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At least 685 records · Page 38Linked to original sources
PubMed1991

The FIS protein binds and bends the origin of chromosomal DNA replication, oriC, of Escherichia coli.

The FIS protein (factor for inversion stimulation) is known to stimulate site-specific recombination processes, such as the inversion of the G segment of bacteriophage Mu, by binding to specific enhancer sequences. It has also been shown to activate transcription from rRNA promoters both in vitro and in vivo. We have identified a specific binding site for FIS in the center of the origin of chromosomal DNA replication, oriC. The DNA bends upon FIS binding. Occupation of the FIS site and binding of DnaA, the initiator protein, to its adjacent binding site (R3) are mutually exclusive. A fis mutant strain can not be efficiently transformed with plasmids which carry and replicate from oriC, suggesting that FIS is required for minichromosome replication.

Bacterial Proteins↗
PubMed1984

Hydroxyurea treatment does not prevent initiation of DNA synthesis in Ehrlich ascites tumour cells and leads to the accumulation of short DNA fragments containing the replication origins.

The ability of EAT cells to initiate DNA synthesis in the presence of high doses of hydroxyurea was examined using the recently developed method for crosslinking DNA in vivo. Since crosslinking blocks elongation but has little effect on initiation (Russev and Vassilev (1982) J. Mol. Biol. 161, 77-87), this approach permits a separate study of the two stages of the DNA replication. We found out that hydroxyurea did not greatly affect the initiation of DNA replication but strongly inhibited the elongation of the already initiated new DNA chains. This resulted in the formation of short fragments enriched in sequences synthesized at and around the sites where DNA initiation began. These fragments were not ligated to the high molecular weight chromosomal DNA and could be released under denaturing conditions in single-stranded form. The reassociation and electrophoretic analysis showed that they contained about 200 nucleotides long interspersed DNA sequences repeated approx. 10(4) times per haploid genome, that probably served as replication origins.

Animals↗
PubMed1996

Crystal structure of the DNA-binding domain of the Epstein-Barr virus origin-binding protein, EBNA1, bound to DNA.

The Epstein-Barr virus nuclear antigen 1 (EBNA1) protein binds to and activates DNA replication from oriP, the latent origin of DNA replication in Epstein-Barr virus. The crystal structure of the DNA-binding domain of EBNA1 bound to an 18 bp binding site was solved at 2.4 A resolution. EBNA1 comprises two domains, a flanking and a core domain. The flanking domain, which includes a helix that projects into the major groove and an extended chain that travels along the minor groove, makes all of the sequence-determining contacts with the DNA. The core domain, which is structurally homologous to the complete DNA-binding domain of the bovine papilloma virus E2 protein, makes no direct contacts with the DNA bases. A model for origin unwinding is proposed that incorporates the known biochemical and structural features of the EBNA1-origin interaction.

Amino Acid Sequence↗
PubMed2005

Cdt1 transgenic mice develop lymphoblastic lymphoma in the absence of p53.

The exact duplication of chromosomal DNA during each cell cycle ensures the correct inheritance of genetic material from mother to daughter cells. In eukaryotic cells, DNA replication can occur only when the origin of DNA replication is accurately marked by a group of proteins termed licensing proteins. One such protein is Cdt1, which is recruited first to the origin of DNA replication followed by cell division cycle 6 (Cdc6) and mini-chromosome maintenance proteins (Mcms). We previously reported that NIH3T3 cells overexpressing Cdt1 readily formed tumors in mice. To further investigate its oncogenic mechanism, we generated transgenic mice expressing Cdt1 in thymocytes. Our studies demonstrated that T-cell-directed Cdt1 transgenic mice showed normal T-cell development. However, such transgenic mice developed thymic lymphoblastic lymphoma when crossed with p53 null mice. Furthermore, tumor cells derived from NIH3T3 cells overexpressing Cdt1 displayed numerical and structural chromosomal aberrations in the form of ploidy, double minutes, translocation, inversion, chromosome end-to-end fusion and robertsonian mutation. Collectively, our studies suggest that Cdt1 overexpression most likely contributes to tumorigenecity by causing genomic instability.

