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Results for “DNA replication”

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At least 163 records · Page 9Linked to original sources
PubMed2005

The cell cycle regulator p27Kip1 interacts with MCM7, a DNA replication licensing factor, to inhibit initiation of DNA replication.

The G1/S phase restriction point is a critical checkpoint that interfaces between the cell cycle regulatory machinery and DNA replicator proteins. Here, we report a novel function for the cyclin-dependent kinase inhibitor p27Kip1 in inhibiting DNA replication through its interaction with MCM7, a DNA replication protein that is essential for initiation of DNA replication and maintenance of genomic integrity. We find that p27Kip1 binds the conserved minichromosome maintenance (MCM) domain of MCM7. The proteins interact endogenously in vivo in a growth factor-dependent manner, such that the carboxyl terminal domain of p27Kip1 inhibits DNA replication independent of its function as a cyclin-dependent kinase inhibitor. This novel function of p27Kip1 may prevent inappropriate initiation of DNA replication prior to S phase.

Binding Sites↗
PubMed2005

Progressive activation of DNA replication initiation in large domains of the immunoglobulin heavy chain locus during B cell development.

In mammalian cells, the replication of tissue-specific gene loci is believed to be under developmental control. Here, we provide direct evidence of the existence of developmentally regulated origins of replication in both cell lines and primary cells. By using single-molecule analysis of replicated DNA (SMARD), we identified various groups of coregulated origins that are activated within the Igh locus. These origin clusters can span hundreds of kilobases and are activated sequentially during B cell development, concomitantly with developmentally regulated changes in chromatin structure and transcriptional activity. Finally, we show that the changes in DNA replication initiation that take place during B cell development, within the D-J-C-3'RR region, occur on both alleles (expressed and nonexpressed).

Animals↗
PubMed1999

The bromodeoxyuridine comet assay: detection of maturation of recently replicated DNA in individual cells.

The single-cell gel electrophoresis (Comet) assay is a relatively simple method of measuring DNA single strand breaks and alkali-labile sites in individual cells. We have combined this with bromodeoxyuridine (BrdUrd) labeling of DNA and immunolocalization of the BrdUrd to assess DNA replicative integrity on a single-cell basis. We show that the existence of strand discontinuities in recently replicated domains of DNA, caused during semiconservative replication or exacerbated by the arrest of replicative polymerases at UV irradiation- or chemical-induced lesions, can be detected in individual cells. Data obtained from BrdUrd-Comets are consistent with biochemical data derived with a range of techniques showing that DNA replication involves the creation of strand breaks or gaps adjacent to recently replicated material, and that DNA damage prolongs the duration of such discontinuities where DNA polymerases are stalled opposite lesions (R. T. Johnson et al, The Legacy of Cell Fusion, pp. 50-67, Oxford: Science Publications, 1994; R. B. Painter, J. Mol. Biol., 143: 289-301, 1980.). Compared with standard biochemical techniques, the BrdUrd-Comet assay is simple and suitable for the accurate and automatable assessment of replicative integrity in very small numbers of mammalian cells, such as may be obtained by biopsy.

Animals↗
PubMed2001

Topoisomerase II can unlink replicating DNA by precatenane removal.

We have analysed the role of topoisomerase II (topo II) in plasmid DNA replication in Xenopus egg extracts, using specific inhibitors and two-dimensional gel electrophoresis of replication products. Topo II is dispensable for nuclear assembly and complete replication of plasmid DNA but is required for plasmid unlinking. Extensive unlinking can occur in the absence of mitosis. Replication intermediates generated in the absence of topo II activity have an increased positive superhelical stress (+DeltaLk), suggesting a deficiency in precatenane removal. The geometry of replication intermediates cut by poisoning topo II with etoposide and purified by virtue of their covalent attachment to topo II subunits demonstrates that topo II acts behind the forks at all stages of elongation. These results provide direct evidence for unlinking replicating DNA by precatenane removal and reveal a division of labour between topo I and topo II in this eukaryotic system. We discuss the role of chromatin structure in driving DNA unlinking during S phase.

Animals↗
PubMed1977

DNA replication in Physarum polycephalum: characterization of DNA replication products made in vivo in the presence of cycloheximide in strains sensitive and resistant to cycloheximide.

Synchronous plasmodia of cycloheximide-sensitive and cycloheximide-resistant strains of Physarum polycephalum were labelled with 3[H]-deoxyadenosine in pulse and pulse-chase experiments in presence and absence of cycloheximide. The replication products were studied with alkaline sucrose gradient sedimentation analysis. We show that the action of cycloheximide on DNA replication in Physarum is mediated through the ribosome, since the ribosomally located resistance also makes the plasmodial DNA replication refractile to the action of cycloheximide. Cycloheximide caused inhibition of three stages in DNA replication in the wild type: first, the formation of primary replication units ("Okazaki" size fragments), secondly, the ligation of primary units into secondary ("Replicon" size) units and thirdly, the ligation of secondary units into mature DNA.

