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PubMed2004

DNA replication initiates at domains overlapping with nuclear matrix attachment regions in the xenopus and mouse c-myc promoter.

Only a very few origins have been mapped in different multicellular organisms, and they do not share detectable consensus sequence elements. Moreover, it is not clear if origins are localized at similar positions in the corresponding locus in genomes of different organisms. Here, we have mapped DNA replication origins in the c-myc locus both in Xenopus and mouse, allowing a comparison of the corresponding sites in three different animal species (Xenopus, mouse, human). An origin of DNA replication is present in the three homologous c-myc loci. In Xenopus, a main DNA replication origin was located 3 kilobases (kb) upstream of the active c-myc promoter, whereas, in mouse, we detected an origin 1 kb upstream of the promoter, as previously mapped in human c-myc. We also identified a nuclear matrix attachment region in both Xenopus and mouse, which is localized to two different regions of the c-myc promoter region. However, in both cases, the nuclear matrix attachment sites are close to the DNA replication origin mapped in the locus. These data suggest that global features of chromatin organization in different organisms may contribute to DNA replication origin localization.

Animals↗
PubMed1995

Eukaryotic replicators and associated protein complexes.

In the past year, genetic studies have provided a detailed understanding of the DNA sequence elements that constitute Saccharomyces cerevisiae origins of DNA replication and have identified larger DNA domains that direct DNA replication in both Schizosaccharomyces pombe and human cells. In vivo studies of the proteins associated with S. cerevisiae origins of DNA replication indicate that there are dynamic changes in origin chromatin structure during the cell cycle and suggest that the Cdc7 protein kinase is among the associated proteins.

Chromatin↗
PubMed1978

Human mitochondrial DNA: analysis of 7S DNA from the origin of replication.

Heat-treated samples of human mitochondrial DNA (mtDNA) exhibited a set of three low molecular weight DNA bands in addition to the major mtDNA band when electrophoresed in polyacrylamide gels. These DNA components were seen only after heat treatment or after relaxation of the mtDNA with a restriction endonuclease. The three components were single stranded and had sizes of 550, 585, and 629 nucleotides, close to the size (600 nucleotides) estimated from contour length measurements for the 7S DNA from the D loop of human mtDNA. Hybridization of the components with restriction endonuclease fragments of known position in the mtDNA confirmed this identification. Digestion of each 7S DNA component with the restriction endonuclease Hae III produced three fragments, two of which were identical in size among the components and the third of which varied. This third fragment, shown to be from the 5' end of each component, differed in length by approximately 35 nucleotides among the components. These results suggest that human 7S mtDNA synthesis is terminated at a distinct position and that it is either initiated at one of three possible sites in the same mtDNA or that the mtDNA population consists of three subpopulations, each differing from the others by the presence or absence of a nucleotide sequence immediately adjacent to the origin of replication.

DNA Restriction Enzymes↗
PubMed1990

Reconstitution of simian virus 40 DNA replication with purified proteins.

Replication of plasmid DNA molecules containing the simian virus 40 (SV40) origin of DNA replication has been reconstituted with seven highly purified cellular proteins plus the SV40 large tumor (T) antigen. Initiation of DNA synthesis is absolutely dependent upon T antigen, replication protein A, and the DNA polymerase alpha-primase complex and is stimulated by the catalytic subunit of protein phosphatase 2A. Efficient elongation of nascent chains additionally requires proliferating cell nuclear antigen, replication factor C, DNA topoisomerase I, and DNA polymerase delta. Electron microscopic studies indicate that DNA replication begins at the viral origin and proceeds via intermediates containing two forks that move in opposite directions. These findings indicate that the reconstituted replication reaction has many of the characteristics expected of authentic viral DNA replication.

Antigens, Polyomavirus Transforming↗
PubMed2001

The Chinese hamster dihydrofolate reductase replication origin beta is active at multiple ectopic chromosomal locations and requires specific DNA sequence elements for activity.