Aging↗
PubMed1984

Human embryonic kidney cells: stable transformation with an origin-defective simian virus 40 DNA and use as hosts for human papovavirus replication.

An origin-defective mutant DNA of simian virus 40 immortalized human embryonic kidney cells, maintaining a T protein which could function for human papovavirus BK DNA replication but not for human papovavirus JC DNA replication. Neither BK virions nor capsid proteins were produced in these cells. This may indicate that the simian virus 40 T protein in human embryonic kidney cells is competent for maintaining transformation and initiating and completing DNA replication for BK but is not competent for switching to late gene functions. Furthermore, it appears that the JC DNA replication origin cannot efficiently use the simian virus 40 T protein for its DNA synthesis, as suggested by its DNA sequence data (R. Frisque, J. Virol. 46:170-176, 1983; T. Miyamura, H. Jikoya, E. Soeda, and K. Yoshiike, J. Virol. 45:73-79, 1983).

Cell Line↗
PubMed1991

Enhancer effect of bovine papillomavirus E2 protein in replication of polyomavirus DNA.

In polyomavirus, both transcription from the early promoter and viral DNA replication initiated at the origin of DNA replication is controlled by binding of proteins to the enhancer region. We have developed a simple model system to study the role of an enhancer binding factor in the initiation of polyomavirus DNA replication. A reporter plasmid was constructed which has the enhancer region replaced by two binding sites for a transcription factor, the E2 protein encoded by bovine papillomavirus type 1. Co-transfection of COP 5 cells which express polyomavirus large T-antigen, with the reporter plasmid and an E2 expression plasmid, resulted in E2-dependent stimulation of replication of the reporter plasmid. The activity of several E2 mutants was also analysed. Mutant proteins with decreased activity in transcriptional trans-activation, resulting from impaired DNA binding or other defects, were found to have quantitatively similar reductions of activity in viral DNA replication. The results suggest that the E2 protein activates transcription and polyomavirus DNA replication by similar mechanism(s).

Antigens, Polyomavirus Transforming↗
PubMed1984

Amplification and excision of integrated polyoma DNA sequences require a functional origin of replication.

Cells transformed by Polyoma virus (Py) can undergo a high rate of excision or amplification of integrated viral DNA sequences, and these phenomena require the presence of homology (i.e., repeats) within the viral insertion as well as a functional viral large T antigen (T-Ag). To determine whether the main role of large T-Ag in excision and amplification was replicative or recombination-promoting, we studied transformed rat cell lines containing tandem insertions of a ts-a Py molecule (encoding a thermolabile large T-Ag) with a deletion of the origin of viral DNA replication. Culturing of these cells at the temperature permissive for large T-Ag function did not result in any detectable excision or amplification of integrated Py sequences. We then introduced into origin-defective lines a recombinant plasmid containing the viral origin of replication and the gene coding for resistance to the antibiotic G418. All G418-resistant clones analyzed readily amplified the integrated plasmid molecules when grown under conditions permissive for large T-Ag function, showing that these cells produced viral large T-Ag capable of promoting amplification in trans of DNA sequences containing the Py origin. These observations strongly suggest that Polyoma large T antigen promotes excision or amplification of viral DNA by initiating replication at the integrated origin, providing a favorable substrate for subsequent recombination.

Animals↗
PubMedDate not supplied

Regulation of DNA replication initiation in mammalian lymphocyte systems.

