Topoisomerase I sites cluster asymmetrically at the ends of the simian virus 40 core origin of replication.

In vivo, topoisomerase I cleavage sites are located predominantly on the strands of simian virus 40 DNA that are the templates for discontinuous synthesis (S.E. Porter and J.J. Champoux, Mol. Cell. Biol. 9:541-550, 1989). This arrangement of sites suggests that topoisomerase I may associate with replication complexes in unique functional orientations at replication forks. We have mapped topoisomerase I cleavage sites in the simian virus 40 origin of replication in vitro under conditions suitable for DNA replication. Numerous sites cluster in the inverted repeat and AT-rich domains at the ends of the core origin and are arranged on the same strands that are cut most frequently in vivo. We propose that cleavage at these sites would allow bidirectional extension of the replication bubble induced by T antigen within the core origin of replication early in the initiation of DNA synthesis. A mutational analysis of the topoisomerase I sites confirms the importance of positions -4 to -1 and +1 in the consensus sequence 5'-A/T-A/G-A/T-T-break-G/A-3'. Surprisingly, more distant nucleotide positions also influence topoisomerase I sites in the inverted repeat and AT-rich domains of the core origin. The effects of distant sequences could be mediated by direct interactions with topoisomerase I or by the conformation of DNA in the core origin.     

Partial purification and characterization of a cellular protein that binds to the SV40 core origin of DNA replication

A monkey cell factor that interacts specifically with double- and single-stranded DNA sequences in the early domain of the simian virus 40 (SV40) core origin of replication was identified using gel-retention assays. The protein was enriched over 1200-fold using ion-exchange and affinity chromatography on single-strand DNA cellulose. Binding of protein to mutant origin DNA restriction fragments was correlated with replication activity of the mutant DNAs. Exonuclease footprint experiments on single-stranded DNA revealed prominent pause sites in the early domain of the core origin. The results suggest that this cellular protein may be involved in SV40 DNA replication.     

Partial purification and characterization of a cellular protein that binds to the SV40 core origin of DNA replication

A monkey cell factor that interacts specifically with double- and single-stranded DNA sequences in the early domain of the simian virus 40 (SV40) core origin of replication was identified using gel-retention assays. The protein was enriched over 1200-fold using ion-exchange and affinity chromatography on single-strand DNA cellulose. Binding of protein to mutant origin DNA restriction fragments was correlated with replication activity of the mutant DNAs. Exonuclease footprint experiments on single-stranded DNA revealed prominent pause sites in the early domain of the core origin. The results suggest that this cellular protein may be involved in SV40 DNA replication.     

Chromatin structure of simian virus 40-pBR322 recombinant plasmids in COS-1 cells.

To study the nucleoprotein structure formed by recombinant plasmid DNA in mammalian cells, nuclei were isolated from COS-1 cells after transfection with a recombinant (pJI1) containing pBR322 sequences and a segment of simian virus 40 containing information for a nuclease-sensitive chromatin structure. The nuclei were incubated with DNase I. DNA fragments which were the size of linear pJI1 DNA were isolated, redigested with restriction enzymes, fractionated by electrophoresis, and detected by hybridization with nick-translated segments prepared from the plasmid DNA. Two DNase I-sensitive sites were detected in the simian virus 40 portion of the plasmid at the same sites that were DNase I sensitive in simian virus 40 chromatin prepared late after infection of African green monkey kidney (BSC-1) cells. One site extended from the viral origin of replication to approximately nucleotide 40. The 21-base pair repeated sequences were relatively DNase I resistant. A second site occurred over the single copy of the 72-base pair segment present in this plasmid. These results indicate that the nuclease-sensitive chromatin structure does not depend on the presence of viral structural proteins. In addition, late viral proteins added to pJI1-transfected COS-1 cells by superinfection with simian virus 40 caused no change in the distribution of DNase I-sensitive sites in plasmid chromatin. Analysis of transfected plasmid DNA may provide a general method applicable to the study of the chromatin structure of cloned segments of DNA.     

Mapping in vivo topoisomerase I sites on simian virus 40 DNA: asymmetric distribution of sites on replicating molecules.

Complexes between simian virus 40 DNA and topoisomerase I (topo I) were isolated from infected cells treated with camptothecin. The topo I break sites were precisely mapped by primer extension from defined oligonucleotides. Of the 56 sites, 40 conform to the in vitro consensus sequence previously determined for topo I. The remaining 16 sites have an unknown origin and were detectable even in the absence of camptothecin. Only 11% of the potential break sites were actually broken in vivo. In the regions mapped, the pattern of break sites was asymmetric. Most notable are the clustering of sites near the terminus for DNA replication and the confinement of sites to the strand that is the template for discontinuous DNA synthesis. These asymmetries could reflect the role of topo I in simian virus 40 DNA replication and suggest that topo I action is coordinated spatially with that of the replication complex.     

