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PubMed1986

Cloning and characterization of oriL2, a large palindromic DNA replication origin of herpes simplex virus type 2.

An origin of replication within the long unique sequence of herpes simplex virus type 2 designated oriL2 has been identified in a position homologous to its type 1 counterpart, oriL1, between map coordinates 0.398 and 0.413. The difficulties encountered in previous attempts to clone both oriL2 and oriL1 in an undeleted form were surmounted by minimizing the growth of the host Escherichia coli, using a recBC sbcB E. coli host, and purifying the full-length plasmid from delected forms by using a novel method which exploits the ability of a palindrome-containing plasmid to adopt a cruciform conformation, thereby decreasing its supercoiling. In a previously developed assay for functional origin activity, oriL2 was localized to a 241-base-pair ApaI-SstII fragment. DNA sequence analysis revealed a 136-base pair, almost perfect palindrome. Comparison with oriL1 showed a very high degree of conservation: the two origins differ in only 16 of the 144-base-pair oriL1 palindromic region. Most significantly, the differences between oriL1 and oriL2 mainly occur in pairs so as to generally preserve the potential for intrastrand base pairing. The central region of oriL2 is homologous with the shorter palindromic structures detected in origins located within the repetitive sequences of the short component of herpes simplex virus type 1 or 2.

Chromosome Deletion↗

PubMed1991

Two spatially distinct genetic elements constitute a bipartite DNA replication origin in the minute virus of mice genome.

Mutations were introduced into plasmid pMM984, a full-length infectious clone of the fibrotropic strain of minute virus of mice, to identify cis-acting genetic elements required for the excision and replication of the viral genome. The replicative capacity of these mutants was measured directly, using an in vivo transient DNA replication assay following transfection of plasmids into murine A9 cells and primate COS-7 cells. Experiments with subgenomic constructs indicated that both viral termini must be present on the same DNA molecule for replication to occur and that the viral nonstructural protein NS-1 must be provided in trans. The necessary sequences were located within 1,084 and 807 nucleotides of the 3' and 5' ends of the minute virus of mice genome, respectively. The inhibitory effect of deletions within the 206-bp 5'-terminal palindrome demonstrated that these sequences comprise a cis-acting genetic element that is absolutely essential for the excision and replication of viral DNA. The results further indicated a requirement for a stem-plus-arms T structure as well as for the formation of a simple hairpin. In addition, the removal of one copy of a tandemly arranged 65-bp repeat found 94 nucleotides inboard of the 5'-terminal palindrome inhibited viral DNA replication in cis by 10- and just greater than 100-fold in A9 and COS-7 cells, respectively. The latter results define a novel genetic element within the 65-bp repeated sequence, distinct from the terminal palindrome, that is capable of regulating minute virus of mice DNA replication in a species-specific manner.

Animals↗

PubMed1991

New pUC-derived cloning vectors with different selectable markers and DNA replication origins.

Four new Escherichia coli cloning vectors are described, pUC6S, pUC21, pUK21 and pOK12. These vectors contain a polylinker or multiple cloning site (MCS) with the recognition sequences for 28 restriction enzymes. Plasmids pUC21, pUK21, and pOK12 contain the MCS in the N-terminal end of the lacZ alpha fragment allowing blue/white screening for inserts. To potentially increase the stability of some inserts that may encode toxic proteins, the strength of the lacZ promoter present on these vectors has been reduced. Plasmids pUC6S and pUC21 carry the bla gene encoding ampicillin resistance, while pUK21 and pOK12 contain the gene encoding kanamycin resistance. Plasmid pOK12 carries the replicon from P15A, resulting in a lower copy number pUC-type vector. Plasmid pUC6S carries the ori and bla gene present on all pUC vectors, but does not contain any lac sequences. Plasmids pUC21 and pUK21 contain the M13 intergenic region allowing for the production of plasmid single-stranded DNA. To improve the yield of ss plasmid DNA, two plasmid cis-acting factors that affect yield were also examined: the effect of plasmid-derived transcription across the M13 ori, and the effect of delecting the M13 minus-strand ori from the plasmid.

Base Sequence↗

PubMed1987

Sequence-specific interactions between cellular DNA-binding proteins and the adenovirus origin of DNA replication.

