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PubMed1996

Replication origins in eukaroytes.

Recent experiments in budding yeast and Xenopus have provided new insights into the regulation of eukaroytic DNA replication. The multi-subunit origin recognition complex plays a key role in initiation, remaining bound at origins of replication during most of the cell cycle. Early in the cell cycle, Cdc6 and the Mcm proteins 'reset' chromatin for another round of DNA replication. Cyclin-dependent kinases appear to play a dual role, both in activating replication origins and blocking the formation of new pre-replicative complexes; thus limiting replication to once per cell cycle.

Animals↗

PubMed1989

Analysis of fidelity mechanisms with eukaryotic DNA replication and repair proteins.

We are investigating the mechanisms for producing or avoiding errors during DNA synthesis catalyzed by DNA replication and repair proteins purified from eukaryotic sources. Using assays that monitor the fidelity of a single round of DNA synthesis in vitro, we have defined the error frequency and mutational specificity of the four classes of animal cell DNA polymerases (alpha, beta, delta, gamma), and the fidelity of an SV40 origin-dependent DNA replication complex in extracts of HeLa cells.

Animals↗

PubMed1996

Sequences within the early and late promoters of archetype JC virus restrict viral DNA replication and infectivity.

Two forms of JC virus (JCV) have been isolated from its human host, an archetype found in kidney tissue and urine of nonimmunocompromised individuals and a rearranged type detected in lymphocytes and brain tissue of patients with and without progressive multifocal leukoencephalopathy. To investigate the hypothesis that alterations to the archetype transcriptional control region yield rearranged forms of the virus exhibiting new tissue tropic and pathogenic potentials, attempts were made to propagate archetype JCV in human renal and glial cell cultures. Although rearranged forms of JCV multiplied in these cells, archetype JCV failed to do so. Through the use of chimeric and mutant viral genomes, and a cell line that constitutively expresses viral T protein, we demonstrated that archetype's inactivity relative to that of rearranged forms was due to differences in the promoter-enhancer and not in the protein coding regions or origin of DNA replication. Additional analyses revealed that the absence of a large tandem duplication and the presence of a 23- and a 66-base pair sequence in the archetype transcriptional control region were responsible for this restricted lytic behavior. We discuss the possibility that deletion and duplication events within the archetype promoter-enhancer might yield more active viral variants via the loss of a negative, or the creation of a positive, transcriptional control signal(s).

Adult↗

PubMed2006

Progesterone blocks estrogen-induced DNA synthesis through the inhibition of replication licensing.

In the uterus, progesterone (P4) acts early in G1 as a physiological inhibitor of estradiol-17beta (E2)-induced epithelial cell proliferation. Gene expression profiling of uterine epithelial cell RNA isolated 3 h after hormonal treatment of ovariectomized mice revealed the co-coordinate down-regulation by P4 of >20 genes whose functions are associated with DNA replication. This group included all of the minichromosome maintenance (MCM) proteins that are required for DNA replication licensing. E2 regulated loading of these MCM proteins onto chromatin in parallel with its induction of DNA synthesis. E2 caused this chromatin loading by retention of MCM proteins in the nucleus and through the induction of the loading factor Cdt1, which is necessary for the MCM heterohexamer to bind to the origin of DNA replication. P4 dramatically reduced the binding of the MCMs to chromatin by a number of mechanisms. First, MCM mRNA and protein abundance was down-regulated. Second, P4 inhibited the E2 induction of Cdt1. Third, P4 treatment sequestered the normally nuclear MCM proteins into the cytoplasm. This reduced MCM binding resulted in the complete inhibition of E2-induced DNA synthesis by P4. These data reveal mechanisms not only for female sex steroid hormone action but also in the regulation of DNA replication licensing.

Animals↗

PubMed1983

Simian virus 40 tumor antigen: isolation of the origin-specific DNA-binding domain.

