DNA sequences required for the initiation of adenovirus type 4 DNA replication in vitro

In-vivo studies have demonstrated that adenovirus type 2 and adenovirus type 4 have different DNA sequence requirements for the initiation of DNA replication. To investigate the basis of these differences an in-vitro system has been developed which will faithfully initiate adenovirus type 4 DNA replication. A plasmid containing 140 base-pairs of the right terminus of adenovirus type 4 supported initiation of DNA replication in vitro, provided that the plasmid was linearized in such a way as to locate the viral terminal sequences at the molecular ends of the DNA. Initiation by adenovirus type 4-infected cell extracts was also supported by a plasmid containing the complete adenovirus type 2 inverted terminal repeat (ITR). Deletion analysis of both adenovirus types 2 and 4 ITRs revealed that only the terminal 18 base-pairs of the genomes (perfectly conserved between the 2 viruses) were required for initiation in vitro. Thus, initiation was not enhanced by the presence of either the NFI site, the NFIII site or both sites together. Fractionation of a HeLa cell nuclear extract, by ion-exchange chromatography, identified a nuclear factor that stimulated the initiation reaction four- to fivefold. The stimulatory factor did not correspond to either of the cellular proteins NFI or NFIII which stimulate adenovirus type 2 DNA replication in vitro. Initiation in vitro was also supported by single-stranded DNA templates, albeit at a lower efficiency. Studies with synthetic oligonucleotides indicated a surprising specificity for initiation: whereas the strand used as template during initiation in vivo was active as a template for initiation in vitro, the complementary strand was inactive.     

The origin of adenovirus DNA replication: minimal DNA sequence requirement in vivo.

Adenovirus mini-chromosomes which contain two cloned, inverted adenovirus termini replicate in vivo when supplied with non-defective adenovirus as a helper. This system has been used to define the minimum cis acting DNA sequences required for adenovirus DNA replication in vivo. Deletions into each end of the adenovirus inverted terminal repeat (ITR) were generated with Bal31 exonuclease and the resulting molecules constructed into plasmids which contained two inverted copies of the deleted ITR separated by the bacterial neomycin phosphotransferase gene. To determine the effect of the deletion in vivo plasmids cleaved to expose the adenovirus termini were co-transfected with adenovirus type 2 DNA into tissue culture cells. The replicative ability of the molecules bearing adenovirus termini was assayed by Southern blotting of extracted DNA which had been treated with DpnI, a restriction enzyme which cleaves only methylated and therefore unreplicated, input DNA. Molecules containing the terminal 45 bp of the viral genome were fully active whereas molecules containing only 36 bp were in-active in this assay. Therefore sequences required for DNA replication are contained entirely within the terminal 45 bp of the viral genome. Thus, both the previously described highly conserved region (nucleotides 9-18) and the binding site for the cellular nuclear factor I (nucleotides 19-48) are essential for adenovirus DNA replication in vivo.     

Identification of critical elements within the JC virus DNA replication origin.

The T antigen of JC virus (JCV) does not interact productively with the simian virus 40 (SV40) origin of replication. In contrast, the SV40 T antigen does drive replication from the JCV origin as well as from its own. The basis for this restricted interaction was investigated by analyzing the structure of the JCV replication origin. The replication activities of JCV-SV40 hybrid origin plasmids were tested in cells constitutively producing either the JCV or SV40 T antigen. Results indicated that a region of the JCV origin critical for interaction with the JCV T antigen was positioned to the late side of the central palindrome of the putative core origin. A mutational analysis of this region indicated that the sequence of the A + T-rich tract was primarily responsible for determining the efficiency with which JCV can initiate replication from its origin. The tandemly repeated pentameric sequence AGGGA located proximal to the A + T-rich tract in the JCV enhancer element was found to stimulate JCV, but not SV40, T antigen-mediated replication. The effect on replication of other elements within the JCV enhancer was also dependent on the T antigen employed for initiation. A plasmid containing the replication origin of prototype BK virus was unable to replicate in cells containing JCV T antigen, again indicating the inflexibility of the JCV T antigen in interacting with heterologous origins.     

Multiple regions within the coxsackievirus and adenovirus receptor cytoplasmic domain are required for basolateral sorting

The coxsackievirus and adenovirus receptor (CAR) mediates attachment and infection by coxsackie B viruses and many adenoviruses. In human airway epithelia, as well as in transfected Madin-Darby canine kidney cells, CAR is expressed exclusively on the basolateral surface. Variants of CAR that lack the cytoplasmic domain or are attached to the cell membrane by a glycosylphosphatidylinositol anchor are expressed on both the apical and basolateral surfaces. We have examined the localization of CAR variants with progressive truncations of the cytoplasmic domain, as well as with mutations that ablate a potential PDZ (PSD95/dlg/ZO-1) interaction motif and a putative tyrosine-based sorting signal. In addition, we have examined the targeting of two murine CAR isoforms, with different C-terminal sequences. The results suggest that multiple regions within the CAR cytoplasmic domain contain information that is necessary for basolateral targeting.     

