Adenovirus type 2 DNA replication. I. Evidence for discontinuous DNA synthesis.

Isolated nuclei from adenovirus type 2-infected HeLa cells catalyze the incorporation of all four deoxyribonucleoside triphosphates into viral DNA. The observed DNA synthesis occurs via a transient formation of DNA fragments with a sedimentation coefficient of 10S. The fragments are precursors to unit-length viral DNA, they are self-complementary to an extent of at least 70%, and they are distributed along most of the viral chromosome. In addition, accumulation of 10S DNA fragments is observed either in intact, virus-infected HeLa cells under conditions where viral DNA synthesis is inhibited by hydroxyurea or in isolated nuclei from virus-infected HeLa cells at low concentrations of deoxyribonucleotides. Under these suboptimal conditions for DNA synthesis in isolated nuclei, ribonucleoside triphosphates determine the size distribution of DNA intermediates. The evidence presented suggests that a ribonucleoside-dependent initiation step as well at two DNA polymerase catalyzed reactions are involved in the discontinuous replication of adenovirus type 2 DNA.     

Bidirectional replication of adenovirus type 2 DNA.

After short periods of labeling with [3H]thymidine, recently completed adenovirus DNA molecules were isolated and cleaved with restriction endonucleases. The strands (heavy and light) of most of the restriction endonuclease fragments were separated. The pattern of labeling clearly shows an asymmetry of radioactivity on the isolated strands of each restriction endonuclease piece. The data is consistent with replication proceeding in the 5' to 3' direction on each strand. Thus, there is an initiation point placed at or near each end of the molecule.     

Adenovirus type 5 DNA binding protein stimulates binding of DNA polymerase to the replication origin

The adenovirus (Ad) DNA-binding protein (DBP) is essential for the elongation phase of Ad DNA replication by unwinding the template in an ATP-independent fashion, employing its capacity to form multimers. DBP also enhances the rate of initiation, with the highest levels obtained at low concentrations of Ad DNA polymerase (Pol). Here, we show that stimulation of initiation depends on the template conformation. Maximal stimulation, up to 15-fold, is observed on double-stranded or viral TP-containing origins. The stimulation is reduced on partially single-stranded origins and DBP does not enhance initiation any more once the origin is completely unwound. This suggests a role for DBP in origin unwinding that is comparable to its unwinding capacity during elongation. However, mutant DBP proteins defective in unwinding and elongation can still enhance initiation on ds templates. DBP also stimulates the binding of nuclear factor I (NFI) to the origin and lowers the K(m) for coupling of the first nucleotide to the precursor terminal protein by Pol. Mobility shift experiments reveal that DBP stimulates the binding of Pol on double-stranded origin and nonorigin DNA but not on single-stranded DNA. This effect is specific for DBP and is also seen with other DNA Pols. Our results suggest that, rather than by origin unwinding, DBP enhances initiation by modulating the origin conformation such that DNA Pol can bind more efficiently.     

Adenovirus origin of DNA replication: sequence requirements for replication in vitro.

The initiation of adenovirus DNA takes place at the termini of the viral genome and requires the presence of specific nucleotide sequence elements. To define the sequence organization of the viral origin, we tested a large number of deletion, insertion, and base substitution mutants for their ability to support initiation and replication in vitro. The data demonstrate that the origin consists of at least three functionally distinct domains, A, B, and C. Domain A (nucleotides 1 to 18) contains the minimal sequence sufficient for origin function. Domains B (nucleotides 19 to 40) and C (nucleotides 41 to 51) contain accessory sequences that significantly increase the activity of the minimal origin. The presence of domain B increases the efficiency of initiation by more than 10-fold in vitro, and the presence of domains B and C increases the efficiency of initiation by more than 30-fold. Mutations that alter the distance between the minimal origin and the accessory domains by one or two base pairs dramatically decrease initiation efficiency. This critical spacing requirement suggests that there are specific interactions between the factors that recognize the two regions.     

Adenovirus types 2 and 5 functions elicit replication and late expression of adenovirus type 12 DNA in hamster cells.

A recombinant plasmid harboring both genomic termini of tupaia herpesvirus (THV) DNA was characterized by restriction enzyme analysis and by determination of the nucleotide sequence. A unique NotI cleavage site was found that is located approximately 19 base pairs upstream of the THV terminal junction. THV DNA fragments from virion DNA were analyzed by using the same restriction enzymes as for the recombinant plasmid. The comparative fine mapping of virion THV DNA revealed heterogeneous molecules of variable lengths with the NotI cleavage site conserved. A number of short direct and inverted repeats and palindromes were found surrounding the THV terminal joint. The THV repetitive sequences were compared with the repeats reported for the DNA termini of herpes simplex virus, varicella-zoster virus, and Epstein-Barr virus and are discussed in respect to signals for a site-specific endonuclease required for packaging.     

Termination sites for adenovirus type 2 DNA replication.