The adenovirus origin of DNA replication contains three functionally distinct sequence domains (A, B, and C) that are essential for initiation of DNA synthesis. Previous studies have shown that domain B contains the recognition site for nuclear factor I (NF-I), a cellular protein that is required for optimal initiation. In the studies reported here, we used highly purified NF-I, prepared by DNA recognition site affinity chromatography (P. J. Rosenfeld and T. J. Kelly, Jr., J. Biol. Chem. 261:1398-1408, 1986), to investigate the cellular protein requirements for initiation of viral DNA replication. Our data demonstrate that while NF-I is essential for efficient initiation in vitro, other cellular factors are required as well. A fraction derived from HeLa cell nuclear extract (BR-FT fraction) was shown to contain all the additional cellular proteins required for the complete reconstitution of the initiation reaction. Analysis of this complementing fraction by a gel electrophoresis DNA-binding assay revealed the presence of two site-specific DNA-binding proteins, ORP-A and ORP-C, that recognized sequences in domains A and C, respectively, of the viral origin. Both proteins were purified by DNA recognition site affinity chromatography, and the boundaries of their binding sites were defined by DNase I footprint analysis. Additional characterization of the recognition sequences of ORP-A, NF-I, and ORP-C was accomplished by determining the affinity of the proteins for viral origins containing deletion and base substitution mutations. ORP-C recognized a sequence between nucleotides 41 and 51 of the adenovirus genome, and analysis of mutant origins indicated that efficient initiation of replication is dependent on the presence of a high-affinity ORP-C-binding site. The ORP-A recognition site was localized to the first 12 base pairs of the viral genome within the minimal origin of replication. These data provide evidence that the initiation of adenovirus DNA replication involves multiple protein-DNA interactions at the origin.

Adenoviruses, Human↗

PubMed1992

EBNA1 can link the enhancer element to the initiator element of the Epstein-Barr virus plasmid origin of DNA replication.

The plasmid origin of DNA replication of Epstein-Barr virus, oriP, is replicated once per cell division, employing cellular replication machinery and only one viral protein. To understand how replication from this origin is initiated and regulated, we purified this viral protein, EBNA1. EBNA1 was expressed in CV-1p cells by using an infectious simian virus 40 vector containing the EBNA1 gene. It was purified in two chromatographic steps to apparent homogeneity. The purified protein is capable of supporting transcription of the luciferase gene from a reporter plasmid carrying the FR enhancer element to which EBNA1 binds. EBNA1 does not have oriP-dependent ATPase activity, indicating that it does not carry out an energy-dependent step in the initiation of DNA replication. However, EBNA1 does mediate an association between the two elements of oriP. We measured this association by binding one of the elements, the enhancer element, to a solid matrix and measuring retention by this element of the other one, the initiator element, in the presence of EBNA1. This retention is specific for DNA fragments containing EBNA1-binding sites. EBNA1 thus can link the two elements of the origin, providing a locally high concentration of EBNA1 at the site of initiation of DNA replication. We propose that this association is important either (i) to affect DNA structure to allow a cellular helicase to initiate DNA strand separation or (ii) to bind replication proteins to bring them to the origin of replication.

Adenosine Triphosphatases↗

PubMed1992

On the nature of origins of DNA replication in eukaryotes.

Chromosomal origins of DNA replication in higher eukaryotes differ significantly from those of E. coli (oriC) and the tumor virus, SV40 (ori sequence). Initiation events appear to occur throughout broad zones rather than at specific origin sequences. Analysis of four chromosomal origin regions reveals that they share common modular sequence elements. These include DNA unwinding elements, pyrimidine tracts that may serve as strong DNA polymerase-primase start sites, scaffold associated regions, transcriptional regulatory sequences, and, possibly, initiator protein binding sites and inherently destabilized regions. Based on the novel organization of chromosomal origin regions, we propose a model for initiation of DNA replication in higher eukaryotes. Unwinding of duplex DNA during initiation may be uncoupled, both temporally and spatially, from DNA synthesis, resulting in transient single-stranded intermediates that function in lieu of conventional replication forks during chromosomal DNA replication. DNA synthesis begins subsequently at multiple sites within the unwound regions rather than at specific origin sequences.

Animals↗

PubMed1978

Similarity of nucleotide sequences around the origin of DNA replication in mouse polyoma virus and simian virus 40.

