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At least 1,495 records · Page 83Linked to original sources
PubMed2003

Analysis of the cruciform binding activity of recombinant 14-3-3zeta-MBP fusion protein, its heterodimerization profile with endogenous 14-3-3 isoforms, and effect on mammalian DNA replication in vitro.

The human cruciform binding protein (CBP), a member of the 14-3-3 protein family, has been recently identified as an origin of DNA replication binding protein and involved in DNA replication. Here, pure recombinant 14-3-3zeta tagged with maltose binding protein (r14-3-3zeta-MBP) at its N-terminus was tested for binding to cruciform DNA either in the absence or presence of F(TH), a CBP-enriched fraction, by electromobility shift assay (EMSA), followed by Western blot analysis of the electroeluted CBP-cruciform DNA complex. The r14-3-3zeta-MBP was found to have cruciform binding activity only after preincubation with F(TH). Anti-MBP antibody immunoprecipitation of F(TH) preincubated with r14-3-3zeta-MBP, followed by Western blot analysis with antibodies specific to the beta, gamma, epsilon, zeta, and sigma 14-3-3 isoforms showed that r14-3-3zeta-MBP heterodimerized with the endogenous beta, epsilon, and zeta isoforms present in the F(TH) but not with the gamma or sigma isoforms. Immunoprecipitation of endogenous 14-3-3zeta from nuclear extracts (NE) of HeLa cells that were either serum-starved (s-s) or blocked at the G(1)/S or G(2)/M phases of the cell cycle revealed that at G(1)/S and G(2)/M, the zeta isoform heterodimerized only with the beta and epsilon isoforms, while in s-s extracts, the 14-3-3zeta/epsilon heterodimer was never detected, and the 14-3-3zeta/beta heterodimer was seldom detected. Furthermore, addition of r14-3-3zeta-MBP to HeLa cell extracts used in a mammalian in vitro replication system increased the replication level of p186, a plasmid bearing the minimal 186-bp origin of the monkey origin of DNA replication ors8, by approximately 3.5-fold. The data suggest that specific dimeric combinations of the 14-3-3 isoforms have CBP activity and that upregulation of this activity leads to an increase in DNA replication.

14-3-3 Proteins↗
PubMed1991

The stringent response blocks DNA replication outside the ori region in Bacillus subtilis and at the origin in Escherichia coli.

When the Bacillus subtilis dnaB37 mutant, defective in initiation, is returned to permissive temperature after growth at 45 degrees C, DNA replication is synchronized. Under these conditions, we have shown previously that DNA replication is inhibited when the Stringent Response is induced by the amino acid analogue, arginine hydroxamate. We have now shown, using DNA-DNA hybridization analysis, that substantial replication of the oriC region nevertheless occurs during the Stringent Response, and that replication inhibition is therefore implemented downstream from the origin. On the left arm, replication continues for at least 190 x 10(3) base-pairs to the gnt gene and for a similar distance on the right arm to the gerD gene. When the Stringent Response is lifted, DNA replication resumed downstream from oriC on both arms, confirming that DNA replication is regulated at a post-initiation level during the Stringent Response in B. subtilis. Resumption of DNA synthesis following the lifting of the Stringent Response did not require protein or RNA synthesis or the initiation protein DnaB. We suggest, therefore, that a specific control region, involving Stringent Control sites, facilitate reversible inhibition of fork movement downstream from the origin via modifications of a replisome component during the Stringent Response. In contrast, in Escherichia coli, induction of the Stringent Response appears to block initiation of DNA replication at oriC itself. No DNA synthesis was detected in the oriC region and, upon lifting the Stringent Response, replication occurred from oriC. Post-initiation control in B. subtilis therefore results in duplication of many key genes involved in growth and sporulation. We discuss the possibility that such a control might be linked to differentiation in this organism.

Arginine↗
PubMed1995

Identification of an origin of bidirectional DNA replication in the ubiquitously expressed mammalian CAD gene.