Termination sites for replication of adenovirus type 2 DNA have been demonstrated at both ends of the viral chromosome by the procedure of Danna and Nathans (1972). Single-stranded DNA from replicating intermediates was also characterized by hybridization with separated strands of viral DNA. The results indicate that both strands are exposed during replication.     

Human Y-chromatin : II. DNA replication

The DNA synthesis of the human Y-chromatin in its various morphological configurations was studied by labeling with tritiated thymidine (³H-TdR) and autoradiography. Continuous terminal and pulse labeling studies revealed that while condensed, the Y-body lagged in DNA synthesis behind the rest of the nucleus. The highest rate of incorporation of ³H-TdR by the Y-body occurred when it was dispersed. These observations are consistent with the replication characteristics of the Y chromosome as determined by conventional late labeling studies and strongly suggest that the Y-body uncoils at the time it replicates its DNA.     

Synthesis of type 2 Adenovirus DNA in the Presence of Cycloheximide

Adenovirus type 2 DNA synthesis, either in permissive human cells or nonpermissive monkey cells, becomes independent of protein synthesis after the appearance of progeny viral DNA. In the presence of cycloheximide, semiconservative replication and initiation of progeny molecules can occur.     

Adenovirus type 12 DNA sequences in primary hamster tumors.

In five out of six primary hamster tumors induced by adenovirus type 12, less than 55% of the adenovirus type 12 genome is present. Various fragments of the integrated viral DNA were present in non-equimolar amounts.     

Adenovirus type 2 coded single-stranded DNA binding protein: in vivo phosphorylation and modification.

The adenovirus type 2-coded single-stranded DNA binding protein (DBP) was shown to be a phosphoprotein and to exist in at least two forms that differ in mobility by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After a 30-min pulse with [35S]methionine or 32PO4, 35S- or 32P-labeled DBP had a nominal molecular weight of 74,000 whereas after a 30-min label followed by a 20-h chase, 35S- and 32P-labeled DBP had a nominal molecular weight of 77,000. Both large and small forms of 35S- and 32P-labeled DBP bound to single-stranded DNA-cellulose columns and were eluted by 0.4 to 0.6 M NaCl; both forms also were immunoprecipitated by antiserum against adenovirus type 1-simian virus 40-induced tumor cells (this antiserum contains antibodies against DBP) and by monospecific antiserum against 95 to 99% purified DBP. With highly purified 32P-DBP labeled 7 to 10 h postinfection, it was shown that the 32P radioactivity was firmly associated with protein material (i.e., not contaminating nucleic acids or phospholipids) and had properties expected of a phosphoester of an amino acid; paper electrophoresis of acid hydrolysates of this preparation identified phosphoserine but not phosphothreonine. Phosphoserine but not phosphothreonine was also identified in acid hydrolysates of another preparation of 32P-DBP labeled for 30 min, chased for 20 h, and then immunoprecipitated by adenovirus type 1-simian virus 40 antiserum.     

Adenovirus type 40 and 41 growth in vitro: host range diversity reflected by differences in patterns of DNA replication.

Subgroup F adenoviruses adapt poorly to cell culture, but the reasons for their fastidious nature are as yet ill defined. In an attempt to gain an overview of the differences in replication between adenovirus type 2 (Ad2) and representative strains of Ad40 and Ad41, cell lines which show different degrees of permissiveness to Ad40 and Ad41 were infected and examined with respect to three key functions in the Ad2 life cycle: host protein shutoff, DNA synthesis, and late antigen synthesis. The complexity of growth patterns exhibited by the subgroup F adenoviruses suggests that defectiveness is a multifactorial phenomenon not easily explainable by a single aberrant function. Furthermore, results suggest that there may be replicative defects in subgroup F adenoviruses which are not shared by both serotypes or by all strains.     

Adenovirus DNA replication in vitro: duplication of single-stranded DNA containing a panhandle structure

Adenovirus DNA replicates by displacement of one of the parental strands followed by duplication of the displaced parental single strand (complementary strand synthesis). Displacement synthesis has been performed in a reconstituted system composed of viral and cellular proteins, employing either the viral DNA-terminal protein complex as template or linearized plasmids containing the origin. Previously, evidence was obtained that in vivo complementary strand synthesis requires formation of a panhandle structure originating from hybridization of the inverted terminal repeats. To study the conditions for complementary strand synthesis in vitro, we have constructed an artificial panhandle molecule that contains a double-stranded inverted terminal repetition (ITR) region and a single-stranded loop derived from the left and right terminal XmaI fragments of Ad2. Such a molecule appeared to be an efficient template and could initiate by the same protein-priming mechanism as double-stranded DNA, employing the precursor terminal protein. The efficiency of both types of template was comparable. Like for replication of the duplex molecule initiation of panhandle replication was stimulated by nuclear factors I and III, proteins that bind to specific double-stranded regions of the ITR. The Ad DNA-binding protein is essential and the 39 kDa C-terminal domain of this protein that harbors the DNA-binding properties is sufficient for its function. These results support the hypothesis that panhandle formation is required for duplication of the displaced strand.     