The nucleotide sequence around the origin of replication in DNA of mouse polyoma virus was determined by 32P labeling of the 3' terminus of the Hap II-5/Alu I-1 DNA fragment, with the use of DNA polymerase. The result coincided with our previous report on the 32P labeling, with the use of polynucleotide kinase, of the 5' terminus of the Hap II-5/Hha I-1 DNA fragment, which corresponds to the large part of the present fragment, Hap II-5/Alu I-1. A symmetrical (A+T)-rich region containing a five-A stretch (or a five-T stretch) was flanked by two small regions with a 2-fold rotational axis of symmetry. On comparison of the sequence near the replication origin of polyoma DNA with that in the corresponding region of simian virus 40 DNA, which was included in the EcoRI-G fragment sequenced by Weissman's group (Subramanian K.N., Dahr, R. & Weissman, S. M. (1977) J. Biol. Chem. 252, 355--367), a considerable similarity was detected. Several possible common sequences for important biological activities such as the starting of DNA replication and RNA synthesis were suggested.

Base Sequence↗

PubMed1988

Sequence-specific interactions between a cellular DNA-binding protein and the simian virus 40 origin of DNA replication.

The core origin of simian virus 40 (SV40) DNA replication is composed of a 64-base-pair sequence encompassing T-antigen-binding site II and adjacent sequences on either side. A 7-base-pair sequence to the early side of T-antigen-binding site II which is conserved among the papovavirus genomes SV40, BK, JC, and SA12 was recently shown to be part of a 10-base-pair sequence required for origin activity (S. Deb, A.L. DeLucia, C.-P. Baur, A. Koff, and P. Tegtmeyer, Mol. Cell. Biol. 6:1663-1670, 1986), but its functional role was not defined. In the present report, we have used gel retention assays to identify a monkey cell factor that interacts specifically with double-stranded DNA carrying this sequence and also binds to single-stranded DNA. DNA-protein complexes formed with extracts from primate cells are more abundant and display electrophoretic mobilities distinct from those formed with rodent cell extracts. The binding activity of the factor on mutant templates is correlated with the replication activity of the origin. The results suggest that the monkey cell factor may be involved in SV40 DNA replication.

Animals↗

PubMed1987

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.

DNA Helicases↗

PubMed1989

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.

Adenine↗

PubMed1978

Construction of a viable SV40 variant containing two functional origins of DNA replication.

Viable variants of simian virus 40 (SV40) have been constructed which contain two functional origins of DNA replication (Or). The variants were made by introducing, at 0.175 on the SV40 map, a segment of DNA containing the viral Or. Two types of experiments demonstrate that the second Or is functional. First, the distribution of radioactivity in pulse-labeled SV40 (I) DNA is dramatically altered in the variants when compared with the parental virus. Second, electron microscopic examination of viral replicative intermediates indicates that while there is one initiation site for DNA synthesis in the parental genome, there are two sites in the variant. It was possible to introduce a deletion which inactivated the original Or at 0.67 map units in this variant. The resulting mutant could be propagated, and its DNA replication originated at the site of the newly inserted Or.

DNA Replication↗

PubMed1991

Simian virus 40 large T antigen untwists DNA at the origin of DNA replication.

Simian virus 40 large tumor antigen (SV40 T antigen) untwists DNA at the SV40 replication origin. In the presence of ATP, T antigen shifted the average linking number of an SV40 origin-containing plasmid topoisomer distribution. The loss of up to two helical turns was detected. The reaction required the presence of the 64-base pair core origin of replication containing T antigen DNA binding site II; binding site I had no effect on the untwisting reaction. The presence of human single-stranded DNA binding protein (SSB) slightly reduced the degree of untwisting in the presence of ATP. ATP hydrolysis was not required since untwisting occurred in the presence of nonhydrolyzable analogs of ATP. However, in the presence of a nonhydrolyzable analog of ATP, the requirement for the SV40 origin sequence was lost. The origin requirement for DNA untwisting was also lost in the absence of dithiothreitol. The origin-specific untwisting activity of T antigen is distinct from its DNA helicase activity, since helicase activity does not require the SV40 origin but does require ATP hydrolysis. The lack of a requirement for SSB or ATP hydrolysis and the reduction in the pitch of the DNA helix by just a few turns at the replication origin distinguishes this reaction from the T antigen-mediated DNA unwinding reaction, which results in the formation of a highly underwound DNA molecule. Untwisting occurred without a lag after the start of the reaction, whereas unwound DNA was first detected after a lag of 10 min. It is proposed that the formation of a multimeric T antigen complex containing untwisted DNA at the SV40 origin is a prerequisite for the initiation of DNA unwinding and replication.

Adenosine Triphosphate↗

PubMed1990

Analysis of the binding sites for the varicella-zoster virus gene 51 product within the viral origin of DNA replication.

