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At least 1,081 records · Page 60Linked to original sources
PubMed1986

Interaction between gene II protein and the DNA replication origin of bacteriophage f1.

The origin of DNA replication of the filamentous bacteriophage f1 binds its initiator protein (gene II protein) in vitro to form a complex that can be trapped on nitrocellulose filters. The binding occurs with both superhelical form DNA and linear DNA fragments. A number of defective mutants of the origin were tested for the ability to bind gene II protein. The region of DNA required for the binding is around a second palindrome downstream from the palindrome that contains the DNA replication initiation site. It overlaps, but is not identical to, the region required for the nicking reaction by the protein. The nicking site itself was dispensable for the binding. In vivo, a number of defective deletion mutants of the origin, when in a plasmid, inhibited growth of superinfecting phage if the intracellular level of gene II protein was low. In addition, these defective origins inhibited the activity of the functional phage origin located on the same replicon. The domain of the DNA sequence required for inhibition in vivo was consistent with that for the binding in vitro.

Bacteriophages↗
PubMed1987

A novel endoribonuclease cleaves at a priming site of mouse mitochondrial DNA replication.

Priming at the mouse mitochondrial origin of heavy-strand DNA replication is effected by transcripts from the light-strand promoter. The transition from RNA synthesis to DNA synthesis occurs at specific locations between 75 and 165 nucleotides downstream from the transcriptional initiation site. We have identified and partially purified an endonucleolytic activity that cleaves RNA accurately near one of these transition sites; this finding implies a role of specific RNA processing in DNA replication. Cleavage products possess 5'-phosphoryl and 3'-hydroxyl termini. Heterologous assays using mouse or human mitochondrial endoribonuclease with substrates containing the sequences of the human or mouse mitochondrial origins of heavy-strand DNA replication suggest that selection of the cleavage site is guided by sequences adjacent to the actual position of cleavage.

Animals↗
PubMed2004

Escherichia coli DNA polymerase I (Klenow fragment) uses a hydrogen-bonding fork from Arg668 to the primer terminus and incoming deoxynucleotide triphosphate to catalyze DNA replication.

Interactions between the minor groove of the DNA and DNA polymerases appear to play a major role in the catalysis and fidelity of DNA replication. In particular, Arg668 of Escherichia coli DNA polymerase I (Klenow fragment) makes a critical contact with the N-3-position of guanine at the primer terminus. We investigated the interaction between Arg668 and the ring oxygen of the incoming deoxynucleotide triphosphate (dNTP) using a combination of site-specific mutagenesis of the protein and atomic substitution of the DNA and dNTP. Hydrogen bonds from Arg668 were probed with the site-specific mutant R668A. Hydrogen bonds from the DNA were probed with oligodeoxynucleotides containing either guanine or 3-deazaguanine (3DG) at the primer terminus. Hydrogen bonds from the incoming dNTP were probed with (1 'R,3 'R,4 'R)-1-[3-hydroxy-4-(triphosphorylmethyl)cyclopent-1-yl]uracil (dcUTP), an analog of dUTP in which the ring oxygen of the deoxyribose moiety was replaced by a methylene group. We found that the pre-steady-state parameter kpol was decreased 1,600 to 2,000-fold with each of the single substitutions. When the substitutions were combined, there was no additional decrease (R668A and 3DG), a 5-fold decrease (3DG and dcUTP), and a 50-fold decrease (R668A and dcUTP) in kpol. These results are consistent with a hydrogen-bonding fork from Arg668 to the primer terminus and incoming dNTP. These interactions may play an important role in fidelity as well as catalysis of DNA replication.

Arginine↗
PubMed1990

Structural analysis of replicating DNA following exposure to cytotoxic drugs: implications for current models of DNA synthesis in mammalian cells.

The polynucleotide length of single-stranded regions in double-stranded DNA may be determined by caffeine gradient elution from benzoylated DEAE-cellulose. On the basis of this principle, analysis has been made of sheared, deproteinized DNA isolated from synchronized lymphoblastoid cells. Two classes of single-stranded regions were detected. A minor fraction of replicating DNA contained single-stranded regions of 200 nucleotides, whilst the major structural discontinuity involved single-stranded regions of 1-4 kilobases. Newly incorporated [3H]thymidine was principally associated with the latter. Using a 'pulse-chase' protocol, the effect of certain cytotoxic drugs (and related compounds) on the proportion of replicating DNA exhibiting single-stranded character was assessed. The effects were variable. The proportion was increased by hydroxyurea and 3-aminobenzamide, but decreased by inhibitors of DNA polymerase and, to a greater extent, by inhibitors of topoisomerase. Caffeine gradient elution associated drug-induced changes with the radiolabelling of long single-stranded regions. The results are consistent with models of DNA replication involving DNA polymerization remote from replicating forks.

