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At least 1,351 records · Page 75Linked to original sources

PubMed1993

Unusual properties of a replication-defective mutant SV40 large T antigen.

The C11A mutant of SV40 large T antigen is unable to support the replication of viral origin containing DNA (ori-DNA) in vivo or in vitro. The mutation within C11A at residue 522 (pro-->ser) is located within the presumptive ATPase region of T antigen. While C11A T antigen was previously reported to be defective in ATPase and DNA helicase activities, it was shown to be capable of binding specifically to DNA containing the viral replication origin. As the positions of many conditional mutations of SV40 T antigen are located within the ATPase domain we asked whether C11A might also exhibit temperature-sensitive defects. We found that several activities of C11A T antigen are conditionally defective. C11A T antigen was able to hydrolyze ATP, assemble into hexamers, and display ATP-dependent alterations in DNA binding and ori-DNA structure at 33 degrees but not 41 degrees. Wild-type T antigen did not exhibit temperature-sensitive defects in these activities. C11A T antigen was completely unable to unwind ori-DNA at either temperature. This defect in unwinding was trans-dominant; C11A T antigen inhibited ori-DNA unwinding by wild-type T antigen. These data show that a mutant displaying a nonconditional defective phenotype may contain a subset of relevant properties that are temperature sensitive.

Adenosine Triphosphatases↗

PubMed1992

Temporal expression of a membrane-associated protein putatively involved in repression of initiation of DNA replication in Bacillus subtilis.

A Bacillus subtilis membrane-associated protein that binds specifically to the origin region of DNA replication may act as an inhibitor of DNA replication (J. Laffan and W. Firshein, Proc. Natl. Acad. Sci. USA 85:7452-7456, 1988). This protein, originally estimated to be 64 kDa, had a slightly lower molecular size (57 kDa), as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis during these studies. The size difference may be due to processing that results in modification of the protein. The protein can be extracted from both cytosol and membrane fractions, and the amounts in these fractions vary during the developmental cycle of B. subtilis. A complex pattern of expression in which significant levels were detected in spores was revealed; levels decreased dramatically during germination and increased after the first round of DNA replication. The decrease during germination was due to protease activity, as demonstrated by the addition of protease inhibitors and radioactive-labeling chase experiments. During vegetative growth, the protein levels increased until stationary phase, after which there was another decrease during sporulation. The decrease during sporulation may be partially due to sequestering of the protein into forespores, since as the putative repressor protein decreased in the mother cell, it increased in the forespores. However, protease activity was also involved in the decrease in the mother cell. The changes in expression of this protein are consistent with its role as a repressor of initiation of DNA replication. Additional studies, including sequence analysis and further antibody analysis, show that this protein is not a subunit of the pyruvate dehydrogenase complex. This relationship had been a possibility based upon the results of others (H. Hemila, A. Pavla, L. Paulin, S. Arvidson, and I. Palva, J. Bacteriol. 172:5052-5063, 1990).

Acetyltransferases↗

PubMed2006

The Rep protein binding elements of the plasmid ColE2-P9 replication origin.

The plasmid ColE2-P9 (ColE2) origin (32bp) is specifically recognized by the plasmid-specified Rep protein that initiates DNA replication. The ColE2 origin is divided into at least three functional subregions (I, II, and III), and three sites (a, b, and c) found in subregions I and II play important roles in Rep protein binding. We performed SELEX experiments of plasmid ColE2 to determine the optimal sequences for specific binding of the Rep protein. From these experiments, we obtained a common 16-bp sequence (5'-TGAGACCANATAAGCC-3'), which corresponds to about one half of the minimal ColE2 origin and contains sites a and b. Gel mobility shift assays using single-point mutant origins and the Rep protein further indicated that high affinity sequence-specific recognition by the Rep protein requires sites a, b, and c, but that mutations in site c were less disruptive to this recognition than those in sites a and b.

Amino Acid Sequence↗

PubMed1977

Temporal sequence of events during the initiation process in Escherichia coli deoxyribonucleic acid replication: roles of the dnaA and dnaC gene products and ribonucleic acid polymerase.