Patterns of DNA replication of human chromosomes. II. Replication map and replication model.

Combining higher resolution chromosome analysis and bromodeoxyuridine (BrdU) incorporation, our study demonstrates that: (1) Human chromosomes synthesize DNA in a segmental but highly coordinated fashion. Each chromosome replicates according to its innate pattern of chromosome structure (banding). (2) R-positive bands are demonstrated as the initiation sites of DNA synthesis in all human chromosomes, including late-replicating chromosomes such as the LX and Y. (3) Replication is clearly biphasic in the sense that late-replicating elements, such as G-bands, the Yh, C-bands, and the entire LX, initiate replication after it has been completed in the autosomal R-bands (euchromatin) with minimal or no overlap. The chronological priority of R-band replication followed by G-bands is also retained in the facultative heterochromatin or late-replicating X chromosome (LX). Therefore, the inclusion of G-bands as a truly late-replicating chromatin type or G(Q)-heterochromatin is suggested. (4) Lateral asymmetry (LA) in the Y chromosome can be detected after less than half-cycle in 5-bromodeoxyuridine (BrdUrd), and the presence of at least two regions of LA in this chromosome is confirmed. (5) Finally, the replicational map of human chromosomes is presented, and a model of replication chronology is suggested. Based on this model, a system of nomenclature is proposed to place individual mitoses (or chromosomes) within S-phase, according to their pattern of replication banding. Potential applications of this methodology in clinical and theoretical cytogenetics are suggested.     

Adenovirus cores can function as templates in in vitro DNA replication

Adenovirus cores prepared by gentle disruption of virus by heating at 56 degrees C in the presence of deoxycholate were able to function as templates in an in vitro DNA replication system, allowing both initiation, indicated by the formation of terminal protein-dCMP complex, and elongation of > 300 nucleotides. Using both cores and DNA-protein complexes as templates, it was also demonstrated that novobiocin, an inhibitor of DNA gyrase, inhibited in vitro DNA replication by preventing formation of the initiation complex.     

Adenovirus preterminal protein synthesized in COS cells from cloned DNA is active in DNA replication in vitro.

Replication of the DNA genome of human adenovirus serotype 2 requires three virus-encoded proteins. Two of these proteins, the preterminal protein (pTP) and the adenovirus DNA polymerase, are transcribed from a single promoter at early times after virus infection. The mRNAs for these proteins share several exons, including one encoded near adenovirus genome coordinate 39. By using plasmids containing DNA fragments postulated to encode the various exons of pTP mRNA, the contributions of each exon to the synthesis of an active pTP have been measured. Only plasmids that contain both the open reading frame for pTP (genome coordinates 29.4 to 23.9) and the HindIII J fragment that contains the exon at genome coordinate 39 can express functional pTP.     

Intracellular Forms of Adenovirus DNA III. Integration of the DNA of Adenovirus Type 2 into Host DNA in Productively Infected Cells

KB cells productively infected with human adenovirus type 2 contain an alkalistable class of viral DNA sedimenting in a broad zone between 50 and 90S as compared to 34S for virion DNA. This type of DNA is characterized as viral by DNA-DNA hybridization. It is extremely sensitive to shear fragmentation. Extensive control experiments demonstrate that the fast-sedimenting viral DNA is not due to artifactual drag of viral DNA mechanically trapped in cellular DNA or to association of viral DNA with protein or RNA. Furthermore, the fast-sedimenting DNA is found after infection with multiplicities between 1 and 1,000 PFU/cell and from 6 to 8 h postinfection until very late in infection (24 h). Analysis in dye-buoyant density gradients eliminates the possibility that the fast-sedimenting viral DNA represents supercoiled circular molecules. Upon equilibrium centrifugation in alkaline CsCl density gradients, the fast-sedimenting viral DNA bands in a density stratum intermediate between that of cellular and viral DNA. In contrast, the 34S virion DNA isolated and treated in the same manner as the fast-sedimenting DNA cobands with viral marker DNA. After ultrasonic treatment of the fast-sedimenting viral DNA, it shifts to the density positions of viral DNA and to a lesser extent to that of cellular DNA. The evidence presented here demonstrates that the 50 to 90S viral DNA represents adenovirus DNA covalently integrated into cell DNA.     

Adenovirus sequences required for replication in vivo.

We have studied the in vivo replication properties of plasmids carrying deletion mutations within cloned adenovirus terminal sequences. Deletion mapping located the adenovirus DNA replication origin entirely within the first 67 bp of the adenovirus inverted terminal repeat. This region could be further subdivided into two functional domains: a minimal replication origin and an adjacent auxillary region which boosted the efficiency of replication by more than 100-fold. The minimal origin occupies the first 18 to 21 bp and includes sequences conserved between all adenovirus serotypes. The adjacent auxillary region extends past nucleotide 36 but not past nucleotide 67 and contains the binding site for nuclear factor I.