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At least 1,657 records · Page 92Linked to original sources
PubMed2001

Epstein-Barr nuclear antigen 1 binds and destabilizes nucleosomes at the viral origin of latent DNA replication.

The EBNA1 protein of Epstein-Barr virus (EBV) activates latent-phase DNA replication by an unknown mechanism that involves binding to four recognition sites in the dyad symmetry (DS) element of the viral latent origin of DNA replication. Since EBV episomes are assembled into nucleosomes, we have examined the ability of Epstein-Barr virus nuclear antigen 1 (EBNA1) to interact with the DS element when it is assembled into a nucleosome core particle. EBNA1 bound to its recognition sites within this nucleosome, forming a ternary complex, and displaced the histone octamer upon competitor DNA challenge. The DNA binding and dimerization region of EBNA1 was sufficient for nucleosome binding and destabilization. Although EBNA1 was able to bind to nucleosomes containing two recognition sites from the DS element positioned at the edge of the nucleosome, nucleosome destabilization was only observed when all four sites of the DS element were present. Our results indicate that the presence of a nucleosome at the viral origin will not prevent EBNA1 binding to its recognition sites. In addition, since four EBNA1 recognition sites are required for both nucleosome destabilization and efficient origin activation, our findings also suggest that nucleosome destabilization by EBNA1 is important for origin activation.

Animals↗
PubMed1995

Mutation of the Oct-1 POU-specific recognition helix leads to altered DNA binding and influences enhancement of adenovirus DNA replication.

To assess which residues of Oct-1 POU-specific (POUs) are important for DNA recognition and stimulation of adenovirus DNA replication we have mutated 10 residues of the POUs helix-turn-helix motif implicated in DNA contact. Seven of these turned out to have reduced DNA binding affinity. Of these, three alanine substituted proteins were found to have a changed specificity using a binding site selection procedure. Mutation of the first residue in the recognition helix, Gln44, to alanine led to a loss of specificity for the first two bases, TA, of the wild-type recognition site TATGC(A/T)AAT. Instead of the A, a T was selected, suggesting a new contact and a novel specificity. A change in specificity was also observed for the T45A mutant, which could bind to TATAC(A/T)AAT, a site hardly recognized by the wild-type protein. Mutation of residue Arg49 led to a relaxed specificity for three consecutive bases, TGC. This residue, which is critical for high affinity binding, is absent from the structurally homologous lambdoid helix-turn-helix motifs. Employing a reconstituted system all but two mutants could stimulate adenovirus DNA replication upon saturation. Mutation of residues Gln27 and Arg49 impairs the ability of the Oct-1 POU domain protein to enhance replication, with a concomitant loss of DNA contacts. Since the POU domain binds the precursor terminal protein-DNA polymerase complex and guides it to the origin, lack of stimulation may be caused by incorrect targetting of the DNA polymerase due to loss of specificity.

Adenoviridae↗
PubMed2004

Lesion (in)tolerance reveals insights into DNA replication fidelity.

The initial encounter of an unrepaired DNA lesion is likely to be with a replicative DNA polymerase, and the outcome of this event determines whether an error-prone or error-free damage avoidance pathway is taken. To understand the atomic details of this critical encounter, we have determined the crystal structures of the pol alpha family RB69 DNA polymerase with DNA containing the two most prevalent, spontaneously generated premutagenic lesions, an abasic site and 2'-deoxy-7,8-dihydro-8-oxoguanosine (8-oxodG). Identification of the interactions between these damaged nucleotides and the active site provides insight into the capacity of the polymerase to incorporate a base opposite the lesion. A novel open, catalytically inactive conformation of the DNA polymerase has been identified in the complex with a primed abasic site template. This structure provides the first molecular characterization of the DNA synthesis barrier caused by an abasic site and suggests a general mechanism for polymerase fidelity. In contrast, the structure of the ternary 8-oxodG:dCTP complex is almost identical to the replicating complex containing unmodified DNA, explaining the relative ease and fidelity by which this lesion is bypassed.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1993

Decatenation activity of topoisomerase IV during oriC and pBR322 DNA replication in vitro.