The control of DNA replication is central to the control of cell proliferation, and defects in S phase regulation have been implicated in senescence and neoplasia. To examine the regulation of DNA replication in lymphocytes, an in vitro system was developed in which lymphocyte derived proteins could regulate the initiation of DNA replication in isolated quiescent nuclei. Cytosolic extracts from mitogen or IL-2 activated lymphocytes as well as lymphoblastoid cell lines produce a factor (Activator of DNA replication; ADR) that can induce DNA synthesis in isolated quiescent nuclei, and DNA synthesis in this system is consistent with DNA replication and not repair. ADR activity is tightly associated with a protease activity and is not detectable in resting cells, but can be induced by a mechanism dependent on serine/threonine and tyrosine phosphorylation. Quiescent cells contain an ADR inhibitor which blocks DNA synthesis in isolated normal nuclei but not in nuclei from transformed cells, a potential factor in the uncontrolled proliferation of neoplastic cells. The control of cellular DNA replication is dependent on the interaction of origin sequences with specific replicative and regulatory proteins. However, mammalian origins of DNA replication are not well defined. Plasmids containing a replication origin within the human rRNA gene can act as replicative templates in our cell-free replication system, thus allowing a detailed molecular dissection of replication initiation in a completely human experimental system.

Aging↗
PubMed1995

Stepwise assembly of initiation complexes at budding yeast replication origins during the cell cycle.

DNA replication is a pivotal event in the cell cycle and, as a consequence, is tightly controlled in eukaryotic cells. The initiation of DNA replication is dependent upon the completion of mitosis and upon the commitment to complete the cell cycle made during G(1). Characterisation of the protein factors required for initiating DNA replication is essential to understand how the cell cycle is regulated. Recent results indicate that initiation complexes assemble in multiple stages during the cell cycle. First, origins are bound by the multisubunit origin recognition complex (ORC) which is essential for DNA replication in vivo. ORC, present at little more than one complete complex per replication origin, binds to origins immediately after initiation in the previous cell cycle. ORC binding occurs by the recognition of a bipartite sequence that includes the essential ARS consensus sequence (ACS) and the functionally important B1 element adjacent to the ACS. A novel pre-replicative complex (pre-RC) assembles at origins at the end of mitosis in actively cycling cells and remains at origins until DNA replication initiates. Finally, Dbf4, which is periodically synthesised at the end of G(1), interacts with replication origins. Dbf4-origin interaction requires an intact ACS strongly suggesting that interaction occurs through ORC. Dbf4 interacts with and is required for the activation of the Cdc7 protein kinase and together, Dbf4 and Cdc7 are required for the G(1)-S transition. Separate regions of Dbf4 are required for Cdc7- and origin-interaction suggesting that Dbf4 may act to recruit Cdc7 to replication origins where phosphorylation of some key component may cause origin firing.

Base Sequence↗
PubMed1986

Nucleosome phasing on a DNA fragment from the replication origin of simian virus 40 and rephasing upon cruciform formation of the DNA.

Nucleosomes were reconstituted in vitro from a fragment of DNA spanning the simian virus 40 minimal replication origin. The fragment contains a 27-base-pair palindrome (perfect inverted repeat). DNA molecules with stable cruciform structures were generated by heteroduplexing this DNA fragment with mutants altered within the palindromic sequence (C. Nobile and R. G. Martin, Int. Virol., in press). Analyses of the structural features of the reconstituted nucleosomes by the DNase I footprint technique revealed two alternative DNA-histone arrangements, each one accurately phased with respect to the uniquely labeled DNA ends. As linear double-stranded DNA, a unique core particle was formed in which the histones strongly protected the regions to both sides of the palindrome. The cruciform structure seemed to be unable to associate with core histones and, therefore, an alternative phasing of the histone octamer along the DNA resulted. Thus, nucleosome positioning along a specific DNA sequence appears to be influenced in vitro by the secondary structure (linear or cruciform) of the 27-base-pair palindrome. The formation of cruciform structures in vivo, if they occur, might therefore represent a molecular mechanism by which nucleosomes are phased.

Animals↗
PubMedDate not supplied

Single molecule analysis of DNA replication.