In the Simian Virus 40 In Vitro Replication System, Start Site Selection by the Polymerase α-Primase Complex Is Not Significantly Altered by Changes in the Concentration of Ribonucleotides

The simian virus 40 (SV40) in vitro replication system was previously used to demonstrate that the human polymerase (Pol) alpha-primase complex preferentially initiates DNA synthesis at pyrimidine-rich trinucleotide sequences. However, it has been reported that under certain conditions, nucleoside triphosphate (NTP) concentrations play a critical role in determining where eukaryotic primase initiates synthesis. Therefore, we have examined whether increased NTP concentrations alter the template locations at which SV40 replication is initiated. Our studies demonstrate that elevated ribonucleotide concentrations do not significantly alter which template sequences serve as initiation sites. Of considerable interest, the sequences that serve as initiation sites in the SV40 system are similar to those that serve as initiation sites for prokaryotic primases. It is also demonstrated that regardless of the concentration of ribonucleotides present in the reactions, DNA synthesis initiated outside of the core origin. These studies provide additional evidence that the Pol alpha-primase complex can initiate DNA synthesis only after a considerable amount of single-stranded DNA is generated.     

Cellular factors required for papillomavirus DNA replication.

In vitro replication of papillomavirus DNA has been carried out with a combination of purified proteins and partially purified extracts made from human cells. DNA synthesis requires the viral E1 protein and the papillomavirus origin of replication. The E2 protein stimulates DNA synthesis in a binding site-independent manner. Papillomavirus DNA replication is also dependent on the cellular factors replication protein A, replication factor C, and proliferating-cell nuclear antigen as well as a phosphocellulose column fraction (IIA). Fraction IIA contains DNA polymerase alpha-primase and DNA polymerase delta. Both of these polymerases are essential for papillomavirus DNA replication in vitro. However, unlike the case with T-antigen-dependent replication from the simian virus 40 origin, purified DNA polymerase alpha-primase and delta cannot efficiently replace fraction IIA in the replication reaction. Hence, additional cellular factors seem to be required for papillomavirus DNA replication. Interestingly, replication factor C and proliferating-cell nuclear antigen are more stringently required for DNA synthesis in the papillomavirus system than in the simian virus 40 in vitro system. These distinctions indicate that there must be mechanistic differences between the DNA replication systems of papillomavirus and simian virus 40.     

Expression of a bacterial repair gene in mammalian cells

The coding sequence of the uvrA gene from Escherichia coli has been fused to the early promoter, enhancer and origin of replication of the simian virus SV40, and was supplemented with splicing and polyadenylation sites arising from the same virus. Introduction of this hybrid gene into simian cos-1 cells results in the synthesis of a full length UvrA protein (114 kD) which has retained its ability to bind to single-stranded DNA.     

Replication and supercoiling of simian virus 40 DNA in cell extracts from human cells.

Soluble extracts prepared from the nucleus and cytoplasm of human 293 cells are capable of efficient replication and supercoiling of added DNA templates that contain the origin of simian virus 40 replication. Extracts prepared from human HeLa cells are less active than similarly prepared extracts from 293 cells for initiation and elongation of nascent DNA strands. DNA synthesis is dependent on addition of purified simian virus 40 tumor (T) antigen, which is isolated by immunoaffinity chromatography of extracts from cells infected with an adenovirus modified to produce large quantities of this protein. In the presence of T antigen and the cytoplasmic extract, replication initiates at the origin and continues bidirectionally. Initiation is completely dependent on functional origin sequences; a plasmid DNA containing an origin mutation known to affect DNA replication in vivo fails to replicate in vitro. Multiple rounds of DNA synthesis occur, as shown by the appearance of heavy-heavy, bromodeoxyuridine-labeled DNA products. The products of this reaction are resolved, but are relaxed, covalently closed DNA circles. Addition of a nuclear extract during DNA synthesis promotes the negative supercoiling of the replicated DNA molecules.     

The T-antigen-binding domain of the simian virus 40 core origin of replication.