Most DNA replication origins in eukaryotes localize to nontranscribed regions, and there are no reports of origins within constitutively expressed genes. This observation has led to the proposal that there may be an incompatibility between origin function and location within a ubiquitously expressed gene. The biochemical and functional evidence presented here demonstrates that an origin of bidirectional replication (OBR) resides within the constitutively expressed housekeeping gene CAD, which encodes the first three reactions of de novo uridine biosynthesis (carbamoyl-phosphate synthetase, aspartate carbamoyltransferase, and dihydroorotase). The OBR was localized to a 5-kb region near the center of the Syrian hamster CAD transcriptional unit. DNA replication initiates within this region in the single-copy CAD gene in Syrian baby hamster kidney cells and in the large chromosomal amplicons that were generated after selection with N-phosphonacetyl-L-aspartate, a specific inhibitor of CAD. DNA synthesis also initiates within this OBR in autonomously replicating extrachromosomal amplicons (CAD episomes) located in an N-phosphonacetyl-L-aspartate-resistant clone (5P20) of CHOK1 cells. CAD episomes consist entirely of a multimer of Syrian hamster CAD cosmid sequences (cCAD1). These data limit the functional unit of replication initiation and timing control to the 42 kb of Syrian hamster sequences contained in cCAD1. In addition, the data indicate that the origin recognition machinery is conserved across species, since the same OBR region functions in both Syrian and Chinese hamster cells. Importantly, while cCAD1 exhibits characteristics of a complete replicon, we have not detected autonomous replication directly following transfection. Since the CAD episome was generated after excision of chromosomally integrated transfected cCAD1 sequences, we propose that prior localization within a chromosome may be necessary to "license" some biochemically defined OBRs to render them functional.

Animals↗
PubMed1984

On the fidelity of DNA replication. Lack of primer position effect on the fidelity of mammalian DNA polymerases.

Mechanisms for the fidelity of DNA replication in eucaryotes are not adequately understood. Certain hypotheses can be tested by examining whether the first nucleotide inserted is incorporated with a significantly higher error rate than subsequent nucleotides. Using synthetic oligodeoxynucleotides, we have measured the effect of primer position on single-base misinsertion frequencies at an amber site in phi X174 DNA. Our results show a lack of position effect, indicating that processivity and the most direct "energy relay" proofreading mechanisms are not important determinants in eucaryotic replication fidelity.

Bacteriophage phi X 174↗
PubMed2003

Inhibition of bacterial DNA replication by zinc mobilization during nitrosative stress.

Phagocytic cells inhibit the growth of intracellular pathogens by producing nitric oxide (NO). NO causes cell filamentation, induction of the SOS response, and DNA replication arrest in the Gram-negative bacterium Salmonella enterica. NO also induces double-stranded chromosomal breaks in replication-arrested Salmonella lacking a functional RecBCD exonuclease. This DNA damage depends on actions of additional DNA repair proteins, the RecG helicase, and RuvC endonuclease. Introduction of a recG mutation restores both resistance to NO and the ability of an attenuated recBC mutant Salmonella strain to cause lethal infection in mice, demonstrating that bacterial DNA replication is inhibited during host-pathogen interactions. Inhibition of DNA replication during nitrosative stress is invariably accompanied by zinc mobilization, implicating DNA-binding zinc metalloproteins as critical targets of NO-related antimicrobial activity.

Animals↗
PubMed1995

Identification and analysis of a putative origin of DNA replication in the Choristoneura fumiferana multinucleocapsid nuclear polyhedrosis virus genome.

A recombinant plasmid containing the Choristoneura fumiferana multinucleocapsid nuclear polyhedrosis virus (CfMNPV) HindIII R fragment (m.u. 2.2-3.9) was shown to undergo CfMNPV infection-dependent DNA replication in Cf-124T cells. Replication of this DNA sequence was detectable by 24 hr p.i. and did not appear to have resulted as a consequence of recombination with the virus genome. Replication was inhibited by mimosine, an inhibitor of eukaryotic DNA replication. These data suggest that HindIII R of CfMNPV DNA contains an origin of DNA replication which we call ori1. HindIII R contains five GC-rich and three AT-rich regions and a 0.9-kb homologous repeat region 1 (hr1). Two short 440- and 740-bp contiguous sequences at the right end of the HindIII R fragment separately exhibited limited ori function. HindIII R subfragments with optimal ori activity contained a cluster of repeated and inverted sequences including nine copies of a 50-bp homologous repeat sequence (hr1a to hr1i) within hr1. The CfMNPV hr1 sequence was somewhat homologous with the homologous repeat (hr) of the putative Autographa californica MNPV (AcMNPV) replication origins. HindIII Y, another CfMNPV DNA fragment containing an hr sequence, hr3, also supported infection-dependent DNA replication, suggesting that it too contains an ori. Although replication of a putative AcMNPV origin (HindIII Q) was detectable in CfMNPV-infected Cf-124T cells, replication of CfMNPV HindIII R was not detectable in AcMNPV-infected Spodoptera frugiperda cells.

Animals↗
PubMed1987

[Characteristics of DNA replication in the long-term culture of human cells at the stationary phase].