Topoisomerase IV (Topo IV), encoded by parC and parE, is required for partition of the daughter chromosomes in Escherichia coli. This enzyme is likely responsible for decatenating the linked daughter chromosomes after replication. In this report, we have examined the action of Topo IV in both pBR322 and oriC DNA replication reconstituted in vitro with purified proteins. Gyrase fails to decatenate the linked daughter molecules under any condition in the oriC system and at physiological salt concentrations in the pBR322 system, whereas Topo IV stimulates generation of monomer product DNA by 7- to 10-fold. Topo IV-catalyzed decatenation of isolated multiply linked DNA dimers was relatively insensitive to salt; it proceeded at 14% of the maximal rate even in the presence of 800 mM potassium glutamate. In contrast, decatenation in vitro by gyrase was inhibited completely under these conditions. Pulse-chase analysis indicated that Topo IV-catalyzed resolution of linked daughter DNA molecules occurred prior to completion of DNA replication, such that multiply linked daughter molecules did not arise. These results suggest that during DNA replication, gyrase acts primarily to relieve accumulated positive supercoiling and Topo IV acts to segregate the daughter chromosomes.

Bacterial Proteins↗
PubMed2002

Evidence that the pre-mRNA splicing factor Clf1p plays a role in DNA replication in Saccharomyces cerevisiae.

Clf1p is an essential, highly conserved protein in S. cerevisiae that has been implicated in pre-mRNA splicing. Clf1p's ortholog in Drosophila, Crn, is required for normal cell proliferation. Cells depleted of Clf1p arrest primarily with large buds, a single nucleus, a 2C DNA content, and a short, intact mitotic spindle. We isolated temperature-sensitive clf1 mutants that exhibit similar mitotic defects when released to the restrictive temperature from an early S-phase block. While these mutants also accumulate unspliced pre-mRNA at the restrictive temperature, the mitotic arrest does not appear to result from a failure to splice tubulin pre-mRNA. Moreover, the same mutants exhibit a delayed entry into S phase when released to the restrictive temperature from a G1 phase block. This delay could not be suppressed by disruption of the S-phase CDK inhibitor SIC1, suggesting that Clf1p is involved in DNA replication. Consistent with this possibility, we find that Clf1p (but not the mutant clf1p) interacts with the DNA replication initiation protein Orc2p in two-hybrid and co-immunoprecipitation assays, that Clf1p preferentially associates with origins of DNA replication, and that this association is Orc2p dependent. These observations suggest that Clf1p plays a direct role in the initiation of DNA replication.

Cell Cycle Proteins↗
PubMed1995

p65cdc18 plays a major role controlling the initiation of DNA replication in fission yeast.

A key problem in the cell cycle is understanding what brings about the initiation of DNA replication and how this is linked with global cell cycle controls. The fission yeast gene cdc18 is required for DNA replication and is transcriptionally activated by the cdc10/res1/res2 control acting at START in late G1. We show here that overexpressing cdc18 is able to bring about repeated rounds of DNA synthesis in the absence of mitosis and of continuing protein synthesis. The level of the cdc18-encoded protein p65cdc18 is periodic in the cell cycle, peaking at the G1 to S phase transition, and p65cdc18 is located in the nucleus when cdc18 is overexpressed. We propose that p65cdc18 acts at the initiation of DNA replication and plays a major role in controlling the onset of S phase.

Cell Cycle Proteins↗
PubMed1999

The Cdc6 nucleotide-binding site regulates its activity in DNA replication in human cells.

