, the initiation and elongation of Ad DNA catalyzed by H5ts149 extracts prepared from cells grown at permissive temperatures was more labile to urea inactivation than extracts prepared from cells infected with wild-type virus. These results, considered together with the mapping of the H5ts149 mutation within an open reading frame approximately large enough to code for the 140,000-dalton DNA polymerase [Gingeras, T. R., Sciaky, D., Gelinas, R. E., Bing-Dong, J., Yen, C. E., Kelly, M. M., Bullock, P. A., Parsons, B. L., O'Neill, K. E. & Roberts, R. J. (1982) J. Biol. Chem. 257, 13475-13491; Alestrom, P., Akusjarui, G., Pettersson, M. & Pettersson, U. (1982) J. Biol. Chem. 257, 13492-13498], suggest that the Pol is a virally encoded protein, as is the pTP.     

Identification of a primosome assembly site in the region of the ori 2 replication origin of the Escherichia coli mini-F plasmid.

A primosome assembly site for F plasmid DNA replication has been identified. This site, which we term rriA (F), is localized to one strand of a 385-base-pair Sau3A restriction fragment very close to ori 2 and within the 2.25-kilobase DNA sequence required for replication and incompatibility of the entire F plasmid. rriA (F) was isolated by cloning into the deletion phage vector M13 delta Elac. This phage forms very faint plaques due to a deletion of the M13 complementary strand origin but forms large wild-type plaques when DNA single-strand initiation determinants are inserted. The single-stranded viral DNA of the Sau3A F-M13 delta Elac recombinant provides an effector site of dATP hydrolysis by the primosomal protein n'. It also provides an assembly site for the Escherichia coli primosome protein complex that directs the in vitro conversion of the single-stranded DNA to a double-stranded form by the same mechanism as that used by phi X174. Homologies of the nucleotide sequence between this F DNA sequence and the previously identified primosome assembly sites in phi X174 phage DNA and in ColE1 plasmid DNA (rriA and rriB) have been found. The sequences 5' G-T-G-A-G-C-G 3' and 5' G-N-G-G-A-A-G-C 3' or variations of these sequences occur from two to five times within each assembly locus. In addition, two distinct 15-base-pair sequences in rriA (F) are perfectly homologous to corresponding sequences in rriA (ColE1).     

Specific interaction between a Saccharomyces cerevisiae protein and a DNA element associated with certain autonomously replicating sequences.

We have isolated a protein from Saccharomyces cerevisiae that binds specifically to a nucleotide sequence associated with the autonomously replicating sequence (ARS) ARS120, located in the telomeric region of a yeast chromosome. "Footprinting" analysis revealed that a 26-base-pair DNA sequence, 5'-CAAGTGCCGTGCATAATGATGTGGGT-3', was protected by this protein from DNase I digestion. A plasmid containing 48 direct tandem repeats of this oligonucleotide was constructed and used to affinity-purify the binding activity. The purified protein, OBF1 (origin binding factor), showed specific binding to ARS120. The 26-base-pair OBF1-protected sequence was sufficient for the recognition and binding of the protein, since the mobility of a DNA fragment containing the synthetic binding site was retarded in agarose gels when incubated with OBF1. By performing competition experiments with a number of different ARSs, we showed that OBF1 binds tightly to some but not all ARSs. Interestingly, OBF1 does not appear to have a discernible affinity for ARS1 or the ARSs associated with mating type loci, HML alpha and HMRa, which are substrates for a DNA-binding activity reported by others. Since OBF1 appears to bind to DNA associated with a number of ARSs, we suggest that this protein may have a function related to ARS activity, perhaps in the initiation of DNA replication at selected ARSs.     

A DNA binding protein specific for an origin of replication of herpes simplex virus type 1.

We have identified a protein that binds specifically to an origin of replication (oris) of the herpes simplex virus type 1 genome. The oris binding protein, detectable only in nuclear extracts of infected cells, shows the same time course of appearance as the herpesvirus-induced DNA polymerase and the DNA binding protein ICP8. The partially purified oris binding protein generates a DNase I "footprint" that spans 18- of the 90-base-pair minimal oris sequence. The oris binding protein may, therefore, be analogous to other origin-specific binding proteins that are required for the initiation of viral and chromosomal DNA replication.     

Purification and properties of the mini-F plasmid-encoded E protein needed for autonomous replication control of the plasmid.

Mini-F plasmid encodes a protein, E protein, that is indispensable for its autonomous replication. We have constructed a plasmid that overproduces the E protein and have purified the protein to apparent homogeneity. Using nitrocellulose filter binding and nuclease digestion assays, we demonstrated that the E protein binds to three unique regions of the mini-F DNA sequence: the replication origin (ori2) and an incompatibility locus (incB), another incompatibility locus (incC), and the promoter for the E gene. These binding sites have a common 8-base-pair sequence. These findings suggest the direct role of the E protein in initiation of mini-F replication and copy number control. They are also in line with the in vivo evidence that the incompatibility phenotype caused by incB and incC DNA is due to titration of a factor(s) indispensable for replication and that the production of the E initiator protein of the mini-F plasmid is under autoregulatory control.     

A host-encoded DNA-binding protein promotes termination of plasmid replication at a sequence-specific replication terminus.

We have purified approximately 6600-fold an approximately 40-kDa protein (Ter protein) encoded by Escherichia coli that specifically binds to two sites at the 216-base-pair replication terminus (tau) of the plasmid R6K. Chemical footprinting experiments have shown that the Ter protein binds to two 14- to 16-base-pair sequences that exist as inverted repeats in the tau fragment. Site-directed mutagenesis of one of the terminus sequences (tau R) resulted in a mutant tau R that failed to bind to the Ter protein. The same mutant terminus also failed to terminate DNA replication in vivo. These experiments strongly suggest that the interaction of the Ter protein with tau sequences plays an essential role in the termination of DNA replication, specifically at tau.