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M Zannis-Hadjopoulos

Publications and source records attributed to M Zannis-Hadjopoulos.

At least 19 recordsLinked to original sources
PubMed1998

Oct-1 enhances the in vitro replication of a mammalian autonomously replicating DNA sequence.

A 186-base pair fragment of ors8, a mammalian autonomously replicating DNA sequence isolated by extrusion of nascent monkey DNA in early S phase, has previously been identified as the minimal sequence required for replication function in vitro and in vivo. This 186-base pair fragment contains, among other sequence characteristics, an imperfect consensus binding site for the ubiquitous transcription factor Oct-1. We have investigated the role of Oct-1 protein in the in vitro replication of this mammalian origin. Depletion of the endogenous Oct-1 protein, by inclusion of an oligonucleotide comprising the Oct-1 binding site,inhibited the in vitro replication of p186 to approximately 15-20% of the control, whereas a mutated Oct-1 and a nonspecific oligonucleotide had no effect. Furthermore, immunodepletion of the Oct-1 protein from the HeLa cell extracts by addition of an anti-POU antibody to the in vitro replication reactioninhibited p186 replication to 25% of control levels. This inhibition of replication could be partially reversed to 50-65% of control levels, a two- to threefold increase, upon the addition of exogenous Oct-1 POU domain protein. Site-directed mutagenesis of the octamer binding site in p186 resulted in a mutant clone, p186-MutOct, which abolished Oct-1 binding but was still able to replicate as efficiently as the wild-type p186. The results suggest that Oct-1 protein is an enhancing component in the in vitro replication of p186 but that its effect on replication is not caused through direct binding to the octamer motif.

Animals↗
PubMed1997

Circular YAC vectors containing a small mammalian origin sequence can associate with the nuclear matrix.

Three different mammalian origins of DNA replication, 343, S3, and X24, have been cloned into a 15.8 kb circular yeast vector pYACneo. Subsequent transfection into HeLa cells resulted in the isolation of several stably maintained clones. Two cell lines, C343e2 and CS3e1, were found to have sequences maintained as episomes in long-term culture with a stability per generation of approximately 80%. Both episomes also contain matrix attachment region (MAR) sequences which mediate the binding of DNA to the nuclear skeleton and are thought to play a role in DNA replication. Using high salt extraction of the nucleus and fluorescent in situ hybridization, we were able to demonstrate an association of the 343 episome with the nuclear matrix, most probably through functional MAR sequences that allow an association with the nuclear matrix and associated regions containing essential replication proteins. The presence of functional MARs in small episomal sequences may facilitate the replication and maintenance of transfected DNA as an episome and improve their utility as small episomal constructs, potential microchromosomes.

Animals↗
PubMed1997

Differential DNA replication origin activities in human normal skin fibroblast and HeLa cell lines.

A modification of the extrusion method for the isolation of nascent DNA from mammalian cells and a PCR-based assay has been used in order to compare the in vivo activities of DNA replication origins in different cell lines. Conventional PCR was firstly applied to detect the chromosomal activities of several known (origins associated with c-myc, hsp70, beta-globin, immunoglobulin mu-chain enhancer) and putative DNA replication origins (autonomously replicating sequences obtained from enriched libraries of human origins of DNA replication from normal and transformed cells) in four human cell lines (HeLa, NSF, WI-38 and SK-MG-1). Then, in nascent DNA samples from normal skin fibroblast (NSF) and HeLa cells, abundance of DNA sequences in the regions of five of these origins was determined by competitive PCR. Our results suggest that autonomously replicating sequences NOA3, S14, S3 and F15 are associated with functional chromosomal origins of replication. Quantitative comparison of origin activities demonstrates that origins associated with c-myc and NOA3 are approximately twice as active in HeLa cells as in NSF cells. The described approach can facilitate the identification of origins which may be differentially active in normal cells and transformed cells or in different cell types.

Cell Line, Transformed↗
PubMed1997

Deletion analysis of ors12, a centromeric, early activated, mammalian origin of DNA replication.

