Definition of the simian virus 40 early promoter region and demonstration of a host range bias in the enhancement effect of the simian virus 40 72-base-pair repeat.

The simian virus 40 (SV40) origin region includes the viral replication origin and the early and late promoters and consists of a few palindromes, a 17-base-pair (bp) A + T-rich sequence, three copies of a G + C-rich 21-bp repeat, and two copies of a 72-bp repeat. We have made sequential deletions in the SV40 origin region and determined the early promoter efficiencies of these truncated DNA segments by connecting them in the correct orientation with the coding regions of selectable marker genes and assaying the expression of the chimeric marker genes in vivo in different host cell lines. A truncated SV40 early promoter segment containing only the TATA box and the major in vivo mRNA initiation sites has essentially no promoter efficiency. We have located the major component of the SV40 early promoter within the 21-bp repeated sequences, which consist of an alternating and mutually overlapping array of two C-rich oligonucleotides having the consensus sequences Y-Y-C-C-G-C-C-C (Y = pyrimidine nucleoside) and G-C-C-C-(C)-TA-AT-A(T)-C-T. Between one and two copies of the 21-bp repeat were adequate for gene expression under conditions in which the enhancement effect of the 72-bp repeat was minimal. We also find that the SV40 72-bp repeat exhibits a pronounced host range in its enhancement of gene expression; the enhancement is only 2-fold in the nonpermissive mouse cells but amounts to 10- or 20-fold in the permissive monkey cells or the semipermissive human cells, respectively.     

Mapping of the late promoter of simian virus 40.

Mapping of the simian virus 40 (SV40) late promoter was carried out in the absence of the viral early protein, large tumor (T) antigen, and replication of the viral DNA template. SV40 late control region DNA fragments, containing specific deletions, were cloned directly upstream from the coding region of the herpes simplex virus-1 (HSV-1) thymidine kinase (TK; ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21) gene (tk). The promoter activities of the fragments were determined by measuring the tk transformation frequencies of the chimeric tk constructs in mouse L TK- APRT- (adenine phosphoribosyltransferase-negative) and human 143B TK- cell lines. The following results were obtained. (i) The SV40 control region functions with equal efficiencies in the early and late promoter orientations. (ii) A major late control element has been localized within the G+C-rich 21-base-pair (bp) repeat. Thus, in conjunction with our earlier results, the 21-bp repeat is a bidirectional promoter element functioning as a major component of both the early and late promoters and is an element that enhances the replication efficiency of SV40 DNA. (iii) Minor late promoters have been localized within the minimal replication origin and the 72-bp repeat. (iv) The minimal replication origin is not per se a constituent of the major late promoter; however, both the minimal replication origin and the 21-bp repeat are required for obtaining high levels of late gene expression observed at late times after infection by SV40. (v) The 72-bp repeat exerts a 4- to 5-fold enhancement of late promoter expression.     

Mouse primase initiation sites in the origin region of simian virus 40.

The sites of initiation of DNA synthesis by purified mouse DNA primase in the origin-of-replication region of simian virus 40 (SV40) were examined. Using as template the separated strands of a cloned fragment of SV40 approximately equal to 300 base pairs (bp) long that includes the origin, we observed specific sites of initiation on the two strands. On the early strand that is the template for early mRNA synthesis, the primary starts are at four positions within 10 nucleotides of each other around nucleotide 5215 and an additional site around nucleotide 5147 that is used at one-sixth the frequency of the major sites. The major start sites on the early strand are within the 65-bp minimal origin of replication and lie between tumor antigen binding sites I and II. On the late strand that is the template for late mRNA synthesis, six major initiation sites were observed, each within the 3' C-C-C-G-C-C 5' sequence in the template that is repeated twice within each of the three 21-bp repeats that lie adjacent to the minimal origin, on its late side. A 6-bp deletion in the 65-bp minimal origin that eliminates its function as an origin reduced the major initiations around nucleotide 5215 on the early strand by 90% but did not affect initiations at the minor start site on the early strand or initiations on the late strand. Mouse DNA primase is able to recognize specific regions on the SV40 DNA. Those on the early strand are within the minimal origin of replication and those on the late strand are within the 21-bp repeat region necessary for maximum replication.     

The simian virus 40 minimal origin and the 72-base-pair repeat are required simultaneously for efficient induction of late gene expression with large tumor antigen.