Peculiarities of DNA replication in cultured human diploid fibroblasts in logarithmic and stationary phases were studied using DNA autoradiography. The rate of DNA replication fall from 30-36 mu/hour at active proliferative phase to 18-20 mu/hour at late stationary phase. This phenomenon is characteristic of stationary cultures after stimulation to proliferate by changing medium as well as by culturing without stimulation. Possible mechanisms of DNA replication rate alteration in senescent human cells are discussed.

Autoradiography↗
PubMed2002

The human cruciform-binding protein, CBP, is involved in DNA replication and associates in vivo with mammalian replication origins.

We previously identified and purified from human (HeLa) cells a 66-kDa cruciform-binding protein, CBP, with binding specificity for cruciform DNA regardless of its sequence. DNA cruciforms have been implicated in the regulation of initiation of DNA replication. CBP is a member of the 14-3-3 family of proteins, which are conserved regulatory molecules expressed in all eukaryotes. Here, the in vivo association of CBP/14-3-3 with mammalian origins of DNA replication was analyzed by studying its association with the monkey replication origins ors8 and ors12, as assayed by a chromatin immunoprecipitation assay and quantitative PCR analysis. The association of the 14-3-3beta, -epsilon, -gamma, and -zeta isoforms with these origins was found to be approximately 9-fold higher, compared with other portions of the genome, in logarithmically growing cells. In addition, the association of these isoforms with ors8 and ors12 was also analyzed as a function of the cell cycle. Higher binding of 14-3-3beta, -epsilon, -gamma, and -zeta isoforms with ors8 and ors12 was found at the G(1)/S border, by comparison with other stages of the cell cycle. The CBP/14-3-3 cruciform binding activity was also found to be maximal at the G(1)/S boundary. The involvement of 14-3-3 in mammalian DNA replication was analyzed by studying the effect of anti-14-3-3beta, -epsilon, -gamma, and -zeta antibodies in the in vitro replication of p186, a plasmid containing the minimal replication origin of ors8. Anti-14-3-3epsilon, -gamma, and -zeta antibodies alone or in combination inhibited p186 replication by approximately 50-80%, while anti-14-3-3beta antibodies had a lesser effect ( approximately 25-50%). All of the antibodies tested were also able to interfere with CBP binding to cruciform DNA. The results indicate that CBP/14-3-3 is an origin-binding protein, acting at the initiation step of DNA replication by binding to cruciform-containing molecules, and dissociates after origin firing.

Base Sequence↗
PubMed2002

Unifying themes in DNA replication: reconciling single molecule kinetic studies with structural data on DNA polymerases.

Structural data suggest that DNA polymerases, from at least three different families, employ common strategies for carrying out DNA replication. Universal features include a large conformational change in the enzyme-template complex and a conserved active-site geometry that imposes a sharp kink at the 5 end of the template strand. Recent single molecule experiments have shown that stretching the DNA template markedly alters the rate of DNA synthesis catalyzed by these motor enzymes. From these data, it was previously inferred that T7 DNA polymerase and two related enzymes convert two or four (depending on the enzyme) single-stranded (ss) template bases to double helix geometry in the polymerase active site during each catalytic cycle. We discuss structural data on related DNA polymerases, which suggest that only one (ss) template base is contracted to dsDNA geometry during the rate-limiting step of each replication cycle. Previous interpretations relied upon the global stretching curves for DNA polymers alone (with no reference to the enzyme or the structure of the transition state). In contrast, we present a structurally guided model that presumes the force dependence of the replication rate is governed chiefly by local interactions in the immediate vicinity of the enzyme s active site. Our analysis reconciles single molecule kinetic studies with structural data on DNA polymerases.

Binding Sites↗
PubMed2004

Requirement of a 12-base-pair TATT-containing sequence and viral lytic DNA replication in activation of the Kaposi's sarcoma-associated herpesvirus K8.1 late promoter.

Kaposi's sarcoma-associated herpesvirus (KSHV) K8.1 late promoter consists of a minimal 24-bp sequence, with a TATA-like, 12-bp promoter core, AATATTAAAGGG, and is active on a reporter only in butyrate-induced KSHV-infected cells. The activity of the K8.1 promoter can be enhanced (>15-fold) by the KSHV left-end lytic origin of DNA replication (oriLyt-L) sequence while providing inefficient replication of plasmid DNA and is inhibited by viral DNA replication inhibitors, suggesting that activation of the K8.1 promoter on the reporter is involved in KSHV lytic DNA replication largely by trans.

Base Sequence↗
PubMed2003

Common domains in the initiators of DNA replication in Bacteria, Archaea and Eukarya: combined structural, functional and phylogenetic perspectives.