We have generated a panel of deletion mutants of ors12 (812-bp), a mammalian origin of DNA replication previously isolated by nascent strand extrusion from early replicating African Green monkey (CV-1) DNA. The deletion mutants were tested for their replication activity in vivo by the bromodeoxyuridine substitution assay, after transfection into HeLa cells, and in vitro by the Dpnl resistance assay, using extracts from HeLa cells. We identified a 215-bp internal fragment as essential for the autonomous replication activity of ors12. When subcloned into the vector pML2 and similarly tested, this subfragment was capable of autonomous replication in vivo and in vitro. Several repeated sequence motifs are present in this 215-bp fragment, such as TGGG(A) and G(A)AG (repeated four times each); TTTC, AGG, and CTTA (repeated 3 times each); the motifs CACACA and CTCTCT, and two imperfect inverted repeats. 22 and 16 bp long, respectively. The overall sequence of the 215-bp fragment is G/C-rich (50.2%), by comparison to the 186-bp (33.5% G/C-rich) minimal sequence required for the autonomous replication activity of ors8, another functional ors that was similarly isolated and characterized.

Base Sequence↗
PubMed1996

The effect of regulatory sequence elements upon the initiation of DNA replication of the minute virus of mice.

The minute virus of mice (MVM) genome is a linear single-stranded length of approximately 5000 nucleotides of DNA with unique terminal palindromic sequences at both ends. The left(3') hairpin is used to prime the initiation of DNA synthesis on parental single-strand DNA while the right (5') hairpin or stem-plus-arms structure can also prime the initiation of DNA synthesis during synthesis of dimer and higher oligomers as well as synthesis of progeny single strands. Previous studies have shown that if viral duplex DNA was input into an in vitro DNA replication system using extracts from uninfected HeLa cells, the 5' end of the molecule was able to form a hairpin and initiate DNA synthesis by DNA polymerase delta (Cossons et al. (1996), Virology 216, 258-264). In this study, the effect of the deletion of known cis-acting genetic elements upon the initiation of DNA replication was studied using a series of MVM mutants with deletions within the 5' terminal region. Mutants containing deletions of elements A (nucleotides 4489-4636), B (nucleotides 4636-4695), and either one or both of the 65-bp repeats (nucleotides 4720-4785 and 4785-4849) were used as template in the in vitro DNA replication system. When element A was deleted, the efficiency of initiation decreased significantly. Subsequent removal of element B, leaving just the two 65-bp repeats, restored levels of initiation back to those seen in the wild-type genome. In the absence of either A or B both 65-bp repeats were necessary for efficient initiation, and removal of one of these repeats caused a decrease in efficiency. Thus, element B appeared to have a negative regulatory effect (in the absence of element A), and element A appeared to have a positive regulatory effect, at least in the presence of element B. These data demonstrate, for the first time, a complex interaction between these cis-acting regulatory elements which can function as both positive or negative regulators in the initiation of MVM DNA replication.

Animals↗
PubMed1996

Identification of a putative DNA replication origin in the gamma-aminobutyric acid receptor subunit beta3 and alpha5 gene cluster on human chromosome 15q11-q13, a region associated with parental imprinting and allele-specific replication timing.

The region containing the GABAA receptor beta3 and alpha5 subunit-encoding genes is subject to parental imprinting and is organized in different allele-specific replication timing domains. A 60-kb domain displaying a maternal early/paternal late pattern of allele-specific replication timing asynchrony is nested within a larger region displaying the opposite pattern. The proximal portion of this maternal early replicating domain is incorporated into phage clone lambda84. In order to identify DNA structures which may be associated with the boundary between the replication domains, phage lambda84 has been subcloned into smaller fragments and several of these have been analyzed by nucleotide sequencing. A plot of helical stability for 13kb of contiguous sequence reveals several A + T-rich regions which display potential DNA unwinding. The plasmid subclones from phage lambda84 have been analyzed for bent DNA and one of these, p82, contains bent DNA and overlaps with the region of highest potential helical instability. Of the seven plasmids tested, only p82 shows strong autonomous replication activity in an in vitro replication assay, with replication initiating within the genomic insert. These results suggest that a putative origin of DNA replication contained within p82 may play a role in establishing the allele-specific replication timing domains in the GABAA receptor subunit gene cluster.

