Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “DNA replication”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,765 records · Page 98Linked to original sources
PubMed1993

Inhibition of initiation of DNA replication in Xenopus egg extracts by a phosphatase inhibitor, calyculin A.

The protein phosphatase inhibitor calyculin A was found to strongly inhibit the DNA replication activity of Xenopus egg extracts in a dose-dependent manner. Calyculin A (0.8 nM) completely prevented the oscillation in DNA replication activity of mitotic extracts released by the addition of CaCl2. In contrast, in the case of extracts prepared from eggs activated by calcium ionophore A23187, calyculin A (0.8 nM) had no effect on the first peak of the oscillation in activity but inhibited the second peak of the oscillation. These results strongly suggest that calyculin A inhibits a protein phosphatase, which is involved in the initiation of DNA replication in the Xenopus egg extracts.

Animals↗
PubMed1984

Chromatin structure and DNA replication.

The structure of (3H) thymidine pulse labeled, replicating chromatin differs from that of non replicating chromatin by several operational criteria which are related to the higher nuclease sensitivity of replicating chromatin. We summarize the structural changes that we observe using replicating chromatin as substrate for micrococcal nuclease. The data suggest a more extended configuration of replicating compared to non replicating chromatin. We use these data to discuss a model of the chromatin structure in the vicinity of replication forks. Finally, we present data to show that the reversion of structural changes in replicative chromatin depends on continued DNA replication.

Animals↗
PubMed1985

Mutagenesis of a herpes simplex virus origin of DNA replication and its effect on viral interference.

Supercoiled plasmid molecules containing cloned copies of a DNA fragment which includes a functional herpes simplex virus type 1 origin of DNA replication were cleaved preferentially at two positions within the viral insert by nuclease S1. Plasmids with molecular linker insertions at these sites were constructed, and analysis of two representative plasmids demonstrated the presence of palindromic DNA sequences at the preferred cleavage positions. One of these palindromic sequences occurred within a 90 bp region in which the cis-acting sequences essential for viral origin function had previously been located. Insertion of a linker at this position abolished origin activity, demonstrating an essential role for sequences within the palindrome in the initiation of DNA synthesis. In transfection assays, plasmids containing a functional viral origin of DNA replication markedly interfered with the infectivity of non-defective viral DNA even in the absence of viral encapsidation signals. Inactivation of the origin greatly reduced this effect on DNA infectivity, suggesting that viral interference may be mediated by a mechanism involving the replicative machinery.

Base Sequence↗
PubMed1993

Identification of EBNA1 amino acid sequences required for the interaction of the functional elements of the Epstein-Barr virus latent origin of DNA replication.

Epstein-Barr nuclear antigen 1 (EBNA1) activates DNA replication from the Epstein-Barr virus latent origin, oriP. This activation involves the direct interaction of EBNA1 dimers with multiple sites within the two noncontiguous functional elements of the origin, the family of repeats (FR) element and the dyad symmetry (DS) element. The efficient interaction of EBNA1 dimers bound to these two elements in oriP results in the formation of DNA loops in which the FR and DS elements are bound together through EBNA1. In order to elucidate the mechanism by which EBNA1 induces oriP DNA looping, we have investigated the DNA sequences and EBNA1 amino acids required for EBNA1-mediated DNA looping. Using a series of truncation mutants of EBNA1 produced in baculovirus and purified to apparent homogeneity, we have demonstrated that the EBNA1 DNA binding and dimerization domain is not sufficient to mediate oriP DNA looping and that an additional region(s) located between amino acids 346 and 450 is required. Single EBNA1-binding sites, separated by 930 bp of plasmid DNA, were also shown to support EBNA1-mediated looping, indicating that the formation of large EBNA1 complexes, such as those observed on oriP FR and DS elements, is not a requirement for looping.

Amino Acid Sequence↗
PubMed1995

Expression of human polyomavirus JC T antigen by an adenovirus hybrid vector and its binding to DNA sequences encompassing the JC virus origin of DNA replication.

In the search for factors that influence the outcome of human polyomavirus JC (JCV) infection, the roles not only of host-related immunological control but also of virus-dependent regulatory steps have to be taken into account. Besides cell-specific control of early expression of the multifunctional virus protein large tumour antigen (T Ag), control mechanisms involve individual steps of the DNA replication process. For the analysis of T Ag DNA binding, the protein was expressed by an adenovirus hybrid vector in the 293 cell line to provide saturating amounts of JCV T Ag. After determination of the size and immunoreactivity, functional activity was analysed by specific DNA binding. To avoid the interference of cellular proteins, T Ag was immunoprecipitated prior to the reaction. Binding to T Ag-binding sites I and II within a 141 bp DNA segment in the control region was analysed using deletion mutants of a JCV subtype from brain tissue of a patient with fatal central nervous system disease. The specificity of the binding was confirmed by recombinant T Ag binding to origin of DNA replication (ori) sequences of wild-type JCV genomes. These data document that recombinant T Ag overexpressed by the adenovirus vector in eukaryotic cells was JCV-specific, had the expected length and exhibited specific ori-binding activity, thus providing the essential tool for future analysis of virus-host interactions at the level of viral DNA replication.