Antineoplastic Agents↗
PubMed2002

An ACF1-ISWI chromatin-remodeling complex is required for DNA replication through heterochromatin.

The mechanism by which the eukaryotic DNA-replication machinery penetrates condensed chromatin structures to replicate the underlying DNA is poorly understood. Here we provide evidence that an ACF1-ISWI chromatin-remodeling complex is required for replication through heterochromatin in mammalian cells. ACF1 (ATP-utilizing chromatin assembly and remodeling factor 1) and an ISWI isoform, SNF2H (sucrose nonfermenting-2 homolog), become specifically enriched in replicating pericentromeric heterochromatin. RNAi-mediated depletion of ACF1 specifically impairs the replication of pericentromeric heterochromatin. Accordingly, depletion of ACF1 causes a delay in cell-cycle progression through the late stages of S phase. In vivo depletion of SNF2H slows the progression of DNA replication throughout S phase, indicating a functional overlap with ACF1. Decondensing the heterochromatin with 5-aza-2-deoxycytidine reverses the effects of ACF1 and SNF2H depletion. Expression of an ACF1 mutant that cannot interact with SNF2H also interferes with replication of condensed chromatin. Our data suggest that an ACF1-SNF2H complex is part of a dedicated mechanism that enables DNA replication through highly condensed regions of chromatin.

3T3 Cells↗
PubMed2004

Initiation of DNA replication in Xenopus egg extracts.

In the last decade, extraordinary advances in our understanding of the initiation step of eukaryotic DNA replication have been achieved. Many factors required for replication initiation have been identified, and an elegant model to explain how DNA replication is restricted to a single round per cell cycle has emerged. Of the many experimental approaches used to study DNA replication, egg extracts from Xenopus laevis are among the most powerful, since they recapitulate a complete round of cell-cycle regulated chromosomal DNA replication in vitro. In this review, we discuss current models for how DNA replication is initiated and regulated in Xenopus eggs, and we highlight similarities and differences seen between this and the other most common experimental organisms, yeast and humans.

Animals↗
PubMed2001

Dissection of the ATP-driven reaction cycle of the bacteriophage T4 DNA replication processivity clamp loading system.

Processive DNA replication requires the loading of a multisubunit ring-shaped protein complex, known as a sliding or processivity clamp, onto the primer-template (p/t) DNA. This clamp then binds to the replication polymerase to form a processive polymerase holoenzyme. The processivity of the holoenzyme derives from the topological properties of the clamp, which encircles the DNA without actually binding to it. Multisubunit complexes known as clamp-loaders utilize ATP to drive the placement of this ring around the DNA. To further understand the role of ATP binding and hydrolysis in driving clamp-loading in the DNA replication system of bacteriophage T4, we report the results of a series of presteady-state and steady-state kinetic ATPase experiments involving the various components of the reconstituted system. The results obtained are consistent with a mechanism in which a slow step, which involves the binary ATP-bound clamp-clamp loader complex, activates this complex and permits p/t DNA to bind and stimulate ATP hydrolysis. ATP hydrolysis itself, as well as the subsequent (after clamp-loading) dissociation of the clamp-loader and the slippage of the loaded clamp from the p/t DNA construct, are shown to be fast steps. A second slow step occurs after ATP hydrolysis. This step involves the dissociated clamp loader complex and may reflect ADP release. Only one molecule of ATP is hydrolyzed per clamp-loading event. Rate constants for each step, and an overall reaction mechanism for the T4 clamp-loading system, are derived from these data and from other results in the literature. The principles that emerge fit into a general framework that can apply to many biological processes involving ATP-driven reaction cycles.

Adenosine Triphosphatases↗
PubMed1999

Human topoisomerase I promotes initiation of simian virus 40 DNA replication in vitro.