Alleles↗
PubMed1996

Application of an in vitro system in the study of chemotherapeutic drug effects on DNA replication.

DNA replication machinery is an important target for chemotherapeutic drugs. We have used an in vitro system to study the effect of drugs on mammalian DNA replication, either by direct interaction with the DNA structure or with replication proteins and machinery. The anthracycline doxorubicin (Dox) showed a dose-dependent inhibitory effect on DNA replication, whether incubated with HeLa cell extracts or with DNA and nucleotides. Earliest-labeled fragment analysis revealed that inhibition of replication began within the origin-containing fragment in both control and Dox-containing reactions in vitro. AraC, a nucleoside analog, had no significant effect on DNA synthesis. In contrast, araCTP was able to inhibit DNA replication in vitro. Since metabolism is diminished in this in vitro system, the degree of phosphorylation of araC was apparently low. Progesterone showed an increase in nucleotide incorporation (sensitive to BuPdGTP inhibition of replication-specific polymerases alpha and delta) after preincubation with HeLa cell extracts, although progesterone receptors were not detectable in the HeLa cell extracts. In addition, we observed an inhibition in DNA replication when progesterone was preincubated with DNA and nucleotides. These results suggest that progesterone may have a mechanism of action that is different from any known to be mediated through progesterone receptors. In conclusion, these results indicate that this mammalian in vitro replication system will be useful for the study of mechanisms and design of therapeutic drugs that inhibit mammalian DNA replication.

Antibiotics, Antineoplastic↗
PubMed1996

DNA polymerase delta-dependent formation of a hairpin structure at the 5' terminal palindrome of the minute virus of mice genome.

The parvovirus the minute virus of mice (MVM) has a linear single-stranded DNA genome with unique palindromic sequences at both termini which enable it to fold back on itself and form hairpin or cruciform-type structures. The purpose of this study was to examine the primary events occurring during MVM replication mediated solely by the host cell replication machinery. In an in vitro DNA replication system using HeLa cell extracts, we found that there was a distinct activity that utilized the 5' terminal palindrome sequence of MVM to produce a secondary structure from a duplex extended form, in a time-dependent fashion. The secondary structure was due to the formation of a hairpin rather than a stem-plus-arms type structure and was associated with initiation of DNA synthesis, performed specifically by DNA polymerase delta. Inhibition of DNA polymerase alpha had no effect upon this activity. Removal of all but 13 base pairs of the hairpin arm abolished the synthesis of DNA, indicating that there is a minimal length requirement for the duplex region of DNA or that this region contains regulatory genetic elements. These data are consistent both with the role of DNA polymerase delta in extending the synthesis of DNA from a DNA primer and with unidirectional continuous DNA synthesis, initiating from a hairpin, as a mode of replication for MVM.

Animals↗
PubMed1996

Inverted repeats, stem-loops, and cruciforms: significance for initiation of DNA replication.

Inverted repeats occur nonrandomly in the DNA of most organisms. Stem-loops and cruciforms can form from inverted repeats. Such structures have been detected in pro- and eukaryotes. They may affect the supercoiling degree of the DNA, the positioning of nucleosomes, the formation of other secondary structures of DNA, or directly interact with proteins. Inverted repeats, stem-loops, and cruciforms are present at the replication origins of phage, plasmids, mitochondria, eukaryotic viruses, and mammalian cells. Experiments with anti-cruciform antibodies suggest that formation and stabilization of cruciforms at particular mammalian origins may be associated with initiation of DNA replication. Many proteins have been shown to interact with cruciforms, recognizing features like DNA crossovers, four-way junctions, and curved/bent DNA of specific angles. A human cruciform binding protein (CBP) displays a novel type of interaction with cruciforms and may be linked to initiation of DNA replication.

Bacteriophages↗
PubMed1995

Anti-cruciform monoclonal antibody and cruciform DNA interaction.