Adenoviridae↗
PubMed1981

The influence of host DNA replication on the formation of infectious and transducing Mu-particles.

We have investigated the influence of bacterial DNA replication on the formation of infectious and transducing Mu-particles. The data obtained agree with the previous findings that growth of phage Mu is independent of the host dnaA gene product (Toussaint and Faelen 1974), but required bacterial replication forks (Fitts and Taylor 1980). The replication of transducing DNA during phage development (Teifel and Schmieger 1979) is controlled by the host and is not a precondition for its packaging. Packaging of transducing DNA does not require a a nearby Mu genome.

Bacteriophage mu↗
PubMed1988

Initiation of adenovirus DNA replication. II. Structural requirements using synthetic oligonucleotide adenovirus templates.

Adenovirus (Ad) virions contain a 55-kDa terminal protein covalently linked to both 5'-ends of the linear duplex DNA genome. The origin of DNA replication is contained within the terminal 50 base pair of the inverted terminal repeats. In the accompanying paper (Kenny, M. K., Balogh, L. A., and Hurwitz, J. (1988) J. Biol. Chem. 263, 9801-9808), it was demonstrated that synthetic oligonucleotide templates which contain the Ad origin, but lack the 55-kDa terminal protein, can serve as templates for the initiation of Ad DNA replication. Partially duplex oligonucleotides that lacked up to 14 nucleotides from the 5'-end of the nontemplate (displaced) strand supported initiation as much as 20-fold more efficiently than fully duplex oligonucleotides. The removal of 18 nucleotides or more from the 5'-end of the displaced strand resulted in a sharp decrease in the ability of the DNA templates to support initiation. The poor template efficiency of certain DNAs could be explained by their inability to bind nuclear factor I. The initiation efficiency observed with other DNAs correlated with their ability to bind the preterminal protein-Ad DNA polymerase complex. At low concentrations of the Ad DNA-binding protein, protein-primed initiation was also observed on single-stranded DNAs. The single-stranded template strand of the Ad origin was at least 5-20-fold better at supporting initiation than other single-stranded DNAs. These findings suggest a model in which the 3'-end of the template strand is rendered single-stranded as a prerequisite for initiation of Ad DNA replication.

Adenosine Triphosphate↗
PubMed2001

Understanding the enzymology of archaeal DNA replication: progress in form and function.

The analysis of completed archaeal genome sequences led to the identification of a set of approximately 10-20 genes whose protein products were inferred to be involved in chromosomal DNA replication. Until recently, however, little was known of the biochemical properties of these proteins. Here, I review recent progress in this area brought about by biochemical and structural analysis. Aside from shedding considerable new light on the molecular machinery of DNA replication in the archaea, the results of these studies also present new opportunities for understanding the molecular events of chromosomal DNA replication in eukaryotic cells.

DNA Replication↗
PubMed1988

Regulation of mouse satellite DNA replication time.

The satellite DNA sequences located near the centromeric regions of mouse chromosomes replicate very late in S in both fibroblast and lymphocyte cells and are heavily methylated at CpG residues. F9 teratocarcinoma cells, on the other hand, contain satellite sequences which are undermethylated and replicate much earlier in S. DNA methylation probably plays some role in the control of satellite replication time since 5-azacytidine treatment of RAG fibroblasts causes a dramatic temporal shift of replication to mid S. In contrast to similar changes accompanying the inactivation of the X-chromosome, early replication of satellite DNA is not associated with an increase in local chromosomal DNase I sensitivity. Fusion of F9 with mouse lymphocytes caused a dramatic early shift in the timing of the normally late replicating lymphocyte satellite heterochromatin, suggesting that trans-activating factors may be responsible for the regulation of replication timing.

Animals↗
PubMed1981

DNAase I digestion of early, middle, and late S phase replicating DNA in murine leukemia L5178Y cells.