Three thermosensitive deoxyribonucleic acid (DNA) initiation mutants of Escherichia coli exposed to the restrictive temperature for one to two generations were examined for the ability to reinitiate DNA replication after returning to the permissive temperature in the presence of rifampin, chloramphenicol, or nalidixic acid. Reinitiation in the dnaA mutant was inhibited by rifampin but not by chloramphenicol, whereas renitiation was not inhibited by rifampin but not by chloramphenicol, whereas reinitiation was not inhibited in two dnaC mutants by either rifampin or chloramphenicol. To observe the rifampin inhibition, the antibiotic must be added at least 10 min before return to the permissive temperature. The rifampin inhibition of reinitiation was not observed when a rifampin-resistant ribonucleic acid ((RNA) polymerase gene was introduced into the dnaA mutant, demonstrating that RNA polymerase synthesizes one or more RNA species required for the initation of DNA replication (origin-RNA). Reinitiation at 30 degrees C was not inhibited by streptolydigin in a stretolydigin-sensitive dnaA muntant. Incubation in the presence of nalidixic acid prevented subsequent reinitiation in the dnaC28 mutant but did not inhibit reinitiation in the dnaA5 muntant. These results demonstrate that the dnaA gene product acts before or during the synthesis of an origin-RNA, RNA polymerase synthesizes this origin RNA, and the dnaC gene product is involved in a step after this RNA synthesis event. Furthermore, these results suggest that the dnaC gene product is involved in the first deoxyribounucleotide polymerization event wheareas the dnaA gene product acts prior to this event. A model is presented describing the temporal sequence of events that occur during initiation of a round of DNA replication, based on results in this and the accompanying paper.

Aminoglycosides↗

PubMed2003

Adenovirus DNA replication.

Replication of the adenovirus genome is catalysed by adenovirus DNA polymerase in which the adenovirus preterminal protein acts as a protein primer. DNA polymerase and preterminal protein form a heterodimer which, in the presence of the cellular transcription factors NFI/CTFI and NFIII/Oct-1, binds to the origin of DNA replication. DNA replication is initiated by DNA polymerase mediated transfer of dCMP onto preterminal protein. Further DNA synthesis is catalysed by DNA polymerase in a strand displacement mechanism which also requires adenovirus DNA binding protein. Here, we discuss the role of individual proteins in this process as revealed by biochemical analysis, mutagenesis and molecular modelling.

Adenoviridae↗

PubMed1990

Characterization of DNA pattern in the site of permanent attachment to the nuclear matrix located in the vicinity of replication origin.

The permanent sites of DNA attachment to the nuclear matrix in the domain of chicken alpha-globin genes originally found in erythrocyte nuclei have also been shown to exist in sperm and cultured fibroblast cells. A primary structure of a 1.7 kb fragment located in 5'-upstream region of chicken alpha-globin gene domain and containing both replication origin and permanent nuclear matrix attachment site has been determined. It was found to possess homologies with papovaviral replication origins and contain short internal repeats and GC-rich motifs.

Animals↗

PubMed1995

A new set of useful cloning and expression vectors derived from pBlueScript.

A new set of cloning vectors derived from pBlueScript (Stratagene, La Jolla, CA, USA) is presented. The ampicillin-resistance-encoding gene (ApR) of pBlueScript has been replaced by genes encoding resistance to either kanamycin (KmR) or tetracycline (TcR). The origin of DNA replication (ori), conferring to pBlueScript a very high-copy-number (500-700 copies/chromosome), has been replaced by the pBR322 ori (15-20 copies/chromosome) or the P15A ori (10-12 copies/chromosome) [Sambrook et al.: Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989]. Therefore, eight new vectors with different drug selection markers and low, medium or high plasmid copy-number were created which are compatible with each other (ColE1 ori and P15A ori) and can be selected to replace one another. These vectors were further modified by the insertion of an expression cassette based on the promoter and AraC repressor/activator of the ara operon, which allows high-level expression, extremely tight regulation and very inexpensive induction. High-level expression of one or two genes within the same cell is demonstrated.

Ampicillin Resistance↗

PubMed2001

RBP1 recruits the mSIN3-histone deacetylase complex to the pocket of retinoblastoma tumor suppressor family proteins found in limited discrete regions of the nucleus at growth arrest.

Retinoblastoma (RB) tumor suppressor family pocket proteins induce cell cycle arrest by repressing transcription of E2F-regulated genes through both histone deacetylase (HDAC)-dependent and -independent mechanisms. In this study we have identified a stable complex that accounts for the recruitment of both repression activities to the pocket. One component of this complex is RBP1, a known pocket-binding protein that exhibits both HDAC-dependent and -independent repression functions. RB family proteins were shown to associate via the pocket with previously identified mSIN3-SAP30-HDAC complexes containing exclusively class I HDACs. Such enzymes do not interact directly with RB family proteins but rather utilize RBP1 to target the pocket. This mechanism was shown to account for the majority of RB-associated HDAC activity. We also show that in quiescent normal human cells this entire RBP1-mSIN3-SAP30-HDAC complex colocalizes with both RB family members and E2F4 in a limited number of discrete regions of the nucleus that in other studies have been shown to represent the initial origins of DNA replication following growth stimulation. These results suggest that RB family members, at least in part, drive exit from the cell cycle by recruitment of this HDAC complex via RBP1 to repress transcription from E2F-dependent promoters and possibly to alter chromatin structure at DNA origins.