Centrifugation, Density Gradient↗
PubMedDate not supplied

Newly replicated DNA is associated with DNA topoisomerase II in cultured rat prostatic adenocarcinoma cells.

DNA topoisomerases have been proposed to function in a variety of genetic processes in both prokaryotes and eukaryotes. Here, we have assessed the role of DNA topoisomerase II in mammalian DNA replication by determining the proximity of newly synthesized DNA to covalent enzyme-DNA complexes generated by treating cultured rat prostatic adenocarcinoma cells with teniposide. Teniposide (VM-26), an epipodophyllotoxin, is known to interact with mammalian DNA topoisomerase II so as to trap the enzyme in a covalent complex with DNA. We have found that the teniposide-induced trapping of such complexes requires MgCl2, is stimulated by ATP and is inhibited by novobiocin. The formation of covalent complexes seems to be reversible on removal of teniposide. Furthermore, analysis of the covalent complexes formed between 3H-thymidine pulse-labelled DNA and topoisomerase II following teniposide treatment reveals a direct association of the enzyme with nascent DNA fragments. Our results suggest that DNA topoisomerase II may interact with newly replicated daughter DNA molecules near DNA replication forks in mammalian cells.

Adenocarcinoma↗
PubMed2003

Effects of sequence on repeat expansion during DNA replication.

Small DNA repeat tracts are located throughout the human genome. The tracts are unstable, and expansions of certain repeat sequences cause neuromuscular disease. DNA expansions appear to be associated with lagging-strand DNA synthesis and DNA repair. At some sites of repeat expansion, e.g. the myotonic dystrophy type 2 (DM2) tetranucleotide repeat expansion site, more than one repeat tract with similar sequences lie side by side. Only one of the DM2 repeat tracts, however, is found to expand. Thus, DNA base sequence is a possible factor in repeat tract expansion. Here we determined the expansion potential, during DNA replication by human DNA polymerase beta, of several tetranucleotide repeat tracts in which the repeat units varied by one or more bases. The results show that subtle changes, such as switching T for C in a tetranucleotide repeat, can have dramatic consequences on the ability of the nascent-strand repeat tract to expand during DNA replication. We also determined the relative stabilities of self-annealed 100mer repeats by melting-curve analysis. The relative stabilities did not correlate with the relative potentials of the analogous repeats for expansion during DNA replication, suggesting that hairpin formation is not required for expansion during DNA replication.

Base Sequence↗
PubMed1992

Identification and mapping of origins of DNA replication within the DNA sequences of the genome of insect iridescent virus type 6.

The origins of DNA replication of the genome (209 kbp) of Chilo iridescent virus (CIV), which is circularly permuted and terminally redundant, were identified. The defined genomic library of CIV, which represents 100% of DNA sequences of the viral genome (e.g., all 32 EcoRI CIV DNA fragments), was used for transfection of Choristoneura fumiferana insect cell cultures (CF-124) that were previously infected with CIV. The plasmid rescue experiments were carried out to select those recombinant plasmids that were amplified during viral replication in CIV-infected cell cultures. It was found that six recombinant plasmids harboring the EcoRI DNA fragments C [13.5 kbp, 0.909-0.974 map units (m.u.)], H (9.8 kbp, 0.535-0.582 m.u.), M (7.25 kbp, 0.310-0.345 m.u.), O (6.5 kbp, 0.196-0.228 m.u.), Q (5.9 kbp, 0.603-0.631 m.u.), and Y (2.0 kbp, 0.381-0.391 m.u.) were able to be amplified under the conditions used. This indicates that the CIV genome possesses six DNA replication origins. Subclones of the EcoRI CIV DNA fragments C and H were screened under the same conditions. It was found that DNA sequences within the EcoRI DNA fragments C and H at the genome coordinates 0.924-0.930 and 0.535-0.548, respectively, contain origins of viral DNA replication. The DNA nucleotide sequences of the EcoRI CIV DNA fragment Y (1986 bp) were determined for identifying the DNA sequence of the corresponding origin of DNA replication. The computer-aided analysis revealed the presence of a 15-mer inverted repeat at nucleotide positions 661-675 and 677-691 (661-TAAATTTAATGAGAA-G-TTCTCATTAAATTTA-692). The analysis of the DNA sequence of the EcoRI DNA fragment H corresponding to the particular region at the genome coordinates 0.535-0.548 (1) showed that this region contains a 16-mer inverted repeat at the nucleotide positions 1315 and 1332 (1315-TAAATTTTAATGGTTA-A-TAACCATTAAAATTTA-1347), which is very similar to the inverted repetition found within the EcoRI DNA fragment Y. The successful recognition and amplification of the single-stranded synthetic DNA sequences of both strands of CIV-ori-Y (nucleotide position 661-691) using phage M13 system in CIV-infected cells is strong evidence that the CIV-ori-Y is bidirectionally active, and this DNA sequence is considered to be the origin of DNA replication within the EcoRI CIV DNA fragment Y.