To localize the origin-specific DNA-binding domain on the simian virus 40 tumor (T) antigen molecule, we used limited proteolysis with trypsin to generate fractional peptides for analysis. A 17,000-Mr peptide was found to be capable of binding not only to calf thymus DNA, but also specifically to the simian virus 40 origin of DNA replication. This approximately 130-amino-acid peptide was derived from the extreme N-terminus of the T antigen and represented less than one-fifth of the entire molecule. The coding sequence for this tryptic peptide was located approximately between 0.51 and 0.67 map units (excluding the intron, which maps between 0.54 and 0.59). Since the first 82 amino acids are shared between large T and small t antigens, and since the latter does not bind DNA, it can be concluded that the sequence between isoleucine 83 and approximately arginine 130 is necessary for origin-specific binding by the T antigen. We also observed that in vivo phosphorylation of the T antigen within this region completely abolished the ability of the 17,000-Mr peptide to bind DNA. This observation is consistent with the idea that DNA binding by the T antigen is regulated by posttranslational modifications.

Animals↗

PubMed1979

Deletion mutants of polyoma virus defining a nonessential region between the origin of replication and the initiation codon for early proteins.

Mutants of polyoma virus with deletions as large as 90 base pairs were isolated by selecting spontaneously arising genomes resistant to endonuclease HaeII or by treating HaeII- or BglI- cleaved linear DNAs with S1 nuclease and exonuclease III. All of the mutants were viable and, therefore, defined a nonessential region in the polyoma genome between the origin of DNA replication and the initiation codon for translation of early proteins. Several mutants with large deletions had altered growth properties, giving smaller plaques and lower virus yields than the parental wild-type virus. These viruses may lack sites that are important for DNA replication or for transcription and translation of early mRNA's. All of the mutants tested could transform BHK-21 cells to anchorage independence.

Animals↗

PubMed1994

Homologous recombination of monkey alpha-satellite repeats in an in vitro simian virus 40 replication system: possible association of recombination with DNA replication.

To study homologous recombination between repeated sequences in an in vitro simian virus 40 (SV40) replication system, we constructed a series of substrate DNAs that contain two identical fragments of monkey alpha-satellite repeats. Together with the SV40-pBR322 composite vector encoding Apr and Kmr, the DNAs also contain the Escherichia coli galactokinase gene (galK) positioned between two alpha-satellite fragments. The alpha-satellite sequence used consists of multiple units of tandem 172-bp sequences which differ by microheterogeneity. The substrate DNAs were incubated in an in vitro SV40 DNA replication system and used to transform the E. coli galK strain DH10B after digestion with DpnI. The number of E. coli galK Apr Kmr colonies which contain recombinant DNAs were determined, and their structures were analyzed. Products of equal and unequal crossovers between identical 172-bp sequences and between similar but not identical (homeologous) 172-bp sequences, respectively, were detected, although those of the equal crossover were predominant among all of the galK mutant recombinants. Similar products were also observed in the in vivo experiments with COS1 cells. The in vitro experiments showed that these recombinations were dependent on the presence of both the SV40 origin of DNA replication and SV40 large T antigen. Most of the recombinant DNAs were generated from newly synthesized DpnI-resistant DNAs. These results suggest that the homologous recombination observed in this SV40 system is associated with DNA replication and is suppressed by mismatches in heteroduplexes formed between similar but not identical sequences.

Animals↗

PubMed1978

Mechanism of phage Mu-1 integration: nalidixic acid treatment causes clustering of Mu-1-induced mutations near replication origin.

Frequencies of Mu-1-induced mutants of Escherichia coli have been compared under two different experimental conditions: cells in exponential growth and the same cells treated with nalidixic acid. The average of values obtained from the nalidixic acid-treated culture is 3 times higher than that obtained from the control. Individual ratios of the frequency of mutants in the two cultures yield decreasing values from 6 to 1, starting from the point of origin of DNA replication to the termini of DNA replication. These results are compatible with the idea that Mu-1 integrates at the replication fork.