Cellular DNA from a synchronized population of cultured mouse L5178Y cells was labeled with [3H]thymidine at three times during the S stage: early, middle and late S phases. The deoxyribonuclease I digestability of DNA from these three periods in the subsequent cell cycle indicated that the early S phase replicating DNA had higher enzymatic susceptibility at G1 and early S phases than at middle and late S phases, while the middle and late S phase replicating DNA demonstrated a low susceptibility throughout the cell cycle. This suggests that most, if not all, of transcriptionally active DNA is replicated very early in the S phase.

Animals↗
PubMed1993

DNA replication in pancreatic islets and adrenals in diabetic Chinese hamsters.

The diabetic syndrome in Chinese hamsters is progressive and there is a decrease in the size of the pancreatic islets. To what extent this is due to impaired DNA replication in their islet cells is unknown. Therefore 3H-thymidine incorporation into islet cells was studied in genetically diabetic (subline L) male hamsters aged 17-20 days, 6 weeks, 3, 6 and 12 months. Because adrenal weight was decreased at almost all ages, studies of adrenal morphology and function were also performed. Age- and sex-matched normal (subline M) hamsters served as controls. Autoradiography showed progressively falling labeling with increasing age in both normal and diabetic animals in both islets and adrenals. At the earliest age studied, the diabetic subline animals were normoglycemic but showed hyperinsulinemia. DNA replication was reduced both in the islet cells and the adrenal cortex. From 6 weeks of age and onwards, the diabetic hamsters were hyperglycemic and usually hyperinsulinemic but DNA replication in both islets and adrenals was not different from that in normals. Histomorphometry showed reduced volume in both the adrenal medulla and cortex in 3-month-old diabetic hamsters. In 12-month-old male and female diabetic hamsters plasma cortisol and aldosterone and 24-hour urinary epinephrine were normal, but urinary norepinephrine and dopamine levels were excessive. Thus, in contrast to what is found in the obese-hyperglycemic mouse, there was no increased islet cell DNA replication in response to the increased functional demand in the diabetic Chinese hamster.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Cortex Hormones↗
PubMedDate not supplied

Escherichia coli contains a DNA replication compartment in the cell center.

The active replication forks of E. coli B/r K cells growing with a doubling time of 210 min have been pulse-labeled with [(3)H] thymidine for 10 min. By electron-microscopic autoradiography the silver grains have been localized in the various length classes. From the known pattern of the DNA replication period in the cell cycle at slow growth and from the average position of grains per length class it was deduced that DNA replication starts in the cell center and that it remains there for a substantial part of the DNA replication period. This suggests the occurrence of a centrally located DNA replication compartment.

Autoradiography↗
PubMed2004

The B-subunit of DNA polymerase alpha-primase associates with the origin recognition complex for initiation of DNA replication.

The B-subunit (p70/Pol12p) of the DNA polymerase alpha-primase (Polalpha-primase) complex is thought to have a regulatory role in an early stage of S phase. We generated a panel of fission yeast thermosensitive mutants of the B-subunit (termed Spb70) to investigate its role in initiation of DNA replication by genetic and biochemical approaches. Here, we show that the fission yeast Spb70 genetically interacts and coprecipitates with origin recognition complex proteins Orp1/Orc1 and Orp2/Orc2 and primase coupling subunit Spp2/p58. A fraction of Spb70 associates with Orp2 on chromatin throughout the cell cycle independent of the other subunits of Polalpha-primase. Furthermore, primase Spp2/p58 subunit preferentially associates with the unphosphorylated Orp2, and the association requires Spb70. Mutations in orp2+ that abolish or mimic the Cdc2 phosphorylation of Orp2 suppress or exacerbate the thermosensitivity of the spb70 mutants, respectively, indicating that an unphosphorylated Orp2 promotes an Spb70-dependent replication event. Together, these results indicate that the chromatin-bound B-subunit in association with origin recognition complex mediates recruiting Polalpha-primase complex onto replication origins in G1 pre-Start through an interaction with primase Spp2/p58 subunit. Our results thus suggest a role for the recruited Polalpha-primase in the initiation of both leading and lagging strands at the replication origins.

Animals↗
PubMed1994

Mitotic checkpoint genes in budding yeast and the dependence of mitosis on DNA replication and repair.