We have studied the temporal regulation of simian virus 40 (SV40) late gene expression by construction and transient expression analysis of plasmids containing the transposon Tn9 chloramphenicol acetyltransferase gene placed downstream from the late control region. The SV40 origin region in the early (but not the late) orientation promotes chloramphenicol acetyltransferase gene expression efficiently in monkey cells lacking large tumor (T) antigen. In monkey cells producing T antigen, the promoter activity of the late control region is induced by approximately 1,000-fold above the basal level. By deletion and point mutagenesis, we define two domains of the late control region required for efficient induction with T antigen. Domain I is the minimal replication origin containing T-antigen binding site II. Domain II consists of the 72-base-pair (bp) repeat and a 19-bp downstream sequence up to nucleotide 270. Domains I and II should act synergistically because the absence of either one or the other decreases induction efficiency by 2 orders of magnitude. Though a complete copy of domain II is optimal, the origin-proximal 22-bp portion of this domain is sufficient. The 21-bp repeat, located between domains I and II, is dispensable for this induction, as are sequences located downstream from nucleotide 270 in the late orientation.     

DNA sequences similar to those around the simian virus 40 origin of replication are present in the monkey genome.

We report the molecular cloning of African green monkey genomic DNA segments that include regions of homology to the origin of replication of simian virus 40 (SV40). Three clearly different cloned segments 14 to 17 kilobase pairs (kb) long were isolated from a genomic library in lambda phage. We estimate that each of the three is repeated fewer than four times in the monkey genome. The SV40-like regions represent a small portion of the cloned segments, and these regions cross hybridize only weakly with one another. One of the three segments is described here in detail. Although the entire segment occurs only once or twice in the monkey genome, it contains DNA sequences (other than the SV40-like sequences) that are repeated elsewhere in the genome including in the other two cloned segments. The homology to SV40 is contained within about 300 base pairs of monkey DNA and is limited to the region around the viral replication origin. The nucleotide sequence of the SV40-like region was determined. It contains a large number of short stretches homologous to three specific noncoding domains around the SV40 origin of replication: the 27-base-pair region of dyad symmetry, the first set of (short) repeats that occur just on the late side of the origin, and, further in the late direction, the two 72-base-pair-long repeats. Although these components are grouped in the monkey DNA, as they are in SV40 DNA, their relative juxtaposition is scrambled.     

DNA binding site for a factor(s) required to initiate simian virus 40 DNA replication.

Efficient initiation of DNA replication in the absence of nonspecific DNA repair synthesis was obtained by using a modification of the system developed by J.J. Li and T.J. Kelly [(1984) Proc. Natl. Acad. Sci. USA 81, 6973-6977]. Circular double-stranded DNA plasmids replicated in extracts of CV-1 cells only when the plasmids contained the cis-acting origin sequence for simian virus 40 DNA replication (ori) and the extract contained simian virus 40 large tumor antigen. Competition between plasmids containing ori and plasmids carrying deletions in and about ori served to identify a sequence that binds the rate-limiting factor(s) required to initiate DNA replication. The minimum binding site (nucleotides 72-5243) encompassed one-half of the simian virus 40 ori sequence that is required for initiation of replication (ori-core) plus the contiguous sequence on the late gene side of ori-core containing G + C-rich repeats that facilitates initiation (ori-auxiliary). This initiation factor binding site was specific for the simian virus 40 ori region, even though it excluded the high-affinity large tumor antigen DNA binding sites.     

Sequence recognition protein for the 17-base-pair A + T-rich tract in the replication origin of simian virus 40 DNA.

A DNA-binding protein has been identified that recognizes runs of deoxyadenines and/or deoxythymines (dA/dT sequences) and purified from a chromatographic fraction containing the multiprotein DNA polymerase alpha-primase complex of HeLa cells by successive steps of chromatography on oligo(dT)-cellulose and Q-Sepharose. Polyacrylamide gel electrophoresis of the purified dA/dT sequence-binding protein in the presence of NaDodSO4 showed a single protein band of 62 kDa. Nitrocellulose filter binding assays using homopolydeoxynucleotides indicated that the purified protein preferentially binds to dA/dT sequences in single-stranded or duplex DNAs. Gel mobility shift assays with a variety of DNAs showed that the purified protein specifically binds to a fragment of simian virus 40 DNA containing the minimal (core) origin for replication. The binding occurred in a protein-dependent manner and in the presence of a vast excess of competing DNAs lacking the simian virus replication origin. The origin binding was reduced, however, when DNA fragments from simian virus 40 deletion mutants containing deletions within the 17-base-pair A + T-rich tract in the core DNA replication origin were used in the assays. These results indicate that the dA/dT sequence-binding protein preferentially binds to the 17-base-pair A + T-rich tract and suggest a possible role for the protein in the initiation of DNA replication.     

Properties of a high-affinity DNA binding site for estrogen receptor.