We describe here a novel approach for the study of DNA replication. The approach is based on a process called molecular combing and allows for the genome wide analysis of the spatial and temporal organization of replication units and replication origins in a sample of genomic DNA. Molecular combing is a process whereby molecules of DNA are stretched and aligned on a glass surface by the force exerted by a receding air/water interface. Since the stretching occurs in the immediate vicinity of the meniscus, all molecules are identically stretched in a size and sequence independent manner. The application of fluorescence hybridization to combed DNA results in a high resolution (1 to 4 kb) optical mapping that is simple, controlled and reproducible. The ability to comb up to several hundred haploid genomes on a single coverslip allows for a statistically significant number of measurements to be made. Direct labeling of replicating DNA sequences in turn enables origins of DNA replication to be visualized and mapped. These features therefore make molecular combing an attractive tool for genomic studies of DNA replication. In the following, we discuss the application of molecular combing to the study of DNA replication and genome stability.

Animals↗
PubMed1991

Emetine allows identification of origins of mammalian DNA replication by imbalanced DNA synthesis, not through conservative nucleosome segregation.

In the presence of emetine, an inhibitor of protein synthesis, nascent DNA on forward arms of replication forks in hamster cell lines containing either single or amplified copies of the DHFR gene region was enriched 5- to 7-fold over nascent DNA on retrograde arms. This forward arm bias was observed on both sides of the specific origin of bidirectional DNA replication located 17 kb downstream of the hamster DHFR gene (OBR-1), consistent with at least 85% of replication forks within this region emanating from OBR-1. However, the replication fork asymmetry induced by emetine does not result from conservative nucleosome segregation, as previously believed, but from preferentially inhibiting Okazaki fragment synthesis on retrograde arms of forks to produce 'imbalanced DNA synthesis'. Three lines of evidence support this conclusion. First, the bias existed in long nascent DNA strands prior to nuclease digestion of non-nucleosomal DNA. Second, the fraction of RNA-primed Okazaki fragments was rapidly diminished. Third, electron microscopic analysis of SV40 DNA replicating in the presence of emetine revealed forks with single-stranded DNA on one arm, and nucleosomes randomly distributed to both arms. Thus, as with cycloheximide, nucleosome segregation in the presence of emetine was distributive.

Animals↗
PubMed1996

RNA-DNA hybrid formation at the human mitochondrial heavy-strand origin ceases at replication start sites: an implication for RNA-DNA hybrids serving as primers.

Critical elements of a mammalian mitochondrial DNA heavy-strand replication origin include a promoter and three downstream conserved sequence blocks (CSBIII, CSBII and CSBI). We found recently that a stable and persistent RNA-DNA hybrid forms during in vitro transcription at Saccharomyces cerevisiae mitochondrial origins; hybrid formation was dependent on the conserved CSBII element. We report here that during in vitro transcription with human mitochondrial RNA polymerase, stable and persistent RNA-DNA hybrid formation is also evident at the human mitochondrial heavy-strand origin. As predicted, hybrid formation was dependent on the GC-rich CSBII element. The human RNA-DNA hybrids terminate within or downstream of CSBI at locations implicated in initiation of mitochondrial DNA replication. Interestingly, efficient hybrid formation in the human system is influenced by sequence 5' to the RNA-DNA hybrid, including the CSBIII element. These results suggest that the RNA-DNA hybrids formed during transcription across the mitochondrial DNA heavy-strand origin provide RNA primers for initiation of mitochondrial DNA replication.

Base Sequence↗
PubMed1984

Multiple initiation sites of DNA replication flanking the origin region of lambda dv genome.