Cruciform DNA structure, as a structural feature, has been associated with regulation of transcription, recombination and replication. Previously used to successfully modify DNA replication and affinity-purify origins and autonomously replicating sequences. Using enzyme protection assays, their binding activity has been localized to the base (elbow) of the cruciform stem. We report here the hydroxyl radical footprinting of 2D3 (kappa IgG1) anti-cruciform monoclonal antibody on a stable cruciform structure created by heteroduplexing fragments from two plasmids, identical except for two centrally located palindromes of different sequence. The footprinting was performed at near-physiological salt concentrations, conditions favouring the stacked X-structure of the cruciform. Our data show that binding by the antibody occurs at the four-way junction (elbows) of the stable cruciform. The binding of the antibody seems also to cause associated structural distortions in the heteroduplex, which generally result in greater sensitivity to hydroxyl radicals at the tips of the cruciforms. The data are consistent to hydroxyl radicals at the tips of the cruciforms. The data are consistent with the binding of a single antibody to an antigen-combining site. The results of this study compare favourably with the hydroxyl radical footprinting studies reported recently for a human cruciform binding protein (CBP), which binds at the base of the stem-loop structure and causes similar distortions of the stable cruciform structure. These studies indicate that the four-way junction of the cruciform possesses certain unique structural qualities that are antigenic; the association of this structural determinant with DNA replication and the existence of a novel cellular protein, CBP, of similar binding specificity as the antibody specificity support a role for cruciforms as important regulatory recognition signals in replication.

Antibodies, Monoclonal↗
PubMed1995

A novel type of interaction between cruciform DNA and a cruciform binding protein from HeLa cells.

We recently identified and enriched a protein (CBP) from HeLa cells with binding specificity for cruciform-containing DNA. We have now studied the interaction of CBP with stable cruciform DNA molecules containing the 27 bp palindrome of SV40 on one strand and an unrelated 26 bp palindrome on the other strand by hydroxyl radical footprinting. The CBP-DNA interaction is localized to the four-way junction at the base of the cruciforms. CBP appears to interact with the elbows of the junctions in an asymmetric fashion. Upon CBP binding, structural distortions were observed in the cruciform stems and in a DNA region adjacent to the junction. These features distinguish CBP from other cruciform binding proteins, which bind symmetrically and display exclusively either contacts with the DNA backbone or structural alterations in the DNA.

Base Sequence↗
PubMed1995

Deletion analysis of minimal sequence requirements for autonomous replication of ors8, a monkey early-replicating DNA sequence.

We have generated a panel of deletion mutants of ors8 (483 bp), a mammalian autonomously replicating DNA sequence, previously isolated by extrusion of nascent monkey (CV-1) DNA from replication bubbles active at the onset of S phase. The deletion mutants were tested for replication function by the DpnI resistance assay, in vivo, after transfection into HeLa cells, and in vitro. An internal fragment of 186-bp that is required for autonomous replication function of ors8 was identified. This fragment, when subcloned into pBR322 and similarly tested, was capable of autonomous replication in vivo and in vitro. The 186-bp fragment contains several repeated sequence motifs, such as the ATTA and ATTTAT motifs, occurring three and five times, respectively, the sequences TAGG and TAGA, occurring three and seven times, respectively, two 5'-ATT-3' repeats, a 44-bp imperfect inverted repeat (IR) sequence, and an imperfect consensus binding element for the transcription factor Oct-1. A measurable sequence-directed DNA curvature was also detected, coinciding with the AT-rich regions of the 186-bp fragment.

Animals↗
PubMed1995

Cofractionation of HeLa cell replication proteins with ors-binding activity.

Ors (origin enriched sequence) 8 is a mammalian autonomously replicating DNA sequence previously isolated by extrusion of nascent monkey (CV-1) DNA in early S phase. A 186 bp fragment of ors 8 has been identified as the minimal sequence required for origin function, since upon its deletion the in vivo and in vitro replication activity of this ors is abolished. We have fractionated total HeLa cell extracts on a DEAE-Sephadex and then on a Affi-Gel Heparin column and identified a protein fraction that interacts with the 186 bp fragment of ors 8 in a specific manner. The same fraction is able to support the in vitro replication of ors 8 plasmid. The ors binding activity (OBA) present in this fraction sediments at approximately 150 kDa in a glycerol gradient. Band-shift elution experiments of the specific protein-DNA complex detect by silver-staining predominantly two protein bands with molecular weights of 146 kDa and 154 kDa, respectively. The fraction containing the OBA is also enriched for polymerases alpha and delta, topoisomerase II, and replication protein A, (RP-A).