It has been shown previously that a short sequence from the 5' regulatory region of the Xenopus laevis vitellogenin gene A2, when appropriately placed, can confer estrogen responsiveness to another gene. Using the Xenopus sequence and similar sequences from the 5' regulatory regions of other estrogen-responsive genes, we derived a consensus sequence 38 nucleotides long. The sequence contains an inverted repeat (5' C-A-G-G-T-C-A-G-A-G-T-G-A-C-C-T-G 3') and an A/T-rich region. Plasmids carrying a single copy of the sequence bound 3-fold-more partially purified estrogen receptor (ER) than did control plasmids when assayed by gel filtration. Maximum specificity for ER binding occurs at 100-150 mM ionic strength and pH 7.5-8.0. Plasmids carrying multiple copies of the sequence bound correspondingly more ER. The dissociation constant for ER bound to the sequence is 0.5 nM. This value is lower by a factor of about 400 than the dissociation constant for ER bound to an equivalent length of plasmid DNA. Portions of the consensus sequence were evaluated for binding efficiency. Plasmids containing the inverted repeat alone bound ER, though less efficiently than did plasmids containing the entire sequence. The A/T-rich region alone was ineffective in binding ER. Linearization of the plasmid DNA did not enhance specific binding efficiency for ER. This model system represents an effective tool for characterization of ER binding to DNA sequences involved in the regulation of gene expression.     

Simian virus 40 large tumor antigen requires three core replication origin domains for DNA unwinding and replication in vitro.

Simian virus 40 (SV40) large tumor antigen (T antigen) unwinds DNA containing the SV40 origin of replication. The origin requirement for unwinding can be satisfied by the 64-base-pair SV40 core origin that supports T-antigen-dependent DNA replication both in vivo and in vitro. The core origin contains three domains with specific DNA sequence features. These include an inverted repeat, a central T-antigen binding domain, and an adenine- and thymine-rich domain containing a DNA bending focus. The domain and spacer requirements of the core origin for DNA unwinding and replication in vitro are strikingly similar to the origin requirements for DNA replication in vivo. Thus, each of the three functional domains of the core origin contributes directly to the initiation of duplex DNA unwinding by T antigen.     

Significant potential secondary structures in the Epstein-Barr virus genome.

This paper identifies all statistically significant dyad symmetry combinations in the Epstein-Barr virus genome. The distribution of long dyad symmetry pairings emphasizes two regions, the 5' third of the 3.1-kilobase-pair (kbp) repeat and the oriP region, the latter essential for Epstein-Barr virus replication during latency. A 600-base-pair (bp) stretch in the 3.1-kbp repeat can establish an extended hairpin loop of stem length in excess of 208 bp of predominantly G + C stacking. Moreover, the 3.1-kbp repeat has the potential to form a wide variety of secondary structures based on juxtapositions of sizable palindromes, close dyad symmetry pairings, and direct repeats. The 3.1-kbp repeat presents several features that portend it as an important control region. The oriP region contains an abundance of statistically significant dyad symmetry combinations that strongly correlate with the "21 X 30 bp" tandem repeat units and four truncated copies of this repeat unit 1 kbp downstream. Each of the units centers on the same approximately 30-bp palindrome. Contrasts in the content and the secondary structure formations associated with the 3.1-kbp repeat units versus those of the oriP region are discussed in relation to viral or cellular function.     

Primary structure of the essential replicon of the plasmid pSC101.

The replicon of the low copy number plasmid pSC101 has an obligatory requirement for the dnaA initiator protein of Escherichia coli as well as a plasmid-encoded initiator protein. We have identified the cistron of the plasmid-encoded initiator by DNA sequence analysis. Fusion of the initiator cistron with the lacZ gene of E. coli yielded a fusion protein of approximately equal to 150 kilodaltons, thus confirming that the open reading frame detected by DNA sequence analysis actually encoded a 37.5-kilodalton protein. Deletion of 26 amino acid residues from the COOH terminus of the plasmid initiator abolished autonomous replication from pSC101 origin. By in vitro deletion analysis we have shown that, although sequences downstream from the initiator cistron are dispensable, a maximum of 400 base pairs immediately upstream from the NH2-terminal region of the initiator is necessary for plasmid replication. These upstream sequences contain an A + T-rich region and three tandem repeats of a 21-base pair sequence; these features are characteristics of other replication origins.     

Mismatch repair in Escherichia coli enhances instability of (CTG)n triplet repeats from human hereditary diseases.