Coliphages↗

PubMed1993

Conformation of the origin of P1 plasmid replication. Initiator protein induced wrapping and intrinsic unstacking.

The origin of plasmid DNA replication in bacteriophage P1 has five 19 base-pair sites that bind the plasmid-encoded initiator, RepA. Here we show, using a DNA band retardation assay, that RepA can bend DNA that carries one or more of the RepA binding sites. RepA binding to supercoiled DNA carrying the five sites, directly repeated and phased two turns of B-DNA apart, absorbs about one positive superhelical turn of DNA as determined by two-dimensional gel electrophoresis. This indicates that the DNA is wrapped around RepA as a consequence of in-phase bending at the individual binding sites. The RepA-DNA complexes did not show elevated sensitivity to KMnO4, a reagent specific for pyrimidine bases (T >> C) in unstacked DNA. The wrapping of the DNA around RepA, therefore, does not lead to significant unwinding of the double helix. Extensive unwinding, suitable for the initiation of DNA replication, most likely requires participation of factors other than RepA. We also noted that the thymine bases of the sequence 5'-ATC-3', of which there are 20 in the origin, all reacted to KMnO4 strongly whether or not RepA was present. The preferential reactivity of ATC sequences was specific to the origin region, as thymine residues including those in the ATC sequences did not display elevated sensitivity to KMnO4 in a DNA fragment from pBR322. On one of two strands of the origin the selective reactivity at the ATC sequences was supercoiling dependent. These results indicate that the origin includes unstacked DNA bases, the significance of which remains to be determined.

Bacterial Proteins↗

PubMed1987

Amplification and expression of foreign genes in cells producing polyoma virus large T-antigen.

Polyoma virus (Py) large T-Antigen (LT) can promote the amplification of viral genomes integrated in the chromosomal DNA of rat fibroblasts, and this phenomenon requires the interaction of the LT protein with the viral origin of DNA replication. To compare the rate and the modality of selectable amplification events promoted by the Py LT with cellular-driven events, we constructed a double expression vector containing a murine dihydrofolate reductase (dhfr) cDNA and the bacterial chloramphenicol acetyl transferase (cat) gene controlled by the viral regulatory region. The plasmid was introduced into a rat cell line constitutively producing a temperature sensitive LT (cl 2), and clones were selected in low concentration of methotrexate (MTX). Three cl 2 transformants and one control cell line lacking LT were propagated at temperatures permissive (33 degrees C) and non permissive (39 degrees C) for LT function and, subsequently, challenged in one step with high MTX dosage at 39 degrees C. While the control line produced the same number of colonies irrespective of the temperature of propagation, the three LT positive lines yielded between 2 and greater than 100 times more colonies following propagation at permissive temperature, indicating that the presence of Py LT considerably increased the rate of amplification of integrated sequences linked to the viral origin of replication. In all cases the amplification event involved the exogenous and not the endogenous dhfr gene, and overexpression of the cat gene occurred as a result of co-amplification with the selectable dhfr sequences. Analysis of the structure of the amplified domain in the various resistant derivatives revealed that, in the presence of the viral protein, amplification occurred within the boundaries of the primary plasmid insert. In the absence of a functional LT protein, amplification both internal or involving adjacent host DNA were observed.

Animals↗

PubMed2000

Specification of chromatin domains and regulation of replication and transcription during development.

It is becoming increasingly clear that transcription control is carried out at several interconnecting levels. Besides nucleosomal organization and interaction between transcription factors and gene promoters and other regulatory elements, long-range organization of chromatin in loops or domains seems to play a role in transcriptional regulation. A similar organization is likely to be crucial in the control of the timing and selection of origins of DNA replication. This review considers the implications of domain organization of eukaryotic genome in developmental control of transcription and replication.

Animals↗

PubMed1975

Recombinant clone heterogeneity in Escherichia coli conjunction: effect of pH and partially replicated recipient deoxyribonucleic acid.