Although DNA replication is the universal process for the transmission of genetic information in all living organisms, until very recently evidence was lacking for a related structure and function in the proteins (initiators) that trigger replication in the three 'Life Domains' (Bacteria, Archaea and Eukarya). In this article new data concerning the presence of common features in the initiators of chromosomal replication in bacteria, archaea and eukaryotes are reviewed. Initiators are discussed in the light of: (i) The structure and function of their conserved ATPases Associated with various cellular Activities (AAA+) and winged-helix domains. (ii) The nature of the macromolecular assemblies that they constitute at the replication origins. (iii) Their possible phylogenetic relationship, attempting to sketch the essentials of a hypothetical DNA replication initiator in the micro-organism proposed to be the ancestor of all living cells.

Amino Acid Sequence↗
PubMed1994

[The relation of the characteristics of DNA replication to cell sensitivity to ionizing radiation in xeroderma pigmentosum in form II (XP2CP)].

Using DNA fiber autoradiography, DNA replication in cultured fibroblasts, derived from normal donors, and from XPII patient with increased sensitivity to ionizing radiation was estimated. Here evidence is provided on the fact that the fork movement, significantly decreased in XPII cells before irradiation, remains the same after exposure to X-rays. The density of replicon clusters simultaneously operating in tandem groups, which is initially much less in XPII cells compared to normal cells, also remains unchanged after exposure to X-rays (5 Gy), since the inhibition of DNA replication occurs to individual replicons only. Our data suggest that the inhibition of DNA replication in normal cells throughout the whole cluster, that drastically reduces the rate of DNA-chain growth, may provide an additional time to restore damaged chromosomes, i. e. it is part of the cellular defence mechanism. It seems likely that XPII cells are deficient in such a defence mechanism.

Autoradiography↗
PubMed1984

RNase H-defective mutants of Escherichia coli: a possible discriminatory role of RNase H in initiation of DNA replication.

Mutants of Escherichia coli completely deficient in RNase H activity were isolated by inserting transposon Tn3 into the structural gene for RNase H, rnh, and its promoter. These rnh- mutants exhibited the following phenotypes; (1) the mutants grew fairly normally, (2) rnh- cells could be transformed with ColE1 derivative plasmids, pBR322 and pML21, though the plasmids were relatively unstable, under non selective conditions, (3) rnh- mutations partially suppressed the temperature-sensitive phenotype of plasmid pSC301, a DNA replication initiation mutant derived from pSC101, (4) rnh- mutations suppressed the temperature-sensitive growth character of dnaAts mutant, (5) rnh- cells showed continued DNA synthesis in the presence of chloramphenicol (stable DNA replication). Based on these findings we propose a model for a role of RNase H in the initiation of chromosomal DNA replication. We suggest that two types of RNA primers for initiation of DNA replication are synthesized in a dnaA/oriC-dependent and -independent manner and that only the dnaA/oriC-dependent primer is involved in the normal DNA replication since the dnaA/oriC independent primer is selectively degraded by RNase H.

DNA Replication↗
PubMed1976

Process of infection with bacteriophage phi chi 174. XL. Viral DNA replication of phi chi 174 mutants blocked in progeny single-stranded DNA synthesis.

Mutation in several different cistrons of bacteriophage phi chi 174 blocks net progeny single-stranded DNA synthesis at the late period of infection (15). For the study of the functions of these cistrons in single-stranded DNA synthesis, asymmetric replication of replicative form DNA was examined at the late period of infection with amber mutants of these cistrons. While the normal, rapid process of asymmetric single-stranded viral DNA synthesis is blocked at the late period of these mutant infections, an asymmetric synthesis of the viral strand of replicative-form DNA is observed in this period, though at a reduced level, together with degradation of prelabeled viral strand. Some intermediate replicative-form molecules were also detected. Asymmetric synthesis of the viral strand of replicative-form DNA at the late period of phi chi infection is completely inhibited in the presence of a low concentration (35mug/ml) of chloramphenicol (which also blocks net single-stranded viral DNA synthesis). These results are discussed in terms of the possible role of the specific viral proteins for normal single-stranded DNA synthesis.

Chloramphenicol↗
PubMed2001

Cdc28 and Ime2 possess redundant functions in promoting entry into premeiotic DNA replication in Saccharomyces cerevisiae.