Long CTG triplet repeats which are associated with several human hereditary neuromuscular disease genes are stabilized in ColE1-derived plasmids in Escherichia coli containing mutations in the methyl-directed mismatch repair genes (mutS, mutL, or mutH). When plasmids containing (CTG)180 were grown for about 100 generations in mutS, mutL, or mutH strains, 60-85% of the plasmids contained a full-length repeat, whereas in the parent strain only about 20% of the plasmids contained the full-length repeat. The deletions occur only in the (CTG)180 insert, not in DNA flanking the repeat. While many products of the deletions are heterogeneous in length, preferential deletion products of about 140, 100, 60, and 20 repeats were observed. We propose that the E. coli mismatch repair proteins recognize three-base loops formed during replication and then generate long single-stranded gaps where stable hairpin structures may form which can be bypassed by DNA polymerase during the resynthesis of duplex DNA. Similar studies were conducted with plasmids containing CGG repeats; no stabilization of these triplets was found in the mismatch repair mutants. Since prokaryotic and human mismatch repair proteins are similar, and since several carcinoma cell lines which are defective in mismatch repair show instability of simple DNA microsatellites, these mechanistic investigations in a bacterial cell may provide insights into the molecular basis for some human genetic diseases.     

Stabilization of diverged tandem repeats by mismatch repair: evidence for deletion formation via a misaligned replication intermediate.

A functional methyl-directed mismatch repair pathway in Escherichia coli prevents the formation of deletions between 101-bp tandem repeats with 4% sequence divergence. Deletions between perfectly homologous repeats are unaffected. Deletion in both cases occurs independently of the homologous recombination gene, recA. Because the methyl-directed mismatch repair pathway detects and excises one strand of a mispaired duplex, an intermediate for RecA-independent deletion of tandem repeats must therefore be a heteroduplex formed between strands of each repeat. We find that MutH endonuclease, which in vivo incises specifically the newly replicated strand of DNA, and the Dam methylase, the source of this strand-discrimination, are required absolutely for the exclusion of "homeologous" (imperfectly homologous) tandem deletion. This supports the idea that the heteroduplex intermediate for deletion occurs during or shortly after DNA replication in the context of hemi-methylation. Our findings confirm a "replication slippage" model for deletion formation whereby the displacement and misalignment of the nascent strand relative to the repeated sequence in the template strand accomplishes the deletion.     

Purification and characterization of replication protein A, a cellular protein required for in vitro replication of simian virus 40 DNA.

The replication of simian virus 40 (SV40) DNA is largely dependent upon cellular replication proteins. To define these proteins we have made use of a cell-free system that is capable of replicating plasmid DNA molecules containing the SV40 origin of replication. Systematic fractionation-reconstitution experiments indicate that there are a minimum of six cellular proteins that are required for efficient viral DNA replication in vitro. We report here the purification of one of these proteins, replication protein A (RP-A), to homogeneity. RP-A is a multisubunit protein that contains four tightly associated polypeptides of 70, 53, 32, and 14 kDa. Partial proteolysis experiments indicate that the 53-kDa polypeptide is closely related to the 70-kDa polypeptide, suggesting that it may be a proteolytic fragment of the larger subunit. RP-A is absolutely required for reconstitution of SV40 DNA replication in vitro. The purified protein binds to single-stranded DNA and is required for the large tumor (T)-antigen-mediated unwinding of DNA molecules containing the SV40 origin of DNA replication. These properties are consistent with the possibility that RP-A plays a central role in the generation of a single-stranded region at the origin prior to initiation of DNA synthesis. The protein may also function to facilitate unwinding of the parental DNA strands during the elongation phase of SV40 DNA replication.     

Simian virus 40 DNA replication in vitro.

Soluble extracts prepared from monkey cells (COS-1 or BSC-40) infected with simian virus 40 (SV40) catalyze the efficient replication of exogenously added plasmid DNA molecules containing the cloned SV40 origin of replication. Extracts prepared from uninfected monkey cells also support origin-dependent replication in vitro but only in the presence of added SV40 large tumor (T) antigen. Very little DNA synthesis is observed when the cloned viral origin contains a 4-base-pair deletion mutation known to abolish SV40 DNA replication in vivo or when the parental plasmid vector lacking SV40 sequences is employed as template. The in vitro replication reaction proceeds via branched intermediates (theta structures) that resemble in vivo replication intermediates. Replication is sensitive to aphidicolin but relatively resistant to dideoxythymidine triphosphate. The product of the reaction consists of covalently closed circular DNA molecules that contain full-length daughter strands hydrogen bonded to the parental template. These observations support the conclusion that replication in the in vitro system closely resembles SV40 DNA replication in vivo. The system provides a biochemical assay for the replication activity of SV40 T antigen and should also facilitate the purification and functional characterization of cellular proteins involved in DNA replication.