Early replicative intermediates of lambda dv plasmid were prepared by an in vitro replication system in the presence of 2',3'-dideoxycytidine 5'-triphosphate, an inhibitor of DNA chain elongation. Short-chain DNAs produced from regions near the replication origin were purified from the intermediates. A fraction of the DNAs was covalently linked to primer RNA. The transition sites from primer RNA to DNA synthesis were mapped along the nucleotide sequence of the genome, by eliminating the RNA by alkaline hydrolysis and labeling the freshly exposed 5' ends of DNA with 32P. The transition sites were found to be located on both sides of the ori region, which includes four 19-base-pair repeats where one of the lambda specific initiator proteins, O, binds. No transition arose within the ori region. The transition sites are multiple on both sides of the ori region and are clustered in one of the two strands in such a way that DNA syntheses from the two sides converge. The frequency of the "leftward" DNA synthesis is several times higher than that of "rightward" synthesis, reflecting the asymmetric bidirectional replication of lambda dv DNA.

Bacteriophage lambda↗
PubMed1992

A single-stranded DNA-binding protein from Crithidia fasciculata recognizes the nucleotide sequence at the origin of replication of kinetoplast DNA minicircles.

A sequence-specific single-stranded DNA-binding protein from the trypanosomatid protozoan Crithidia fasciculata binds to a sequence of 12 nucleotides located at the origin of replication of kinetoplast DNA minicircles. This sequence, termed the universal minicircle sequence (UMS), is conserved in the kinetoplast DNA minicircles among species of the family Trypanosomatidae. The purified protein binds specifically to the heavy strand of the DNA at this site, which consists of the sequence 5'-GGGGTTGGTGTA-3'. Binding analyses using mutated UMS dodecamers have revealed the significant contribution of each of the individual residues at the binding site, with the exception of the 3'-terminal adenine residue, to the generation of specific protein-DNA complexes. The possible role of this sequence-specific single-stranded DNA-binding protein in replication of kinetoplast DNA minicircles and the relation of the UMS to chromosomal telomeric sequences are discussed.

Animals↗
PubMed2001

Intramitochondrial localization of universal minicircle sequence-binding protein, a trypanosomatid protein that binds kinetoplast minicircle replication origins.

Kinetoplast DNA (kDNA), the mitochondrial DNA of the trypanosomatid Crithidia fasciculata, is a unique structure containing 5,000 DNA minicircles topologically linked into a massive network. In vivo, the network is condensed into a disk-shaped structure. Replication of minicircles initiates at unique origins that are bound by universal minicircle sequence (UMS)-binding protein (UMSBP), a sequence-specific DNA-binding protein. This protein, encoded by a nuclear gene, localizes within the cell's single mitochondrion. Using immunofluorescence, we found that UMSBP localizes exclusively to two neighboring sites adjacent to the face of the kDNA disk nearest the cell's flagellum. This site is distinct from the two antipodal positions at the perimeter of the disk that is occupied by DNA polymerase beta, topoisomerase II, and a structure-specific endonuclease. Although we found constant steady-state levels of UMSBP mRNA and protein and a constant rate of UMSBP synthesis throughout the cell cycle, immunofluorescence indicated that UMSBP localization within the kinetoplast is not static. The intramitochondrial localization of UMSBP and other kDNA replication enzymes significantly clarifies our understanding of the process of kDNA replication.

Animals↗
PubMed1988

Herpes simplex virus DNA replication: the UL9 gene encodes an origin-binding protein.

Herpes simplex virus 1 contains seven genes that are necessary and sufficient for origin-dependent DNA synthesis in cultured cells. We have expressed the product of one of these genes, UL9, in insect cells by using a baculovirus expression vector. The apparent size of the UL9 protein, both in insect cells and in herpes simplex virus-infected Vero cells, is 82,000 Da. By using an immunoassay for protein-DNA interaction, we have shown that UL9 protein binds specifically to the herpes simplex virus origins of DNA replication, oriS and oriL. DNase I "footprint" analysis has shown that the UL9 protein interacts with two related sites on oriS, located on each arm of a nearly perfect palindrome. Our data strongly suggest that the origin-binding activity described previously by Elias et al. [Elias, P., O'Donnell, M. E., Mocarski, E. S. & Lehman, I. R. (1986) Proc. Natl. Acad. Sci. USA 83, 6322-6326] is the product of the UL9 gene.

Animals↗