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 δ, we suggest that both polymerases α and β are involved in this system. Three further fractions have been identified. One is a partially purified fraction which, under certain conditons, 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.     

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.     

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.     

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.     

Sequences required for enhancer blocking activity of scs are located within two nuclease-hypersensitive regions.

The Drosophila 87A7 heat shock locus is bordered, on the proximal and distal sides, by two special chromatin structures, scs and scs'. Each structure is characterized by two sets of nuclease-hypersensitive sites, located within moderately G/C-rich DNA, flanking an A/T-rich nuclease-resistant region. scs and scs' have been shown to insulate a white reporter gene from position effects and to prevent enhancer-promoter interactions. These and other properties suggest scs and scs' might function as chromatin domain boundaries. To identify the DNA sequences which are essential for the insulating activity of scs we used an enhancer blocking assay based on the white gene. Sequences capable of suppressing activation of white by its upstream enhancer elements reside within a 900 bp DNA fragment corresponding to the scs chromatin structure. Within this region, DNA fragments associated with the two nuclease-hypersensitive regions are essential for full enhancer blocking activity, while the central A/T-rich region is dispensable. Deletions which remove part of the hypersensitive regions result in intermediate levels of white activity. Insulating activity can, however, be reconstituted by multimerizing DNA fragments from either hypersensitive region. Our results suggest that the scs boundary is assembled from a discrete number of functionally redundant DNA sequences located within both hypersensitive regions and that boundaries act by decreasing the frequency of enhancer-promoter interactions. We also show that certain types of position effects, like those involved in dosage compensation, are not efficiently blocked by scs.     

Identification of two regions within the cytoplasmic domain of the human interferon-gamma receptor required for function.

Functionally active human interferon-gamma (IFN gamma) receptors require the presence of at least two polypeptides: the IFN gamma receptor and an accessory molecule encoded by a gene on human chromosome 21. Here we have used a murine L cell line that stably contains human chromosome 21 (SCC16-5) to determine whether the receptor's cytoplasmic domain is important for receptor function. SCC16-5 stably transfected with the full-length human IFN gamma receptor cDNA bound, internalized, and responded to human IFN gamma. In contrast, SCC16-5 expressing human IFN gamma receptors lacking a cytoplasmic domain bound human IFN gamma but did not internalize or respond to it. Using a family of IFN gamma receptor deletion mutants, two functionally important regions within the intracellular domain were identified: (a) a membrane proximal region (residues 256-303) required for ligand processing and biologic responsiveness and (b) the carboxyl-terminal 39 amino acids (residues 434-472) needed exclusively for biologic responses.     

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.     

A DNA structure is required for geminivirus replication origin function.

The genome of the geminivirus tomato golden mosaic virus (TGMV) consists of two single-stranded circular DNAs, A and B, that replicate through a rolling-circle mechanism in nuclei of infected plant cells. The TGMV origin of replication is located in a conserved 5' intergenic region and includes at least two functional elements: the origin recognition site of the essential viral replication protein, AL1, and a sequence motif with the potential to form a hairpin or cruciform structure. To address the role of the hairpin motif during TGMV replication, we constructed a series of B-component mutants that resolved sequence changes from structural alterations of the motif. Only those mutant B DNAs that retained the capacity to form the hairpin structure replicated to wild-type levels in tobacco protoplasts when the viral replication proteins were provided in trans from a plant expression cassette. In contrast, the same B DNAs replicated to significantly lower levels in transient assays that included replicating, wild-type TGMV A DNA. These data established that the hairpin structure is essential for TGMV replication, whereas its sequence affects the efficiency of replication. We also showed that TGMV AL1 functions as a site-specific endonuclease in vitro and mapped the cleavage site to the loop of the hairpin. In vitro cleavage analysis of two TGMV B mutants with different replication phenotypes indicated that there is a correlation between the two assays for origin activity. These results suggest that the in vivo replication results may reflect structural and sequence requirements for DNA cleavage during initiation of rolling-circle replication.     

Identification of two distinct subfamilies of alpha satellite DNA that are highly specific for human chromosome 15

We report the isolation of two distinct subfamilies of alpha satellite DNA (pTRA-20 and -25) from human chromosome 15. In situ hybridization experiments indicated that both subfamilies are highly specific for this chromosome. Southern analysis of a somatic hybrid cell line carrying human chromosome 15 revealed a likely higher-order genomic band of 2.5 kb for pTRA-20. Similar analysis for pTRA-25 showed multiple higher-order bands of 3.5, 4.5, and 5 kb at moderately high hybridization stringency, but a predominance of the 4.5-kb species at very high stringency. Direct comparison with human genomic DNA confirmed the authenticity of these higher-order structures and demonstrated polymorphic variations using both probes. The origin of the different alphoid subfamilies on chromosome 15 is discussed. These sequences should be useful for the construction of centromere-based genetic linkage maps for human chromosome 15 and, in conjunction with the other alphoid sequences already reported for chromosomes 13, 14, 21, and 22, should allow a concerted analysis of the evolution and the possible etiological role of these DNAs in aberrations commonly seen in these chromosomes.     