At pH 6.8, a substantial fraction of recombinant colonies obtained from conjugation with an HfrH donor contained multiple recombinant classes in a single colony (polygenotype colony). In contrast, when the conjugation was performed at pH 7.6, the number of polygenotypic colonies was drastically reduced, and the recombinant colonies were predominantly monogenotypic or digenotypic. Genetic analysis revealed that the digenotypic recombinants differ in those donor markers near the origin of DNA replication but share those donor markers near the terminus. This integration pattern suggests that the formation of digenotypic recombinants involves recombination of a single copy of the exogenome with a partially replicated recipient DNA molecule. This suggestion was supported by examination of the genotype of recombinant colonies recovered from crosses with an HfrKL96 donor which was derived from HfrH but transfers its chromosome in the reverse direction.

Chromosomes, Bacterial↗

PubMed1995

Herpes simplex virus type 1 amplicon vectors with glucocorticoid-inducible gene expression.

A glucocorticoid-inducible transcription unit, composed of reiterated steroid-responsive cis elements, a synthetic promoter, and the human growth hormone gene (hGH), was inserted into herpes simplex virus type 1 (HSV-1) amplicon vectors to determine if regulated expression can be achieved in cell lines and primary hepatocytes. Initial constructs were prepared in a HSV amplicon vector that contained a 0.96-kb DNA fragment encompassing an origin of DNA replication of the short repeat (ORIs) of HSV-1, similar to ORIs-containing fragments used in many of the existing HSV amplicon vectors. In the absence of glucocorticoids, these constructs produced a high basal level of hGH expression in cells both transfected with naked amplicon DNA and infected with packaged amplicon virus. To lower basal expression, the larger ORIs was replaced with a 237-bp ORIs DNA fragment that was devoid of the many trans-activation elements flanking the larger ORIs, but that could still support DNA replication of amplicon vectors. HSV amplicon vectors with this smaller ORIs produced up to 30-fold hGH induction upon glucocorticoid treatment in virally transduced 293 cells, and up to 50-fold induction in transduced primary rat hepatocytes. The kinetics of hGH accumulation after induction and the dependence of hGH expression with respect to dexamethasone concentration were characterized in both cell types. These vectors have application for experiments in which highly efficient gene transfer and inducible gene expression are required.

Base Sequence↗

PubMed1994

Establishment and characterization of murine macrophage-like cell lines following transformation with simian virus 40 DNA deleted at the origin of replication.

Differentiated mammalian cell lines can be established by introducing viral oncogenes into primary cells. Such lines can retain their original specialised functions while being adapted to prolonged life in culture; but most transformed cell lines obtained in this way characteristically show altered properties compared with the primary cells. The result of these changes is that transformed cell lines no longer provide a good model of the original tissue, and indeed often resemble other transformed lines more than the initial cell type. In our laboratory three murine peritoneal macrophage-like cell lines have been isolated by transforming primary cells with SV40 origin-deleted DNA. These lines have been in continuous culture for approximately 1 year and have been shown to express many macrophage-specific properties throughout this time, including Fc receptors and staining for non-specific esterase. The cell lines phagocytosed IgG-coated particles, they were positive for the murine macrophage-specific marker F4/80 and they showed antigen-presentation function. Lysozyme, acid phosphatase, plasminogen activator, collagenase, prostaglandin E2 and 5'-nucleotidase activities have also been detected in these lines. In this paper the method of DNA transformation will be described as well as some of the assays used for the characterization of the three immortalized cell lines.

Animals↗

PubMed1996

A model for the T-antigen-induced structural alteration of the SV40 replication origin based upon experiments with specific probes for bent, straight, and unwound DNA.