Base Sequence↗
PubMed1994

Anatomy of a DNA replication fork revealed by reconstitution of SV40 DNA replication in vitro.

Complete enzymatic replication of DNA from the simian virus 40 origin has been reconstituted with T antigen and highly purified cellular proteins. DNA polymerase-alpha/primase functions primarily to synthesize RNA-DNA primers for initiation of DNA replication at the origin and for priming each Okazaki fragment. A polymerase switching mechanism requiring replication factor C and the proliferating cell nuclear antigen allows two molecules of DNA polymerase-delta to replicate both strands of the double helix conjointly.

Animals↗
PubMed1997

Transcription of genes encoding DNA replication proteins is coincident with cell cycle control of DNA replication in Caulobacter crescentus.

DNA replication in the dimorphic bacterium Caulobacter crescentus is tightly linked to its developmental cell cycle. The initiation of chromosomal replication occurs concomitantly with the transition of the motile swarmer cell to the sessile stalked cell. To identify the signals responsible for the cell cycle control of DNA replication initiation, we have characterized a region of the C. crescentus chromosome containing genes that are all involved in DNA replication or recombination, including dnaN, recF, and gyrB. The essential dnaN gene encodes a homolog of the Escherichia coli beta subunit of DNA polymerase III. It is transcribed from three promoters; one is heat inducible, and the other two are induced at the transition from swarmer to stalked cell, coincident with the initiation of DNA replication. The single gyrB promoter is induced at the same time point in the cell cycle. These promoters, as well as those for several other genes encoding DNA replication proteins that are induced at the same time in the cell cycle, share two sequence motifs, suggesting that they represent a family whose transcription is coordinately regulated.

Amino Acid Sequence↗
PubMed1994

Stabilization of the EBNA1 protein on the Epstein-Barr virus latent origin of DNA replication by a DNA looping mechanism.

DNA replication from the Epstein-Barr virus latent origin of replication, oriP, is activated by Epstein-Barr nuclear antigen 1 (EBNA1). This activation involves the binding of EBNA1 dimers to multiple sites present in the two noncontiguous functional elements of oriP, the dyad symmetry element (DS) from which replication initiates, and the family of repeats (FR) enhancer element. EBNA1 complexes formed on the FR and DS elements of oriP interact by a DNA looping mechanism. This interaction requires EBNA1 sequences in addition to those required for DNA binding and dimerization. To map the EBNA1 sequences required for the efficient interaction of FR- and DS-bound EBNA1 complexes, we have overproduced in Escherichia coli and purified a series of EBNA1 N-terminal truncation mutants, all of which retain the DNA binding and dimerization domains. The results of electron microscopy and ligation-enhancement assays using these mutants indicated that EBNA1 sequences between amino acids 350 and 361 are required for the efficient interaction of FR- and DS-bound EBNA1 complexes. EBNA1-mediated FR-DS interactions were shown to stabilize EBNA1 binding to the DS element, while EBNA1-mediated DS-DS interactions did not. These results suggest that the stabilization of EBNA1 on the DS element, which occurs as a result of EBNA1-mediate oriP looping, may be important for the activation of DNA replication from the DS element.

Antigens, Viral↗
PubMed1989

Segregation of relaxed replicated dimers when DNA ligase and DNA polymerase I are limited during oriC-specific DNA replication.

An in vitro Escherichia coli oriC-specific DNA replication system was used to investigate the DNA replication pathways of oriC plasmids. When this system was perturbed by the DNA ligase inhibitor nicotinamide mononucleotide (NMN), alterations occurred in the initiation of DNA synthesis and processing of intermediates and DNA products. Addition of high concentrations of NMN soon after initiation resulted in the accumulation of open circular dimers (OC-OC). These dimers were decatenated to open circular monomers (form II or OC), which were then processed to closed circular supercoiled monomers (form I or CC) products. After a delay, limited ligation of the interlinked dimers (OC-OC to CC-OC and CC-CC) also occurred. Similar results were obtained with replication protein extracts from polA mutants. The presence of NMN before any initiation events took place prolonged the existence of nicked template DNA and promoted, without a lag period, limited incorporation into form II molecules. This DNA synthesis was nonspecific with respect to oriC, as judged by DnaA protein dependence, and presumably occurred at nicks in the template DNA. These results are consistent with oriC-specific initiation requiring closed supercoiled molecules dependent on DNA ligase activity. The results also show that decatenation of dimers occurs readily on nicked dimer and represents an efficient pathway for processing replication intermediates in vitro.