The Cdc6 protein of budding yeast and its homologues in other species play an essential role in the initiation of DNA replication. A cDNA encoding a human homologue of Cdc6 (HsCdc6) has been cloned and expressed as a fusion protein in a soluble and functionally active form. The purified protein bound specifically to ATP and slowly hydrolyzed it, whereas HsCdc6 mutants containing amino acid substitutions in the Walker A or B motifs were defective. The mutant proteins retained the ability to bind HsOrc1 and HsCdc6 but displayed aberrant conformations in the presence of nucleotides. Microinjection of either mutant protein into human cells in G1 inhibited DNA replication, suggesting that ATP binding and hydrolysis by HsCdc6 are essential for DNA replication.

Adenosine Triphosphate↗
PubMed1979

Complementation of the temperature-sensitive defect in H5ts125 adenovirus DNA replication in vitro.

Soluble extracts of adenovirus-infected HeLa cell nuclei support DNA replication on exogenous adenovirus DNA templates. Conditions of synthesis using both wild-type and temperature-sensitive extracts have been defined. Nuclear extracts prepared from cells permissively infected with the adenovirus mutant H5ts125 expressed the temperature-sensitive phenotype and could be inactivated at 37 degrees C in vitro. These extracts were completely complemented by the addition of wild-type adenovirus DNA binding protein but not by H5ts125 DNA binding protein. Enhancement by binding protein in the mutant extracts represents replication, as demonstrated by the production of full-sized products and orderly chain elongation originating, as in vivo, at both ends of the linear DNA. Replicative synthesis required the 5'-terminal protein bound covalently to template DNA and could be inhibited by denaturation of this 55,000-dalton protein. Various inhibitors of eukaryotic DNA polymerases, such as aphidicolin and 2',3'-dideoxythymidine triphosphate, inhibited replication of exogenous adenovirus templates in this system as they do in previously reported systems that only elongate endogenous replicating intermediates.

Adenoviruses, Human↗
PubMed1997

Down-regulation of DNA replication in extracts of camptothecin-treated cells: activation of an S-phase checkpoint?

Extracts prepared from camptothecin (CPT)-treated cells have a reduced ability to support SV40 DNA replication in vitro. This reduction derives mainly from a reduction in the frequency of initiation events because DNA chain elongation remains practically unchanged. Mixing of extract from nontreated cells with small amounts of extract of CPT-treated cells indicates that the reduction in DNA replication is due to the synthesis/activation of a dominant inhibitor. The observed reduction in DNA replication activity cannot be attributed to inactivation of Topo I, the molecular target of camptothecin, because levels and activity of this protein remain unchanged in extracts of CPT-treated cells and addition of purified Topo I does not restore replication activity. Although replication protein A (RP-A) is phosphorylated in CPT-treated cells, reduced replication may not be caused by RP-A inactivation, because neither loss of phosphorylation nor the addition of recombinant RP-A restore replication activity. We interpret these observations as biochemical evidence for the activation of a checkpoint in S phase and discuss the ramifications of this activation on the mechanism of CPT-induced cytotoxicity.

Antineoplastic Agents, Phytogenic↗
PubMed1984

Antibodies specific for the phi 29 terminal protein inhibit the initiation of DNA replication in vitro.

The phi 29 DNA-terminal protein serves as a primer for the initiation of DNA replication by covalently binding the first nucleotide in the DNA chain. Two distinct antibodies were used for functional analysis of this protein. One antibody was raised against sonicated phi 29 DNA-protein complex isolated from phage virions (anti-TP). The other antibody was raised against a conjugate of bovine serum albumin and a synthetic peptide corresponding to the carboxy-terminal of the phi 29 terminal protein (anti-gp3C), which was predicted from the nucleotide sequence of phi 29 DNA. Both antibodies react with native phi 29 terminal protein as determined by immunoprecipitation and enzyme-linked immunosorbent assay. Both antibodies specifically inhibit the complex-forming reaction between the phi 29 terminal protein and dAMP, the first nucleotide of phi 29 DNA.