Genetic identification of two distinct DNA polymerases, DnaE and PolC, that are essential for chromosomal DNA replication in Staphylococcus aureus

We isolated and characterized temperature-sensitive mutants for two genes, dnaE and polC, that are essential for DNA replication in Staphylococcus aureus. DNA replication in these mutants had a slow-stop phenotype when the temperature was shifted to a non-permissive level. The dnaE gene encodes a homolog of the alpha-subunit of the DNA polymerase III holoenzyme, the replicase essential for chromosomal DNA replication in Escherichia coli. The polC gene encodes PolC, another catalytic subunit of DNA polymerase, which is specifically found in gram-positive bacteria. The wild-type dnaE or polC gene complemented the temperature-sensitive phenotypes of cell growth and DNA replication in the corresponding mutant. Single mutations resulting in amino-acid exchanges were identified in the dnaE and polC genes of the temperature-sensitive mutants. The results indicate that these genes encode two distinct DNA polymerases which are both essential for chromosomal DNA replication in S. aureus. The number of viable mutant cells decreased at non-permissive temperature, suggesting that inactivation of DnaE and PolC has a bactericidal effect and that these enzymes are potential targets of antibiotics.     

Interactions between the RepB initiator protein of plasmid pMV158 and two distant DNA regions within the origin of replication

Plasmids replicating by the rolling circle mode usually possess a single site for binding of the initiator protein at the origin of replication. The origin of pMV158 is different in that it possesses two distant binding regions for the initiator RepB. One region was located close to the site where RepB introduces the replication-initiating nick, within the nic locus; the other, the bind locus, is 84bp downstream from the nick site. Binding of RepB to the bind locus was of higher affinity and stability than to the nic locus. Contacts of RepB with the bind and nic loci were determined through high-resolution footprinting. Upon binding of RepB, the DNA of the bind locus follows a winding path in its contact with the protein, resulting in local distortion and bending of the double-helix. On supercoiled DNA, simultaneous interaction of RepB with both loci favoured extrusion of the hairpin structure harbouring the nick site while causing a strong DNA distortion around the bind locus. This suggests interplay between the two RepB binding sites, which could facilitate loading of the initiator protein to the nic locus and the acquisition of the appropriate configuration of the supercoiled DNA substrate.     

Recognition of the adenovirus type 2 origin of DNA replication by the virally encoded DNA polymerase and preterminal proteins.

Initiation of adenovirus DNA synthesis is preceded by the assembly of a nucleoprotein complex at the origin of DNA replication containing three viral proteins, preterminal protein, DNA polymerase and DNA binding protein, and two cellular proteins, nuclear factors I and III. While sequence specific interactions of the cellular proteins with their cognate sites in the origin of DNA replication are well characterized, the question of how the viral replication proteins recognize the origin has remained unanswered. Preterminal protein and DNA polymerase were therefore purified to homogeneity from recombinant baculovirus infected insect cells. Gel filtration demonstrated that while DNA polymerase existed in monomeric and dimeric forms, preterminal protein was predominantly monomeric and when combined the proteins formed a stable heterodimer. In a gel electrophoresis DNA binding assay each of the protein species recognized DNA within the origin of DNA replication with unique specificity. Competition analysis and DNase I protection experiments revealed that although each protein could recognize the origin, the heterodimer did so with enhanced specificity, protecting bases 8-17 from cleavage with the nuclease. Thus the highly conserved 'core' of the origin of DNA replication, present in all human adenoviruses, is recognized by the preterminal protein--DNA polymerase heterodimer.     

Identification of two distinct regions of p38 MAPK required for substrate binding and phosphorylation

The mechanism by which different mitogen activated protein kinases (MAPKs) distinguish between different substrates is poorly understood. For example, p38 and SAPK4 are two closely related p38 MAPKs that both phosphorylate ATF2 and MBP. However, p38 phosphorylates MAPKAPK-2 and -3, whereas SAPK4 does not. In this study, we have used mutagenesis to determine the regions of p38 required for substrate selection. Alanine scanning mutagenesis identified one region of p38 that was required for its ability to phosphorylate MAPKAPK-2 and -3, but that did not significantly affect its binding to these substrates. Chimeras of p38 and SAPK4 identified a second region of p38 that affected the ability of p38 to both bind and phosphorylate MAPKAPK-2 and -3. Hence, we show for the first time that MAPKs contain two distinct regions for recognizing and phosphorylating protein substrates.