To identify cis-acting genetic elements essential for mammalian chromosomal DNA replication, a 5.8-kb fragment from the Chinese hamster dihydrofolate reductase (DHFR) locus containing the origin beta (ori-beta) initiation region was stably transfected into random ectopic chromosomal locations in a hamster cell line lacking the endogenous DHFR locus. Initiation at ectopic ori-beta in uncloned pools of transfected cells was measured using a competitive PCR-based nascent strand abundance assay and shown to mimic that at the endogenous ori-beta region in Chinese hamster ovary K1 cells. Initiation activity of three ectopic ori-beta deletion mutants was reduced, while the activity of another deletion mutant was enhanced. The results suggest that a 5.8-kb fragment of the DHFR ori-beta region is sufficient to direct initiation and that specific DNA sequences in the ori-beta region are required for efficient initiation activity.

Animals↗
PubMed1997

Formation of herpes simplex virus type 1 replication compartments by transfection: requirements and localization to nuclear domain 10.

During infection, the seven essential herpes simplex virus type 1 (HSV-1) replication proteins are found in globular nuclear structures called replication compartments. Replication compartments form adjacent to ND10, nuclear matrix-bound domains which are present in most cell types but whose function is unknown (G. G. Maul, I. M. Ishov, and R. D. Everett, Virology 217:67-75, 1996). We now demonstrate that replication compartments can be formed by cotransfecting Vero cells with constructs expressing the seven essential viral replication proteins and a plasmid containing an HSV-1 origin of DNA replication. Like replication compartments in infected cells, replication compartments formed by cotransfection contain all of the essential viral replication proteins, are sites of DNA synthesis, and are found adjacent to ND10. However, neither the viral origin-binding protein nor a plasmid containing an HSV-1 origin of DNA replication is individually required for the formation of transfection replication compartments, although the presence of each increases the efficiency of replication compartment formation. Further, we provide evidence that UL29 independently localizes adjacent to ND10 and so may play a role in directing replication compartments to these preexisting nuclear structures.

Animals↗
PubMed2006

DNA recombination-initiation plays a role in the extremely biased inheritance of yeast [rho-] mitochondrial DNA that contains the replication origin ori5.

Hypersuppressiveness, as observed in Saccharomyces cerevisiae, is an extremely biased inheritance of a small mitochondrial DNA (mtDNA) fragment that contains a replication origin (HS [rho(-)] mtDNA). Our previous studies showed that concatemers (linear head-to-tail multimers) are obligatory intermediates for mtDNA partitioning and are primarily formed by rolling-circle replication mediated by Mhr1, a protein required for homologous mtDNA recombination. In this study, we found that Mhr1 is required for the hypersuppressiveness of HS [ori5] [rho(-)] mtDNA harboring ori5, one of the replication origins of normal ([rho(+)]) mtDNA. In addition, we detected an Ntg1-stimulated double-strand break at the ori5 locus. Purified Ntg1, a base excision repair enzyme, introduced a double-stranded break by itself into HS [ori5] [rho(-)] mtDNA at ori5 isolated from yeast cells. Both hypersuppressiveness and concatemer formation of HS [ori5] [rho(-)] mtDNA are simultaneously suppressed by the ntg1 null mutation. These results support a model in which, like homologous recombination, rolling-circle HS [ori5] [rho(-)] mtDNA replication is initiated by double-stranded breakage in ori5, followed by Mhr1-mediated homologous pairing of the processed nascent DNA ends with circular mtDNA. The hypersuppressiveness of HS [ori5] [rho(-)] mtDNA depends on a replication advantage furnished by the higher density of ori5 sequences and on a segregation advantage furnished by the higher genome copy number on transmitted concatemers.

Alloxan↗
PubMed1999

DNA replication of human papillomavirus type 31 is modulated by elements of the upstream regulatory region that lie 5' of the minimal origin.