The C-terminal 322 amino acids of the varicella-zoster virus (VZV) gene 51 product were expressed in Escherichia coli and shown to bind to specific DNA sequences within the VZV origin of DNA replication. The gene 51 product and its herpes simplex virus type 1 homolog (the UL9 protein) are capable of recognizing identical DNA sequences but the arrangement of binding sites within the origin regions of the two viruses differs. Three binding sites for the VZV gene 51 protein were identified within the VZV origin region and these lie in the same orientation and on the same side of the origin palindromic DNA sequence. DNA replication assays in transfected tissue culture cells demonstrated that the site closest to the palindrome is essential for origin activity whereas the most distal site is dispensable. The middle binding site may play an auxiliary role in DNA synthesis.

Base Sequence↗

PubMed1988

A model for initiation at origins of DNA replication.

Many prokaryotic origins resemble E. coli oriC in possessing essential AT-rich sequences, tandemly repeated. The role of these repeats may be in the initial opening of the duplex by the initiator protein, as has been found for the 13-mers in oriC and is implied for the 11-mers of the lambda origin. Regulatory influences on the effective action of the initiator protein of E. coli (dnaA protein) include transcriptional activation of the origin, nucleotide binding and membrane attachment of the protein, and interactions leading to the introduction of helicases to start replication forks.

Adenosine Triphosphate↗

PubMed1991

Cloning, sequencing, and functional analysis of a Marek's disease virus origin of DNA replication.

Previously, we isolated a replicon from a defective Marek's disease virus (MDV), analogous to defective herpes simplex viruses (amplicons). Defective viruses contain cis-acting elements required for DNA synthesis and virus propagation such as an origin of DNA replication and a packaging-cleavage signal site. In this report, the MDV replicon was utilized to locate an origin of MDV DNA replication. A comparison of MDV replicon sequences with other herpesvirus replication origin sequences revealed a 90-bp sequence containing 72% identity to the lytic origin (oris) of herpes simplex virus type 1. This 90-bp sequence displayed no similarity to betaherpesvirus or gammaherpesvirus replication origins. The 90-bp sequence is arranged as an imperfect palindrome centered around an A+T-rich region. This sequence also contains a 9-bp motif (5'CGTTCGCAC3') highly conserved in alphaherpesvirus replication origins. To test functionality of the 90-bp putative MDV replication origin, we conducted DpnI replication assays with subclones generated from the 4-kbp MDV replicon. A 700-bp MDV replicon subfragment containing the 90-bp putative MDV replication origin sequence is capable of replicating in chicken embryo fibroblast cells cotransfected with helper virus DNA. In conclusion, we identified a functional origin of DNA replication in MDV. Similarity of MDV origin sequences to those of alphaherpesviruses supports the current contention that MDV is more closely related to alphaherpesviruses than to gammaherpesviruses.

Animals↗

PubMed1992

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.

Adenoviruses, Human↗

PubMed1985

Expression of an immediate early polypeptide and activation of a viral origin of DNA replication in cells containing a fragment of herpes simplex virus DNA.

A thymidine kinase cotransformation procedure has been used to introduce the sequences encoding the herpes simplex virus type 1 (HSV-1) immediate early protein, Vmw175, into permissive cells either in the presence or the absence of the adjacent origin of viral DNA replication. Cells transformed by either origin-plus or origin-minus DNA were capable of expressing functional Vmw175 as indicated by their ability to complement the growth at the nonpermissive temperature of an HSV-1 mutant, ts K, containing a temperature-sensitive lesion in the Vmw175 gene. A proportion of the virus yield from cells transformed with the origin-plus, but not the origin-minus, plasmid exhibited a ts+ phenotype. The generation of ts+ virus correlated with an amplification of input plasmid DNA sequences which occurred following superinfection, suggesting that recombination between the ts mutant and the amplified viral DNA sequences had taken place. Encapsidation of the amplified DNA sequences was also detected, suggesting that in addition to a functional origin of replication and Vmw175 gene the transformed cells also retain the viral DNA packaging signals.

Capsid↗

PubMed1992

Guide to identification of origins of DNA replication in eukaryotic cell chromosomes.

Several experimental approaches for identification of origins of DNA replication have been developed recently that allow, for the first time, unique initiation sites in mammalian chromosomes to be mapped at single-copy loci. A brief description of the rationale, advantages, and limitations has been provided for each approach, as well as information that can help the reader choose the method(s) most suitable for a particular system. The various methods are divided into three groups: (1) analysis of nascent DNA strands, (2) analysis of DNA structures, and (3) analysis of origin activity (i.e., ability to support autonomous replication). It is hoped that this information will serve as a practical guide for identifying new origins of replication.

Animals↗