Addition of purified human topoisomerase I (topo I) to simian virus 40 T antigen-driven in vitro DNA replication reactions performed with topo I-deficient extracts results in a greater than 10-fold stimulation of completed molecules as well as a more than 3-fold enhancement of overall DNA replication. To further characterize this stimulation, we first demonstrate that bovine topo I but not Escherichia coli topo I can also enhance DNA replication. By using several human topo I mutants, we show that a catalytically active form of topo I is required. To delineate whether topo I influences the initiation or the elongation step of replication, we performed delayed pulse, pulse-chase, and delayed pulse-chase experiments. The results illustrate that topo I cannot promote the completion of partially replicated molecules but is needed from the beginning of the reaction to initiate replication. Competitive inhibition experiments with the topo I binding T antigen fragment 1-246T and a catalytically inactive topo I mutant suggest that part of topo I's stimulation of replication is mediated through a direct interaction with T antigen. Collectively, our data indicate that topo I enhances the synthesis of fully replicated DNA molecules by forming essential interactions with T antigen and stimulating initiation.

Animals↗
PubMed2003

Mechanical link between cohesion establishment and DNA replication: Ctf7p/Eco1p, a cohesion establishment factor, associates with three different replication factor C complexes.

CTF7/ECO1 is an essential yeast gene required for the establishment of sister chromatid cohesion. The findings that CTF7/ECO1, POL30 (PCNA), and CHL12/CTF18 (a replication factor C [RFC] homolog) genetically interact provided the first evidence that the processes of cohesion establishment and DNA replication are intimately coupled-a link now confirmed by other studies. To date, however, it is unknown how Ctf7p/Eco1p function is coupled to DNA replication or whether Ctf7p/Eco1p physically associates with any components of the DNA replication machinery. Here, we report that Ctf7p/Eco1p associates with proteins that perform partially redundant functions in DNA replication. Chl12p/Ctf18p combines with Rfc2p to Rfc5p to form one of three independent RFC complexes. By chromatographic methods, Ctf7p/Eco1p was found to associate with Chl12/Ctf18p and with Rfc2p, Rfc3p, Rfc4p, and Rfc5p. The association between Ctf7p/Eco1p and this RFC complex is biologically relevant in that (i) Ctf7p/Eco1p cosediments with Chl12p/Ctf18p in vivo and (ii) rfc5-1 mutant cells exhibit precocious sister separation. Previous studies revealed that Rfc1p or Rad24p associates with Rfc2p to Rfc5p to form two other RFC complexes independent of Ctf18p-RFC complexes. These Rfc1p-RFC and Rad24p-RFC complexes function in DNA replication or repair and DNA damage checkpoint pathways. Importantly, Ctf7p/Eco1p also associates with Rfc1p and Rad24p, suggesting that these RFC complexes also play critical roles in cohesion establishment. The associations between Ctf7p/Eco1p and RFC subunits provide novel evidence regarding the physical linkage between cohesion establishment and DNA replication. Furthermore, the association of Ctf7p/Eco1p with each of three RFC complexes supplies new insights into the functional redundancy of RFC complexes in cohesion establishment.

Acetyltransferases↗
PubMed2006

Nucleolar protein upstream binding factor is sequestered into adenovirus DNA replication centres during infection without affecting RNA polymerase I location or ablating rRNA synthesis.

When human adenovirus infects human cells there is disruption of rRNA biogenesis. This report examines the effect of adenovirus infection on the nucleolar protein, upstream binding factor (UBF) which plays a major role in regulating rRNA synthesis. We determined that early after infection, UBF associates with the replication of viral DNA, preferentially associating with the ends of the linear viral genome, and that addition of anti-UBF serum to in vitro replication assays markedly reduced viral DNA replication. Regions of UBF important to these observations are also established. Interestingly, sequestering the majority of UBF from the nucleolus did not lead to the ablation of rRNA synthesis or the sequestration of RNA pol I. In infected cells the bulk of RNA synthesis was RNA pol I associated and distinct from the location of most of the detectable UBF. We propose that UBF plays a role in viral DNA replication, further strengthening the role of nucleolar antigens in the adenovirus life cycle.

Adenoviridae↗
PubMed1994

The role of a pentanucleotide repeat sequence, AGGGAAGGGA, in the regulation of JC virus DNA replication.