The viral replication factors E1 and E2 of papillomaviruses are necessary and sufficient to replicate plasmids containing the minimal origin of DNA replication in transient assays. Under physiological conditions, the upstream regulatory region (URR) governs expression of the early viral genes. To determine the effect of URR elements on E1 and E2 expression specifically, and on the regulation of DNA replication during the various phases of the viral life cycle, we carried out a systematic replication study with entire genomes of human papillomavirus type 31 (HPV31), a high-risk oncogenic type. We constructed a series of URR deletions, spacer replacements, and point mutations to analyze the role of the keratinocyte enhancer (KE) element, the auxiliary enhancer (AE) domain, and the L1-proximal end of the URR (5'-URR domain) in DNA replication during establishment, maintenance, and vegetative viral DNA amplification. Using transient and stable replication assays, we demonstrate that the KE and AE are necessary for efficient E1 and E2 gene expression and that the KE can also directly modulate viral replication. KE-mediated activation of replication is dependent on the position and orientation of the element. Mutation of either one of the four Ap1 sites, the single Sp1 site, or the binding site for the uncharacterized footprint factor 1 reduced replication efficiency through decreased expression of E1 and E2. Furthermore, the 5'-URR domain and the Oct1 DNA binding site are dispensable for viral replication, since such HPV31 mutants are able to replicate efficiently in a transient assay, maintain a stable copy number over several cell generations, and amplify viral DNA under vegetative conditions. Interestingly, deletion of the 5'-URR domain leads to increased transient and stable replication levels. These findings suggest that elements in the HPV31 URR outside the minimal origin modulate viral replication through both direct and indirect mechanisms.

Binding Sites↗
PubMed1985

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.

Adenoviruses, Human↗
PubMed1984

Protein-primed replication of plasmids containing the terminus of the adenovirus genome. I. Characterization of an in vitro DNA replication system dependent on adenoviral DNA sequences.

An in vitro system which replicates plasmid DNA containing the replication origin of adenovirus DNA has been established. Replication of plasmid pLA1 DNA, which contains the left-hand terminus (0-9.4 map units) of adenovirus serotype 5 DNA but which lacks the 55,000-dalton terminal protein, is initiated by a protein-primed mechanism in a manner similar to that found with adenovirus DNA. Initiation of DNA replication using plasmid pLA1 as a template requires (i) that the cloned adenovirus sequence be present at the terminus of a linearized (form III) DNA molecule ( Tamanoi , F., and Stillman , B. W. (1982) Proc. Natl. Acad. Sci. U. S. A., 79, 2221-2225; van Bergen, B. G. M., van der Ley , P. A., van Driel , W., van Mansfield , A. D. M., and van der Vliet , P. A. (1983) Nucleic Acid Res. 11, 1975-1979), and (ii) the presence of the 80,000-dalton precursor to the 55,000-dalton terminal protein and the adenovirus coded DNA-dependent DNA polymerase. In the presence of the four deoxy-nucleoside triphosphates, the preterminal protein, the adenovirus coded DNA binding protein, and an extract prepared from uninfected HeLa nuclei, the adenovirus DNA polymerase can elongate the preterminal-protein dCMP initiation complex formed on pLA1 DNA to full length (6.6 kilobase) DNA molecules. These results suggest that the 55,000-dalton terminal protein covalently linked to the 5' termini of adenovirus DNA is not essential for the replication of this DNA.

Adenoviridae↗
PubMed1993

Cell cycle-regulated nuclear localization of MCM2 and MCM3, which are required for the initiation of DNA synthesis at chromosomal replication origins in yeast.