Bacterial Proteins↗
PubMed1988

Identification of human gene complementing ts AlS9 mouse L-cell defect in DNA replication following DNA-mediated gene transfer.

The temperature-sensitive (ts) mouse L-cell, ts AlS9, is defective in a gene required for nuclear DNA replication early in the S phase of the cell cycle. Human DNA sequences were introduced into ts AlS9 cells together with the plasmid pSV2neo, which can confer resistance to the drug geneticin. Cotransformants, expressing both the plasmid-derived neomycin gene and the transferred human AlS9 gene, were selected for growth in the presence of the drug at the nonpermissive temperature (npt). The resulting transformants retained a common set of human-specific Alu repetitive DNA sequences. These are likely to be accommodated within, or in proximity to, the transferred human AlS9 gene. The results obtained provide the basis for cloning human genes required for DNA replication.

Animals↗
PubMed2006

Interactions of the cellular CCAAT displacement protein and human papillomavirus E2 protein with the viral origin of replication can regulate DNA replication.

Previously, we and others have shown that CCAAT displacement protein (CDP) negatively regulates the papillomavirus promoters. Overexpression of CDP has been shown to inhibit high-risk human papillomavirus virus (HPV) and bovine papillomavirus DNA replication in vivo presumably through reduction in expression of viral replication proteins, E1 and E2. Sequence analysis of the HPV origin indicates several potential CDP-binding sites with one site overlapping the E1-binding site. Therefore, CDP could also negatively regulate papillomavirus replication directly by preventing the loading of the initiation complex. We show here that purified CDP inhibits in vitro HPV DNA replication. Footprint analysis demonstrated that CDP binds the E1-binding site and the TATA box, and that the binding of purified CDP to the E1-binding site is decreased by the addition of purified E2 protein. Consistent with this, E2-independent in vitro HPV replication is inhibited by CDP to a greater extent than E2-dependent replication. These results suggest that binding of E2 at the E2-binding site may play an important role in overcoming the inhibition of E1 initiation complex formation caused by the binding of negative regulators like CDP to the origin of replication.

Base Sequence↗
PubMed1985

1 alpha,25-Dihydroxyvitamin D3 enables regenerating liver cells to make functional ribonucleotide reductase subunits and replicate DNA in thyroparathyroidectomized rats.

Between 16 and 20 h after partial (70%) hepatectomy (HPX) in normal rats, the remaining liver cells accumulate ribonucleotide reductase subunits, assemble these into active holoenzyme, and initiate DNA replication. These late prereplicative activities did not occur in most of the liver cells remaining after HPX in rats which had been thyroparathyroidectomized (TPTX) 72 h previously. However, one intraperitoneal injection of 400 or 600 ng 1 alpha,25-dihydroxyvitamin D3/100 g body weight at the time of HPX enabled the remaining liver cells in such TPTX rats to make functional ribonucleotide reductase subunits, assemble these subunits into active CDP-reducing holoenzymes, and replicate their DNA, though they started to do so 4 to 16 h later than in normal animals.

Animals↗
PubMed1998

Spatial and temporal dynamics of DNA replication sites in mammalian cells.

Fluorescence microscopic analysis of newly replicated DNA has revealed discrete granular sites of replication (RS). The average size and number of replication sites from early to mid S-phase suggest that each RS contains numerous replicons clustered together. We are using fluorescence laser scanning confocal microscopy in conjunction with multidimensional image analysis to gain more precise information about RS and their spatial-temporal dynamics. Using a newly improved imaging segmentation program, we report an average of approximately 1,100 RS after a 5-min pulse labeling of 3T3 mouse fibroblast cells in early S-phase. Pulse-chase-pulse double labeling experiments reveal that RS take approximately 45 min to complete replication. Appropriate calculations suggest that each RS contains an average of 1 mbp of DNA or approximately 6 average-sized replicons. Double pulse-double chase experiments demonstrate that the DNA sequences replicated at individual RS are precisely maintained temporally and spatially as the cell progresses through the cell cycle and into subsequent generations. By labeling replicated DNA at the G1/S borders for two consecutive cell generations, we show that the DNA synthesized at early S-phase is replicated at the same time and sites in the next round of replication.

3T3 Cells↗