Amino Acid Sequence↗
PubMed1989

Evidence of a ter specific binding protein essential for the termination reaction of DNA replication in Escherichia coli.

Activity binding specifically to the 22 bp of the DNA replication terminus (ter) sequence on plasmid R6K and the Escherichia coli genome was detected in the crude extract of E. coli cells. This activity was inactivated by heat or by protease but not by RNase treatments. Overproduction of the ter binding activity was observed when the extract was prepared from the cell carrying a plasmid with a chromosomal-derived 5.0 kb EcoRI fragment, on which one of the four terC sites, terC2, was also located. By mutagenesis of the 5.0 kb fragment on the plasmid with transposon Tn3 and subsequent replacement of the corresponding chromosomal region with the resulting mutant alleles, we isolated tau- mutants completely defective in ter binding activity. These mutants simultaneously lost the activity to block the progress of the DNA replication fork at any ter site, on the genome or the plasmid. It would thus appear that the ter binding protein plays an essential role in the termination reaction, at the ter sites.

Autoradiography↗
PubMed1984

The functional origin of bacteriophage f1 DNA replication. Its signals and domains.

The origin of DNA replication of bacteriophage f1 functions as a signal, not only for initiation of viral strand synthesis, but also for its termination. Viral (plus) strand synthesis initiates and terminates at a specific site (plus origin) that is recognized and nicked by the viral gene II protein. Mutational analysis of the 5' side (upstream) of the origin of plus strand replication of phage f1 led us to postulate the existence of a set of overlapping functional domains. These included ones for strand nicking, and initiation and termination of DNA synthesis. Mutational analysis of the 3' side (downstream) of the origin has verified the existence of these domains and determined their extent. The results indicate that the f1 "functional origin" can be divided into two domains: (1) a "core region", about 40 nucleotides long, that is absolutely required for plus strand synthesis and contains three distinct but partially overlapping signals, (a) the gene II protein recognition sequence, which is necessary both for plus strand initiation and termination, (b) the termination signal, which extends for eight more nucleotides on the 5' side of the gene II protein recognition sequence, (c) the initiation signal that extends for about ten more nucleotides on the 3' side of the gene II protein recognition sequence; (2) a "secondary region", 100 nucleotides long, required exclusively for plus strand initiation. Disruption of the secondary region does not completely abolish the functionality of the f1 origin but does drastically reduce it (1% residual biological activity). We discuss a possible explanation of the fact that this region can be interrupted (e.g. f1, M13 cloning vectors) by large insertions of foreign DNA without significantly affecting replication.

Base Sequence↗
PubMed1986

A new membrane-associated DNA replication protein, the gene 69 product of bacteriophage T4, shares a patch of homology with the Escherichia coli dnaA protein.

A new phage T4 DNA replication protein, gp69, is found to be associated with membrane fractions, as predicted by the translated base sequence of gene 69. In addition, gp69 shares a patch of homology with a segment of the Escherichia coli dnaA initiation protein. The patchy homology of dnaA protein and gp69 suggests that they may serve some similar functions, such as interactions with the same E. coli components in bacterial and viral DNA replication. We have shown before that gene 69 spans an origin of T4 DNA replication, and that this origin is preferentially associated with membrane fractions. We suggest the possibility that gp69 is involved in the attachment of this origin to the bacterial envelope.

Amino Acid Sequence↗
PubMed2006

Replication-associated strand asymmetries in vertebrate genomes and implications for replicon size, DNA replication origin, and termination.

Strand compositional asymmetry has been observed in prokaryotes and used in predicting prokaryotic DNA replication origins and termini. However, it was not found in eukaryotic genomes by the same methods. We propose that transcription-associated strand asymmetries mask the replication-associated ones. By analyzing the nucleotide composition of intergenic sequences larger than 50 kb by cumulative skew diagrams (CSD), we found replication-associated strand asymmetry in vertebrate genomes. Furthermore, we found that the most common replicon sizes in vertebrates are 50-100 kb, and show evidence that the replication origin and termination regions of vertebrate genomes range from a discrete site to a broad zone.