The simian virus 40 origin of replication contains a 27-base-pair palindrome with the sequence 5'-CA-GAGGC-C-GAGGC-G-GCCTC-G-GCCTC-TG-3'. The four 5'-GAGGC-3'/5'-GCCTC-3' pentanucleotides are known contact sites for simian virus 40 T-antigen binding in vitro. We used oligonucleotide-directed cassette mutagenesis to identify features of this palindrome that are important for the initiation of DNA replication in vivo. Each base pair of a pentanucleotide is crucial for DNA replication. In contrast, sequences adjacent to pentanucleotides have little or no effect on replication. Thus, the pentanucleotide is the basic functional unit, not only for T-antigen binding but also for DNA replication. All four pentanucleotides are indispensable in the initiation process. The spacing of pentanucleotides is crucial because duplication of the single base pair between binding sites has a far greater effect on replication than does substitution of the same base pair. Inversion of any pentanucleotide blocks DNA synthesis. Thus, the pentanucleotide is not a functionally symmetrical unit. We propose that each pentanucleotide positions a monomer of T antigen at the proper distance, rotation, and orientation relative to other T-antigen monomers and to other origin domains and that such positioning leads to subsequent events in replication.     

Spacing is crucial for coordination of domain functions within the simian virus 40 core origin of replication.

The simian virus 40 core origin of replication is composed of distinct domains that are bracketed by DNA spacers. We created a matched set of insertion mutations in spacer sites to study the spatial relationships among origin domains. Insertions larger than a single base pair severely inhibit replication regardless of the helical phasing between domains. Replication-defective mutations reduce T-antigen binding and T-antigen-induced KMnO4 modifications of DNA to various extents. Mutations in the early half of the origin reduce T-antigen functions in the entire origin, whereas mutations in the late half reduce functions only in that half. Surprisingly, some mutations that severely inhibit DNA replication reduce T-antigen-induced melting and other structural changes within origin DNA to only a limited extent. In contrast, all replication-defective origin mutations prevent T antigen from extending the primary replication bubble beyond the limits of the core origin of replication. We conclude, therefore, that T-antigen-induced events within the core origin must be spatially coordinated for conversion of T-antigen hexamers bound to the core origin into mobile helicase units.     

Assembly of nascent DNA into nucleosome structures in simian virus 40 chromosomes by HeLa cell extract.

A soluble system was developed that could support DNA replication in simian virus 40 (SV40) chromosomes. DNA synthesis in this system required the presence of purified SV40 large tumor antigen, SV40 chromosomes prepared from virus-infected monkey cells, a crude extract from HeLa cells, and several low-molecular-weight components. In comparison to the replication of purified SV40 form I DNA, the rate of DNA synthesis was 15 to 20% in this system. DNA synthesis started near the replication origin of SV40 and proceeded bidirectionally in a semiconservative manner. Micrococcal nuclease digestion experiments revealed that the replicated DNA produced in this system became organized into a regularly spaced array of nucleosome core particles when an appropriate amount of purified HeLa core histones was added to the reaction mixture. SV40 form I DNA replicating under the same conditions was also assembled into nucleosomes, which were arranged in a rather dispersed manner and formed an aberrant chromatin structure.     

Murine polyomavirus and simian virus 40 large T antigens produce different structural alterations in viral origin DNA.

Murine polyomavirus (Py) and simian virus (SV40) encode homologous large T antigens (T Ags) and also have comparable sequence motifs in their core replication origins. While the ability of SV40 T Ag to produce specific distortions within the SV40 core replication origin (ori) in a nucleotide-dependent fashion has been well documented, little is known about related effects of Py T Ag on Py ori DNA. Therefore, we have examined viral origin DNA binding in the presence of nucleotide and the resulting structural changes induced by Py and SV40 T Ags by DNase I footprinting and KMnO4 modification assays. The structural changes in the Py ori induced by Py T Ag included sites within both the A/T and early side of the core origin region, consistent with what has been shown for SV40. Interestingly, however, Py T Ag also produced sites of distortion within the center of the origin palindrome and at several sites within both the early and late regions that flank the core ori. Thus, Py T Ag produces a more extensive and substantially different pattern of KMnO4 modification sites than does SV40 T Ag. We also observed that both T Ags incompletely protected and distorted the reciprocal ori region. Therefore, significant differences in the interactions of Py and SV40 T Ags with ori DNA may account for the failure of each T Ag to support replication of the reciprocal ori DNA in permissive cell extracts.     

Characterization of the DNA-binding properties of the origin-binding domain of simian virus 40 large T antigen by fluorescence anisotropy

The affinity of the origin-binding domain (OBD) of simian virus 40 large T antigen for its cognate origin was measured at equilibrium using a DNA binding assay based on fluorescence anisotropy. At a near-physiological concentration of salt, the affinities of the OBD for site II and the core origin were 31 and 50 nM, respectively. Binding to any of the four 5'-GAGGC-3' binding sites in site II was only slightly weaker, between 57 and 150 nM. Although the OBD was shown previously to assemble as a dimer on two binding sites spaced by 7 bp, we found that increasing the distance between both binding sites by 1 to 3 bp had little effect on affinity. Similar results were obtained for full-length T antigen in absence of nucleotide. Addition of ADP-Mg, which promotes hexamerization of T antigen, greatly increased the affinity of full-length T antigen for the core origin and for nonspecific DNA. The implications of these findings for the assembly of T antigen at the origin and its transition to a non-specific DNA helicase are discussed.     