The human polyomavirus JCV differs from other papovaviruses in its narrow host range and tissue tropism for human glial cells. It is believed that the cell-specific tropism of JCV to glial cells rests, at least in part, in transcription of the viral early gene that encodes the large tumor antigen (T-antigen). The secondary stage, however, which restricts the replication cycle of JCV to primate cells, is controlled at the level of viral DNA replication. In this study, we demonstrate that a cis-acting transcription regulatory element encompassing the pentanucleotide repeat sequence AGGGAAGGGA (penta), which is located in close proximity to the origin of DNA replication, plays an important role in the replication of viral DNA mediated by the JCV T-antigen, but not T-antigen derived from SV40. Analysis of DNA structure by diethyl pyrocarbonate (DEPC) has revealed that mutations within the penta which affect DNA replication also alter the structure of the neighboring A+T-rich region. These results suggest that, in addition to the regulatory role in viral gene expression, the penta may function as a DNA structural element which is important for JCV DNA replication mediated by the JCV T-antigen.

Animals↗
PubMed1988

[Preferential modification of replicating DNA by benz(a)pyrene)].

The nuclei of cells from regenerating rat liver were incubated with benzo(a)pyrene and the concentrations of the metabolites that covalently bound to DNA of different nuclear fractions were compared. It appeared that DNA associated with nuclear matrix (containing replicating DNA) is modified most intensively. The synchronized mouse embryo cells were incubated with benzo(a)pyrene during S phase and the levels of modifications in short and long single-stranded DNA fragments were compared. It has been observed that replicating DNA is represented in short fragments. These short DNA fragments were found to be modified by benzo(a)pyrene 4-9 times more intensively than total DNA. The possible mechanisms of both the increase in the number of DNA modifications in proliferating cells and the reason for the enhancement of carcinogenic effect on dividing cells are being discussed.

Animals↗
PubMed2005

Arabidopsis RPA2: a genetic link among transcriptional gene silencing, DNA repair, and DNA replication.

Transcriptional gene silencing (TGS) controls the expression of transposable elements and of endogenous genes containing promoter repeats, and it is associated with increased DNA methylation. TGS-deficient mutants impaired in siRNA accumulation and/or chromatin modification (ago4, bru1, cmt3, dcl3, ddm1, drd1, drm2, fas1, fas2, hda6, hog1, met1, mom1, nrpd1a, nrpd1b, nrpd2a, rdr2, suvh2, and suvh4) have been identified, but not all mutations affect the same subset of targets. Here, we identify Arabidopsis RPA2, a conserved protein with DNA replication and DNA repair motifs, as a novel TGS component that is dispensable for endogenous small RNA accumulation. bru1, cmt3, ddm1, fas1, fas2, hda6, hog1, met1, mom1, and rpa2 mutants are impaired in TGS of dispersed Athila/TSI retrotransposons and of the transgene repeat locus L5, but unlike bru1, cmt3, ddm1, fas1, fas2, hda6, hog1, and met1, the rpa2 and mom1 mutants do not affect the accumulation of 5S-derived siRNAs. Like BRU1, FAS1, FAS2, and MOM1, RPA2 is dispensable for DNA methylation, and rpa2, bru1, fas1, and fas2, but not mom1, mutants are hypersensitive to the DNA damage agent MMS. These results suggest a coordination of the TGS machinery with DNA replication, repair, or recombination machinery at some loci, and they emphasize the diversification of the TGS pathway.

Arabidopsis↗
PubMed1993

Superhelical path of the DNA in the nucleoprotein complex that activates the initiation of phage phi 29 DNA replication.

Initiation of bacteriophage phi 29 DNA replication is activated by protein p6, a viral double-stranded DNA-binding protein that forms a nucleoprotein complex at the viral replication origins. This complex consists of a DNA right-handed superhelix wrapped around a multimeric protein p6 core with protein p6 dimers regularly bound every 24 base-pairs (bp). In this paper, we have constructed a concatemer formed by direct repeats of a 24 bp sequence previously proposed to act as a signal for protein p6 binding at a phi 29 replication origin. DNase I footprinting shows that protein p6 binds to the concatemer in a similar way to the phi 29 DNA replication origins but with higher affinity, indicating that the 24 bp sequence is a recognition signal for protein p6. Furthermore, the concatemer was cloned in a plasmid and, by electron microscopy, it was shown to be the highest-affinity protein p6 binding region present in the plasmid. Based on these observations, the linking number change restrained by protein p6 has been measured in a series of plasmids containing concatemers with different numbers of 24 bp repeats; from the values obtained the linking number change restrained by a single protein p6 dimer has been estimated (delta Lkd = 0.1). In addition, when protein p6-DNA complexes fixed with glutaraldehyde were analysed by electron microscopy, it was observed that protein p6 compacts 4.2-fold the length of naked DNA. These data, together with the previously known value of the surface-related DNA helical repeat in the complex (12 bp), completely define the superhelical path of the DNA in the complex: one superhelical turn approximately involves 63 bp and 2.6 protein p6 dimers, and the DNA superhelix has a diameter of 6.6 nm and a slope of 14 degrees. The data obtained also indicate that the DNA in the protein p6-DNA complex is undertwisted (11.5 bp/turn) and strongly bent (66 degrees/12 bp). These DNA conformational changes might contribute to the activation of phi 29 DNA initiation of replication by protein p6.