Animals↗
PubMed2005

Mutational analysis of the direct tandem repeat sequences at the origin of DNA replication of porcine circovirus type 1.

Mutational analysis was conducted to investigate the role of the nucleotide sequences flanking the stem-loop palindromic structure at the origin of DNA replication of porcine circovirus type 1 (PCV1) with respect to self-DNA replication and progeny virus generation. The results demonstrated that the A-rich sequence to the left of the palindrome is non-essential for virus replication. Although a set of four hexanucleotide (H) sequences to the right of the palindrome (organized in two tandem repeats: the proximal H1/H2 and the distal H3/H4) are binding sites for the viral Rep-associated proteins in vitro, only a proximal tandem (H/H or h-like/H) is essential for PCV1 DNA replication. In the presence of H1/H2, mutations engineered into H3/H4 were preserved in the progeny viruses. Mutations engineered into H1/H2 were invariably deleted so that the downstream H3/H4 was placed next to the palindrome. Viral genome with mutations engineered into both H1/H2 and H3/H4 underwent extensive nucleotide reorganization to yield progeny viruses containing either H3/H4, h-like/H4, or h-like/H3/H4 sequences.

Animals↗
PubMed1995

Stimulation of DNA replication by truncated Fos and Jun proteins in a cell-free extract of Xenopus eggs.

The effects of truncated Fos (residue 116-211) and/or Jun (residue 224-334) proteins on the DNA replication of plasmid with or without an AP-1 binding site were examined in a cell-free extract of Xenopus eggs. These truncated proteins, which are depleted of the domains necessary for transcriptional activation, stimulated semiconservative DNA replication only in combination and in the presence of plasmid with an AP-1 binding site. These results suggest that truncated Fos and Jun proteins act together to stimulate DNA replication and that activation depends on the presence of an AP-1 binding site in the Xenopus cell-free DNA replication system.

Animals↗
PubMed1986

Inhibition of SV40 DNA replication by benzo[a]pyrene diol epoxide adducts: two recovery modes.

Anti-benzo[a]pyrene diol epoxide (BPDE) adducts produced in vitro in SV40 initially inhibit SV40 DNA replication in vivo, in cells unexposed to BPDE. A single adduct in a replicon is probably sufficient to block DNA replication. The recovery process appears to begin immediately after infection. The rate of recovery of replicative capacity is inversely related to the initial adduct number. Holding the infected cells temporarily under conditions that prevent viral DNA replication results subsequently in increased recovery, proportional to the holding time. The mechanism of recovery appears to be constitutive and prereplicative. In addition, there is a second mode of recovery which is induced by pretreatment of the host cells with BPDE before infection. The effect of pretreatment is similar to that of extending the holding time before replication: the first molecules begin to replicate earlier but the subsequent rate of recovery is unchanged. The induced mechanism may be either a limited stoichiometric repair process or a slow replicative bypass.

Animals↗
PubMed1982

Characterization of a temperature-sensitive DNA replication mutant of Staphylococcus aureus.

A temperature-sensitive DNA replication mutant of Staphylococcus aureus NCTC 8325 has been isolated and characterized. After transfer to the non-permissive-temperature (42 degrees C), DNA synthesis continued for 30 min and the mean DNA content increased by 56%. The amount of residual DNA synthesis was not reduced when the non-permissive temperature was raised, nor when chloramphenicol was added at the time of the temperature shift. During incubation at 42 degrees C, mutant bacteria accumulated the capacity to synthesize DNA after return to the permissive temperature (30 degrees C) in the presence of chloramphenicol. This capacity was lost when chloramphenicol was present at 42 degrees C. The properties of the mutant are consistent with a defect in the initiation of DNA replication at 42 degrees C.

Chloramphenicol↗