The origin of bidirectional DNA replication in polyoma virus.

The nucleotide locations of RNA-p-DNA covalent linkages in polyoma virus (PyV) replicating DNA were mapped in the region containing the genetically required origin of DNA replication (ori). These linkages mark the initiation sites for RNA-primed DNA synthesis. A clear transition was identified between the presence of these linkages (discontinuous DNA synthesis) and their absence (continuous DNA synthesis) on each strand of ori. This demonstrated that PyV DNA replication, like simian virus 40 (SV40), is semi-discontinuous, and thus revealed the location of the origin of bidirectional DNA replication (OBR). The transition site on the template encoding PyV late mRNA occurred at the junction of ori-core and T-antigen binding site A. This was essentially the same site as previously observed in SV40 (Hay and DePamphilis, 1982). However, in contrast to SV40, the transition site on the template encoding PyV early mRNA was displaced towards the late gene side of ori. This resulted in a 16 nucleotide gap within ori in which no RNA-p-DNA linkages were observed on either strand. A model for the initiation of PyV DNA replication is presented.     

DNA primase-DNA polymerase alpha from simian cells: sequence specificity of initiation sites on simian virus 40 DNA.

Unique single-stranded regions of simian virus 40 DNA, phage M13 virion DNA, and several homopolymers were used as templates for the synthesis of (p)ppRNA-DNA chains by CV-1 cell DNA primase-DNA polymerase alpha. Intact RNA primers, specifically labeled with an RNA capping enzyme, were typically 6 to 8 ribonucleotides long, although their lengths ranged from 1 to 9 bases. The fraction of intact RNA primers 1 to 4 ribonucleotides long was 14 to 73%, depending on the template used. RNA primer length varied among primers initiated at the same nucleotide, as well as with primers initiated at different sites. Thus, the size of an RNA primer depended on template sequence. Initiation sites were identified by mapping 5' ends of nascent RNA-DNA chains on the template sequence, identifying the 5'-terminal ribonucleotide, and partially sequencing one RNA primer. A total of 56 initiation events were identified on simian virus 40 DNA, an average of 1 every 16 bases. Some sites were preferred over others. A consensus sequence for initiation sites consisted of either 3'-dCTTT or 3'-dCCC centered within 7 to 25 pyrimidine-rich residues; the 5' ends of RNA primers were complementary to the dT or dC. High ATP/GTP ratios promoted initiation of RNA primer synthesis at 3'-dCTTT sites, whereas low ATP/GTP ratios promoted initiation at 3'-dCCC sites. Similarly, polydeoxythymidylic acid and polydeoxycytidylic acid were the only effective homopolymer templates. Thus, both template sequence and ribonucleoside triphosphate concentrations determine which initiation sites are used by DNA primase-DNA polymerase alpha. Remarkably, initiation sites selected in vitro were strikingly different from initiation sites selected during simian virus 40 DNA replication in vivo.     

Mapping initiation sites for simian virus 40 DNA synthesis events in vitro.

Primer RNA-DNA, a small (approximately 30-nucleotide) RNA-DNA hybrid molecule, was identified in recent studies of simian virus 40 DNA synthesis in vitro. The available evidence indicates that primer RNA-DNA is the product of the polymerase alpha-primase complex. Primer RNA-DNA is formed exclusively on lagging-strand DNA templates; it is synthesized initially in the vicinity of the simian virus 40 origin and at later times at sites progressively distal to the origin. To further characterize initiation events, template sequences encoding the 5' ends of both primer RNA and primer DNA, formed during a 5-s pulse, have been determined. Analyses of these sequences demonstrate the existence of an initiation signal for lagging-strand synthesis. At any given position, the initiation signal is located within those template sequences encoding primer RNA, situated proximal to the nucleotide encoding the 5' end of the RNA primer. In most instances, the sequence 5'-TTN-3' (where N encodes the nucleotide at the 5' end of the primer) is a feature of the initiation signal. Initiation signals are present, on average, once every 19 nucleotides. These results are discussed in terms of the mechanism of Okazaki fragment formation and possible links between prokaryotic and eukaryotic initiation events.