In the budding yeast Saccharomyces cerevisiae initiation and progression through the mitotic cell cycle are determined by the sequential activity of the cyclin-dependent kinase Cdc28. The role of this kinase in entry and progression through the meiotic cycle is unclear, since all cdc28 temperature-sensitive alleles are leaky for meiosis. We used a "heat-inducible Degron system" to construct a diploid strain homozygous for a temperature-degradable cdc28-deg allele. We show that this allele is nonleaky, giving no asci at the nonpermissive temperature. We also show, using this allele, that Cdc28 is not required for premeiotic DNA replication and commitment to meiotic recombination. IME2 encodes a meiosis-specific hCDK2 homolog that is required for the correct timing of premeiotic DNA replication, nuclear divisions, and asci formation. Moreover, in ime2Delta diploids additional rounds of DNA replication and nuclear divisions are observed. We show that the delayed premeiotic DNA replication observed in ime2Delta diploids depends on a functional Cdc28. Ime2Delta cdc28-4 diploids arrest prior to initiation of premeiotic DNA replication and meiotic recombination. Ectopic overexpression of Clb1 at early meiotic times advances premeiotic DNA replication, meiotic recombination, and nuclear division, but the coupling between these events is lost. The role of Ime2 and Cdc28 in initiating the meiotic pathway is discussed.

Animals↗
PubMed2006

p53-Dependent p21 mRNA elongation is impaired when DNA replication is stalled.

We have previously reported that when DNA replication is blocked in some human cell lines, p53 is impaired in its ability to induce a subset of its key target genes, including p21(WAF1/CIP1). Here, we investigated the reason for this impairment by comparing the effects of two agents, hydroxyurea (HU), which arrests cells in early S phase and impairs induction of p21, and daunorubicin, which causes a G(2) block and leads to robust activation of p21 by p53. HU treatment was shown to inhibit p21 mRNA transcription rather than alter its mRNA stability. Nevertheless, chromatin immunoprecipitation assays revealed that HU impacts neither p53 binding nor acetylation of histones H3 and H4 within the p21 promoter. Furthermore, recruitment of the TFIID/TATA-binding protein complex and the large subunit of RNA polymerase II (RNA Pol II) are equivalent after HU and daunorubicin treatments. Relative to daunorubicin treatment, however, transcription elongation of the p21 gene is significantly impaired in cells treated with HU, as evidenced by reduced occupancy of RNA Pol II at regions downstream of the start site. Likewise, in the p21 downstream region after administration of HU, there is less of a specifically phosphorylated form of RNA Pol II (Pol II-C-terminal domain serine 2P) which occurs only when the polymerase is elongating RNA. We propose that while the DNA replication checkpoint is unlikely to regulate the assembly of a p21 promoter initiation complex, it signals to one or more factors involved in the process of transcriptional elongation.

Acetylation↗
PubMed1976

Mechanism of nuclear DNA replication in radicles of germinating cotton.

The mechanism of nuclear DNA replication in radicles of germinating cotton (Gossypium barbadense) was investigated. Pulse-labeling with [(3)H]thymidine indicates that replication intermediates are of small molecular weight (4-10S) and behave as if single-stranded. Prolonged labeling indicates that intermediates are of discrete size, suggesting a mechanism of discontinuous replication. Electron microscopy of nuclear DNA demonstrates a complex architecture which may include "eye forms" and "forks" similar to those reported in prokaryotes and animal systems. The possible universality of DNA replication mechanisms is discussed.

Journal Article↗
PubMed2006

The 32 kDa subunit of replication protein A (RPA) participates in the DNA replication of Mung bean yellow mosaic India virus (MYMIV) by interacting with the viral Rep protein.

Mung bean yellow mosaic India virus (MYMIV) is a member of genus begomoviridae and its genome comprises of bipartite (two components, namely DNA-A and DNA-B), single-stranded, circular DNA of about 2.7 kb. During rolling circle replication (RCR) of the DNA, the stability of the genome and maintenance of the stem-loop structure of the replication origin is crucial. Hence the role of host single-stranded DNA-binding protein, Replication protein A (RPA), in the RCR of MYMIV was examined. Two RPA subunits, namely the RPA70 kDa and RPA32 kDa, were isolated from pea and their roles were validated in a yeast system in which MYMIV DNA replication has been modelled. Here, we present evidences that only the RPA32 kDa subunit directly interacted with the carboxy terminus of MYMIV-Rep both in vitro as well as in yeast two-hybrid system. RPA32 modulated the functions of Rep by enhancing its ATPase and down regulating its nicking and closing activities. The possible role of these modulations in the context of viral DNA replication has been discussed. Finally, we showed the positive involvement of RPA32 in transient replication of the plasmid DNA bearing MYMIV replication origin using an in planta based assay.

Biological Assay↗