The T-antigen-induced structural changes of the SV40 replication origin were probed with three DNA-reactive antitumor agents: (+)-CC-1065, bizelesin, and pluramycin. (+)-CC-1065 is an N3 adenine minor groove alkylating agent that selectively reacts with AT-rich DNA sequences with a bent conformation; bizelesin also reacts with the minor groove of AT-rich sequences but is selective for a conformation; bizelesin also reacts with the minor groove of AT-rich sequences but is selective for a straight DNA conformation. Pluramycin is an intercalative guanine alkylator whose reactivity is increased by unwinding and decreased by compression of the minor and/or major grooves of DNA. We show that while binding of T-antigen reduced the ability of (+)-CC-1065 to alkylate the AT tract in the SV40 replication origin, it did not interfere with bizelesin modification of the same sequence. These unexpected results suggest that when T-antigen binds to the SV40 origin the AT tract is in a straight DNA conformation. High-resolution DNase I footprinting experiments indicate that at least three helically in-phase T-antigen binding sites exist in the GC box region located immediately downstream of the AT tract. The binding of T-antigen enhances the reactivity of (+)-CC-1065 to the two 5'-AGTTA(asterisk) (the asterisk indicates the covalent bonding site) drug modification sites in the GC box region, demonstrating that these sites are in a bent conformation. In contrast, T-antigen inhibited the reactivity of pluramycin at sequences within the GC box region that are known not to bind T-antigen. These data, in combination with the DNase I footprinting results, suggest that T-antigen binding induces a conformational change in the DNA that no longer favors pluramycin intercalation. Based on our results, we propose that T-antigen binds tightly to the upstream region of the AT tract of SV40 replication origin forming double hexamers. In the downstream region, binding of T-antigen to the helically in-phase sites in the GC box region induces DNA bending in the opposite direction of the natural AT tract bending, while simultaneously transforming the naturally bent AT tract DNA into a straight conformation.

Aminoglycosides↗

PubMed1985

Site-directed mutagenesis of the simian virus 40 large T-antigen gene: replication-defective amino acid substitution mutants that retain the ability to induce morphological transformation.

We used a heteroduplex deletion loop mutagenesis procedure for directing sodium bisulfite-induced mutations to specific sites on viral or plasmid DNA to generate a series of SV40 large T-antigen point mutants. The mutations were directed to a region of the T-antigen gene, 0.5 map units, that is thought to be important for interaction of the protein with the viral origin of DNA replication. Of the 16 mutants reported here, 10 had lost the ability to replicate their DNA, and 3 others showed a reduced level of replication compared to wild type. All of the mutants tested were capable of transforming rat cells in culture by the dense focus assay. We conclude that the sequences of the early region around 0.5 map units are critical for the replication of viral DNA but not for the transformation function of T antigen.

Animals↗

PubMed2000

Chromosome replication, nucleoid segregation and cell division in archaea.

Recent progress in cell cycle analysis of archaea has included the identification of putative chromosome replication origins, novel DNA polymerases and an unusual mode of cell cycle organization featuring multiple copies of the chromosome and asymmetric cell divisions. Genome sequence data indicate that in crenarchaea, the 'ubiquitous' FtsZ/MinD-based prokaryotic cell division apparatus is absent and division therefore must occur by unique, as-yet-unidentified mechanisms. The evolutionary and functional relationships between the archaeal Cdc6 protein and bacterial and eukaryal replication initiation factors are discussed.

Archaea↗

PubMed1987

Replication origin (oric) on the complementary DNA strand of Escherichia coli phage G4: biological properties of mutants.

Phage G4 origin of complementary DNA strand synthesis (oric) consists of three stable secondary loop structures. In a cloned 274-bp DNA fragment that is active as an ori in the filamentous phage cloning vector R199, insertion mutants have been constructed by introducing EcoRI and HindIII linkers at the base of loop III. The in vivo activity of these oric mutants (conversion of single-strand form to replicative form in the presence of rifampicin) was significantly reduced (50-70%) but not completely abolished. Nucleotide sequences and/or potential secondary structure of loop III centered at the AvaII site are therefore an important functional part of oric.

Base Sequence↗