Binding Sites↗

PubMed1993

Activation of eukaryotic transcriptional promoters by the bovine papillomavirus E1-replication factor.

It has been suggested that the bovine papillomavirus type 1 E1 replication factor may inhibit E2-conditional gene expression from viral promoter P89. To study the possible role of the E1 protein in gene expression, HeLa cells were transfected with E2-conditional or E2-independent reporter plasmids and with vectors expressing the E1 and E2 open reading frames. The data show that: (i) replication factor E1 stimulates gene expression from a variety of eucaryotic transcriptional promoters; (ii) activation of gene expression by E1 also occurs in the absence of viral activator E2-TA; (iii) expression of a recombinant plasmid containing the human papillomavirus type 18 origin of DNA replication is stimulated, but not repressed, by E1, and (iv) E1-dependent activation of gene expression does not reflect the amplification of transfected plasmids.

Bovine papillomavirus 1↗

PubMed1994

Mammalian artificial chromosomes: a new tool for gene therapy.

Effective therapy by in vivo delivery of DNA requires efficient delivery, long-term maintenance of the DNA that is delivered and physiological levels of expression of the therapeutic gene. Full levels of physiologically controlled expression can be obtained after transfer of intact genes on fragments of DNA hundreds of kilobases in size, as has been demonstrated by the transfer of yeast artificial chromosomes into transgenic mice. Long-term maintenance of input DNA could be achieved if the DNA carried replication origins, a centromere and telomeres to allow maintenance and segregation in mammalian cells, and there has been recent progress towards cloning these elements. These features could be combined as a mammalian artificial chromosome which would confer full levels of controlled expression as well as being maintained in any cell into which it was introduced. Methods which would allow delivery of such large fragments of DNA include liposomes and receptor-mediated uptake, both of which have been shown to work in vivo, making such large constructs potentially applicable for use in gene therapy.

Animals↗

PubMedDate not supplied

[Spatial organization of replicons in the eukaryotic nucleus: attachment of replication initiation regions to the nuclear skeleton].

The DNA fraction enriched in replication origins was isolated from chicken erythroblastosis cells with the aid of nascent DNA strand extrusion method. In renaturation experiments, the sequence specificity of this DNA fraction (oriDNA) was compared to the sequence specificity of the nuclear matrix DNA. The oriDNA was shown to comprise a specific subset of unique genes all of which being represented also in the nuclear matrix DNA. The same unique DNA sequences were found to be depleted from the oriDNA and from the nuclear matrix DNA. A conclusion has been drawn that replication origins are located at the basements of DNA loops. The putative positions of replication origins within the domain of chicken alpha globin genes were mapped by hybridization experiments and were found to coincide with the positions of stable (not depending on the transcriptional state of globin genes) sites of DNA attachment to the nuclear skeleton.

Animals↗

PubMed1995

Multiple regions within EBNA1 can link DNAs.

Epstein-Barr virus nuclear antigen 1 (EBNA1) can bind specifically to two clusters of sites within the Epstein-Barr virus plasmid origin of DNA replication (oriP). EBNA1 activates DNA replication mediated by oriP and can also activate transcription and retain DNA in cells when bound site specifically. EBNA1 bound to oriP physically links the two clusters of EBNA1-binding sites, resulting in loop formation by the intervening DNA. To elucidate the contribution of DNA linking by EBNA1 to its biological activities, we identified regions within it that can independently link DNAs to which they are bound. An electrophoretic mobility shift assay was used to detect this activity. Proteins which link DNA aggregate that DNA into large lattices. Proteins which cannot link DNA but still bind to DNA retard the mobility of that DNA but do not cause it to form lattices. Amino-terminal truncations were used to map the amino-terminal limit of a minimal DNA-linking domain approximately to amino acid 372 of EBNA1. To map the carboxy-terminal limit of this minimal domain, fusion proteins containing the DNA-binding domain of GAL4 and fragments of EBNA1 were generated and studied. This approach identified the carboxy-terminal limit of this minimal domain to be approximately amino acid 391 and verified its amino-terminal limit. Internal deletions within a truncated EBNA1 derivative verified the importance of this region. Two additional fragments of EBNA1, each of which independently conferred DNA-linking activity on the domain of GAL4 which binds DNA, were identified within amino acids 54 to 89 and amino acids 331 to 361. Therefore, EBNA1 contains at least three regions that can act independently to link DNAs and that may act in concert within intact EBNA1.

Amino Acid Sequence↗

PubMed1996

Solution structure of the origin DNA-binding domain of SV40 T-antigen.