MCM2 and MCM3 are two genetically interacting and structurally related proteins essential for growth in Saccharomyces cerevisiae. Mutants defective in these proteins affect the stability of minichromosomes in general, but the severity of the defect is dependent on the autonomously replicating sequence (ARS) that drives the replication of that plasmid. In this paper we show by two-dimensional gel electrophoresis that the initiation of DNA synthesis at chromosomal replication origins is also reduced in frequency in these mutants. We show further that the nuclear and subnuclear localizations of the MCM2 and MCM3 proteins are temporally regulated with respect to the cell cycle. These proteins enter the nucleus at the end of mitosis, persist there throughout G1 phase, and disappear from it at the beginning of S phase. Once inside the nucleus, a fraction of the MCM2 and MCM3 proteins becomes tightly associated with DNA. The association of MCM2 and MCM3 with chromatin presumably leads to the initiation of DNA synthesis, and their subsequent disappearance from the nucleus presumably prevents reinitiation of DNA synthesis at replication origins. This temporally and spatially restricted localization of MCM2 and MCM3 in the nucleus may serve to ensure that DNA replication occurs once and only once per cell cycle.

Cell Cycle↗
PubMed1986

The adenine-thymine domain of the simian virus 40 core origin directs DNA bending and coordinately regulates DNA replication.

The simian virus 40 origin of replication contains a 20-base-pair adenine-thymine-rich segment with the sequence 5'-TGCATAAATAAAAAAAATTA-3'. The continuous tract of eight adenines is highly conserved among polyomaviruses. We used single-base substitutions to map structural and functional features of this DNA. Mutations in the AAA and AAAAAAAATT sequences significantly reduce DNA replication and thus identify two sequence-specific functional domains or a single domain with two parts. The AAAAAAAATT sequence also determines a DNA conformation that is characteristic of DNA bending. Single-base mutations in this domain change the degree of net bending, presumably by altering the length and location of the bending sequence. Thus, DNA bending in the correct conformation and location may be a structural signal for replication in polyomavirus origins and perhaps in other origins of replication with consecutive runs of adenines. The first five base pairs (TGCAT) of the 20-base-pair segment and the T between the AAA and AAAAAAAATT domains serve a sequence-independent function that may establish proper spacing within the core origin.

Adenine↗
PubMed2003

DNA replication of mitotic chromatin in Xenopus egg extracts.

Prereplication complexes are assembled at eukaryotic origins of DNA replication in the G1 phase of the cell cycle, and they are activated in S phase by cyclin-dependent kinase (Cdk)2/cyclin E and Cdk2/cyclin A. Previous experiments using Xenopus nuclear assembly egg extracts suggested that Cdk1/cyclin A, which is normally active in early mitosis, can replace the function of Cdk2 in driving DNA replication, whereas Cdk1/cyclin B, which functions later in mitosis, cannot. Here, we use a completely soluble replication system derived from Xenopus egg extracts to show that Cdk1/cyclin B also can support DNA replication. The ability of mitotic Cdks to drive DNA replication raises the question of whether DNA replication is possible in mitosis. To address this question, chromatin containing prereplication complexes was driven into mitosis with Cdk1/cyclin B. Strikingly, upon addition of a replication extract, the chromatin underwent a complete round of DNA replication. Replicating mitotic chromosomes became visibly decondensed, and, after DNA replication was complete, they recondensed. Our results indicate that there is extensive overlap in the substrate specificity of the major metazoan Cdk/cyclin complexes and that mitosis is not fundamentally incompatible with DNA replication. The results suggest that origins that fail to initiate DNA replication in S phase might still be able to do so in mitosis.

Animals↗
PubMed1987

A DNA binding protein showing sequence specificity for a region containing the replication origin of Xenopus laevis mitochondrial DNA.

In Xenopus laevis mitochondria up to 14 different polypeptides with affinity for the DNA, have been identified by the protein blotting technique. Under stringent binding conditions only one polypeptide displayed specific affinity for a restriction fragment containing the H strand origin of replication of the Xenopus laevis mt chromosome. The proteins were fractionated by double stranded DNA cellulose chromatography. Under conditions which favor high affinity interactions between proteins and DNA, a protein of the 2M NaCl step shows specific binding to the DNA fragments containing the D-loop region. Some physical properties of the protein have been studied. It has a MW of 21.5 Kd and a globular shape as can be inferred from the relationship between MW and sedimentation coefficient (2.7 S). It binds non cooperatively to DNA and forms relatively stable complexes as demonstrated by DNA competition experiments.

Animals↗