Base Sequence↗
PubMed1994

Autonomous replication in vivo and in vitro of clones spanning the region of the DHFR origin of bidirectional replication (ori beta).

Plasmids containing the origin of bidirectional replication (ori beta) of the Chinese hamster dihydrofolate reductase-encoding gene (DHFR) were tested for autonomous replication in vivo and in vitro. The results show that plasmids pX24 and pneoS13, that contain a 4.8- and a 11.5-kb fragment, respectively, spanning the ori beta region, are able to replicate autonomously in human cells and in a cell-free system that uses human cell extracts. Another plasmid, pX14, containing a 4.8-kb fragment that is immediately adjacent to the ori beta region, also replicated in these two assays.

Animals↗
PubMed1994

Cruciform DNA binding protein in HeLa cell extracts.

We have analyzed by band-shift assays HeLa cell protein-DNA interactions on a stable cruciform DNA molecule. The stable cruciform was formed by heteroduplexing the HindIII-SphI fragment of SV40 virus DNA that contains the origin of replication with a derivative mutant containing a heterologous substitution at the central inverted repeat. We have identified a novel binding activity in HeLa cell extracts with specificity for the cruciform-containing DNA and no apparent sequence specificity. The activity is protein-dependent, void of detectable nuclease activity, and distinct from that reported for HMG1. A cruciform binding protein (CBP) with an apparent molecular weight of 66 kDa was enriched from HeLa cell extracts. In addition to the CBP, we have detected sequence-specific binding activities to sites proximal to the cruciform. Binding to one such site is increased in the cruciform-containing heteroduplex DNA by comparison to its linear homoduplex counterpart, suggesting transmission of structural effects by the stem-loops to their local environment.

Base Sequence↗
PubMed1994

A reproducible method for identification of human genomic DNA autonomously replicating sequences.

We demonstrate a method for the isolation of autonomously replicating sequences from pools of clones obtained from genomic DNA libraries constructed using affinity purification of cruciform DNA. The selection of autonomously replicating sequences was based on their differential ability to replicate as episomes after transfection of pools of plasmid clones into human HeLa cells. Two separate libraries containing affinity-purified cruciform DNA were used, one prepared from DNA of log phase primary human genital fibroblasts and the other prepared from DNA of log phase SW48 colon adenocarcinoma cells. Representative samples of the entire phage libraries were converted to phagemid clones by filamentous helper phage-mediated mass excision to produce pBluescript libraries in Escherichia coli. Clones were grown up individually and the bacteria pooled into groups of 48 for recovery of plasmid DNA. Plasmid pools of 48 independent clones (120 micrograms total) were then transfected by calcium phosphate coprecipitation onto log phase HeLa cells, which were allowed to grow for 3 days before recovery of plasmid by Hirt lysis. The recovery of plasmid from each transfection was estimated to range from 10 to 60 ng. DpnI digestion was then used to digest plasmids which had not been replicated and therefore retained a bacterial methylation pattern which was sensitive to digestion. We estimated from agarose electrophoresis gels that 40-200 pg of recovered plasmid DNA per transfected pool of DNA was resistant to DpnI and therefore was capable of transforming competent E. coli cells. The DpnI-resistant fraction yielded from one to seven independent clones from each pool, with genomic DNA inserts ranging in size from 0.35 to 3.4 kb.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA↗
PubMed1994

Electron microscopic analysis of in vitro replication products of ors 8, a mammalian origin enriched sequence.

Electron microscopy was used to map the initiation site of ors 8 DNA replication in vitro in a system that is capable of initiating and supporting one round of semiconservative replication of cloned mammalian DNA origin-enriched sequences (ors). Using unique restriction sites in ors 8 plasmid DNA, we have mapped the replication bubble within the monkey DNA sequence. In addition to site-specific initiation within the ors, the results also indicate bidirectional replication.

Animals↗