In eukaryotes a cell-cycle control termed a checkpoint causes arrest in the S or G2 phases when chromosomes are incompletely replicated or damaged. Previously, we showed in budding yeast that RAD9 and RAD17 are checkpoint genes required for arrest in the G2 phase after DNA damage. Here, we describe a genetic strategy that identified four additional checkpoint genes that act in two pathways. Both classes of genes are required for arrest in the G2 phase after DNA damage, and one class of genes is also required for arrest in S phase when DNA replication is incomplete. The G2-specific genes include MEC3 (for mitosis entry checkpoint), RAD9, RAD17, and RAD24. The genes common to both S phase and G2 phase pathways are MEC1 and MEC2. The MEC2 gene proves to be identical to the RAD53 gene. Checkpoint mutants were identified by their interactions with a temperature-sensitive allele of the cell division cycle gene CDC13; cdc13 mutants arrested in G2 and survived at the restrictive temperature, whereas all cdc13 checkpoint double mutants failed to arrest in G2 and died rapidly at the restrictive temperature. The cell-cycle roles of the RAD and MEC genes were examined by combination of rad and mec mutant alleles with 10 cdc mutant alleles that arrest in different stages of the cell cycle at the restrictive temperature and by the response of rad and mec mutant alleles to DNA damaging agents and to hydroxyurea, a drug that inhibits DNA replication. We conclude that the checkpoint in budding yeast consists of overlapping S-phase and G2-phase pathways that respond to incomplete DNA replication and/or DNA damage and cause arret of cells before mitosis.

Alleles↗
PubMedDate not supplied

[Rate of DNA replication and size of replicons in human diploid cells].

The rate of DNA replication and the distances between initiation sites (size of replicons) have been studied in human cultured fibroblasts. The modified Huberman and Riggs technique of DNA fiber autoradiography has been used: the pulse-labelled regions were analysed in DNA fibers preliminarily labelled along the whole length. This enabled us: a) to analyse the arrangement of replicons along the length of labelled DNA fibers with the lengths of 200-750 micron, reaching 2700 micron in some cases; b) to select only single DNA molecules for the analysis. This technique decreases the danger of a mistake when minor labelled regions belonging to different DNA molecules are referred to the same one. The rate of DNA replication varies from 0.2 to 1.2 micron/min, the average of 0.6 micron/min. This conforms with findings of other authors. The distances between initiation sites vary from 15 to 140 micron with the modal interval of 50-60 micron. This value is twice higher than those obtained by other authors. The possible reasons for such difference are discussed.

Animals↗
PubMed1982

Instability of transposase activity: evidence from bacteriophage mu DNA replication.

Transposition of genetic elements involves coupled replication and integration events catalyzed in part by a class of proteins called transposases. We have asked whether the transposase activity of bacteriophage Mu (the Mu A protein) is stable and capable of catalyzing multiple rounds of coupled replication/integration, or whether its continued synthesis is required to maintain Mu DNA replication. Inhibition of protein synthesis during the lytic cycle with chloramphenicol inhibited Mu DNA synthesis with a half-life of approximately 3 min, demonstrating a need for continued protein synthesis to maintain Mu DNA replication. Synthesis of specific Mu-encoded proteins was inhibited by infecting a host carrying a temperature-sensitive suppressor, at permissive temperature, with Mu amber phages, then shifting to nonpermissive temperature. When Aam phages were used, Mu DNA replication was inhibited with kinetics essentially identical to those with chloramphenicol addition; hence, it is likely that continued synthesis of the Mu A protein is required to maintain Mu DNA replication. The data suggest that the activity of the Mu A protein is unstable, and raise the possibility that the Mu A protein and other transposases may be used stoichiometrically rather than catalytically.

Bacteriophage mu↗
PubMed1991

T-antigen binding to site I facilitates initiation of SV40 DNA replication but does not affect bidirectionality.

SV40 origin auxiliary sequence 1 (aux-1) encompasses T-antigen (T-ag) binding site I and facilitates origin core (ori-core) activity in whole cells or cell extracts. Aux-1 activity depended completely upon its sequence, orientation and spacing relative to ori-core. Aux-1 activity was lost either by inserting 10 base pairs between aux-1 and ori-core or by placing either orientation of aux-1 on the opposite side of ori-core. Reversing the orientation of aux-1 in its normal position actually inhibited replication. Easily unwound DNA sequences that stimulate yeast or E. coli origins of replication could not replace aux-1. Aux-1 did not affect bidirectional replication. Replication remained bidirectional even when aux-1 was inactivated, and deletion of aux-1 did not affect selection of RNA-primed DNA synthesis initiation sites in the origin region: the transition from discontinuous to continuous DNA synthesis that marks the origin of bidirectional replication occurred at the same nucleotide locations in both wild-type and aux-1 deleted origins. These results support a model for initiation of SV40 DNA replication in which T-ag binding to aux-1 (T-ag binding site I) facilitates the efficiency with which T-ag initiates replication at ori-core (T-ag binding site II) without affecting the mechanism by which initiation of DNA replication occurs.

Antigens, Polyomavirus Transforming↗
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↗