Bacillus Phages↗
PubMed2003

ATPase/helicase motif mutants of Escherichia coli PriA protein essential for recombination-dependent DNA replication.

BACKGROUND: PriA protein, a DEXH-type helicase with C2C2 zinc-finger motifs, plays essential roles in RecA-dependent modes of Escherichia coli chromosomal DNA replication, namely inducible and constitutive stable DNA replication (iSDR and cSDR respectively, which may be initiated from a D-loop or R-loop structure), and in repair of double-stranded DNA breaks generated by various genotoxic agents or spontaneously during the course of DNA replication. However, the roles of ATPase/DNA helicase activities in functions of PriA are not well understood. RESULTS: We have generated and characterized mutants of PriA protein carrying amino acid substitutions in its conserved ATPase/DNA helicase motifs, namely the Walker A, B and QXXGRXGR motifs. All these mutants were deficient in ATP hydrolysis and DNA helicase activities, but showed wild-type levels of D-loop DNA binding, except for the Walker B mutant which showed reduced DNA binding activity, suggesting that the helicase motifs are not directly involved in the DNA binding activity of PriA protein. They also rescued the low viability and UV-sensitivity of priA null cells. However, they did not rescue iSDR or cSDR-alternative modes of chromosomal DNA replication of the E. coli genome dependent on recombination functions-to the full extent. CONCLUSIONS: ATPase/DNA helicase activities of PriA protein are required for full-level DNA synthesis in recombination-dependent modes of DNA replication in E. coli.

Adenosine Triphosphatases↗
PubMed2006

Differential requirement of DNA replication factors for subtelomeric ARS consensus sequence protosilencers in Saccharomyces cerevisiae.

The establishment of silent chromatin requires passage through S-phase, but not DNA replication per se. Nevertheless, many proteins that affect silencing are bona fide DNA replication factors. It is not clear if mutations in these replication factors affect silencing directly or indirectly via deregulation of S-phase or DNA replication. Consequently, the relationship between DNA replication and silencing remains an issue of debate. Here we analyze the effect of mutations in DNA replication factors (mcm5-461, mcm5-1, orc2-1, orc5-1, cdc45-1, cdc6-1, and cdc7-1) on the silencing of a group of reporter constructs, which contain different combinations of "natural" subtelomeric elements. We show that the mcm5-461, mcm5-1, and orc2-1 mutations affect silencing through subtelomeric ARS consensus sequences (ACS), while cdc6-1 affects silencing independently of ACS. orc5-1, cdc45-1, and cdc7-1 affect silencing through ACS, but also show ACS-independent effects. We also demonstrate that isolated nontelomeric ACS do not recapitulate the same effects when inserted in the telomere. We propose a model that defines the modes of action of MCM5 and CDC6 in silencing.

Cell Cycle Proteins↗
PubMed1985

Accumulation of 10-kilobase DNA replication intermediates in cells treated with 3-aminobenzamide.

During eukaryotic DNA synthesis there is formation of, in addition to Okazaki fragments, discrete 10-kilobase (kb) DNA replication intermediates. We have investigated the ligation of 10-kb DNA replication intermediates to high molecular weight DNA, using the drug 3-aminobenzamide, an inhibitor of poly(ADP-ribose) synthetase. In human melanoma cells treated with this inhibitor, there is an accumulation of 10-kb DNA. In contrast, in cells treated with aphidicolin, which inhibits DNA polymerase alpha, there is continued ligation of 10-kb DNA to high molecular weight DNA. Furthermore, using sequential treatment with aphidicolin and 3-aminobenzamide, one can observe the conversion of radiolabeled Okazaki fragments into 10-kb intermediates. The 10-kb DNA pieces are, however, not ligated to high molecular weight DNA in the presence of 3-aminobenzamide. Our results imply that functioning poly(ADP-ribose) synthetase is necessary for the ligation process.

Aphidicolin↗