The structure of the domain from simian virus 40 (SV40) large T-antigen that binds to the SV40 origin of DNA replication (T-ag-OBD131-260) has been determined by nuclear magnetic resonance spectroscopy. The overall fold, consisting of a central five-stranded antiparallel beta-sheet flanked by two alpha-helices on one side and one alpha-helix and one 3(10)-helix on the other, is a new one. Previous mutational analyses have identified two elements, termed A (approximately 152-155) and B2 (203-207), as essential for origin-specific recognition. These elements form two closely juxtaposed loops that define a continuous surface on the protein. The addition of a duplex oligonucleotide containing the origin recognition pentanucleotide GAGGC induces chemical shift changes and slows amide proton exchange in resonances from this region, indicating that this surface directly contacts the DNA.

Amino Acid Sequence↗

PubMed1981

Construction and characterization of recombinant plasmid DNAs containing sequences of the origin of bacteriophage phi X174 DNA replication.

The synthetic DNA fragment (formula, see text) (corresponding to nucleotides 4299-4314 of the phi X DNA sequence) was cloned into either the AmpR gene or the KmR gene of plasmid pACYC 177. The DNA sequence of the KmR gene around the insertion site was determined by nucleotide sequence analysis of the pACYC 177 FnudII restriction DNA fragment N6 (345 b.p.). Of five selected plasmid DNAs, which contained inserted DNA sequences in the antibiotic resistance genes, the nucleotide sequences at and around these insertions were determined. Two recombinant plasmids (pFH 704 and pFH 614) contain the hexadecamer sequence in tandem (tail-to-tail and tail-to-head). In the recombinant plasmids pFH 812, pFH 903 and pFH 807 the DNA sequence homology with the phi X origin region was 14 (No. 4300-4313), 16 (No. 4299-4314) and 20 nucleotides (No. 4299-4318), respectively. None of the supercoiled recombinant plasmid DNAs is nicked upon incubation with phi X gene A protein. Moreover, the recombinant plasmid RFI DNAs cannot act as substitutes for phi X RFI DNA in the in vitro (+) strand synthesizing system. It has been shown earlier that single-stranded DNA, which contains the decamer sequence CAACTTGATA is efficiently nicked by the phi X gene A protein. The present results indicate that for nicking of double-stranded supercoiled DNA nucleotide sequence homology with the phi X origin region of more than 20 nucleotides is required. These results suggest a model for initiation of phi X RF DNA replication, which involves the presence of the recognition sequence CAACTTGATA of the phi X gene A protein as well as a second specific nucleotide sequence which is required for the binding of the phi X gene A protein. This binding causes local unwinding of the DNA double helix and exposure of the recognition sequence in a single-stranded form, which then can be nicked by phi X gene A protein.

Bacteriophage phi X 174↗

PubMed2001

MCM2-7 proteins are essential components of prereplicative complexes that accumulate cooperatively in the nucleus during G1-phase and are required to establish, but not maintain, the S-phase checkpoint.

A prereplicative complex (pre-RC) of proteins is assembled at budding yeast origins of DNA replication during the G1-phase of the cell cycle, as shown by genomic footprinting. The proteins responsible for this prereplicative footprint have yet to be identified but are likely to be involved in the earliest stages of the initiation step of chromosome replication. Here we show that MCM2-7 proteins are essential for both the formation and maintenance of the pre-RC footprint at the origin ARS305. It is likely that pre-RCs contain heteromeric complexes of MCM2-7 proteins, since degradation of Mcm2, 3, 6, or 7 during G1-phase, after pre-RC formation, causes loss of Mcm4 from the nucleus. It has been suggested that pre-RCs on unreplicated chromatin may generate a checkpoint signal that inhibits premature mitosis during S-phase. We show that, although mitosis does indeed occur in the absence of replication if MCM proteins are degraded during G1-phase, anaphase is prevented if MCMs are degraded during S-phase. Our data indicate that pre-RCs do not play a direct role in checkpoint control during chromosome replication.

Cell Cycle Proteins↗

PubMed1993

The hr5 transcriptional enhancer stimulates early expression from the Autographa californica nuclear polyhedrosis virus genome but is not required for virus replication.

Autographa californica nuclear polyhedrosis virus (AcMNPV) contains five homologous regions (hr1 through hr5) interspersed throughout its genome. Analysis of plasmid transfections indicates that the hrs function as transcriptional enhancers and possible origins of viral DNA replication. The role of these repetitive elements in regulating expression from the AcMNPV genome was examined by constructing a series of recombinant viruses that tested the effect of hr5 on expression of the adjacent p35 gene (p35). When embedded within the viral genome, hr5 stimulated transcription from the early p35 promoter in a position- and orientation-independent manner. Moreover, hr5 and the upstream activating region of p35 were functionally interchangeable. A 28-bp imperfect palindrome, repeated six times within hr5, was the minimal sequence required for p35 promoter activation. hr5 also stimulated another early AcMNPV promoter but not a late promoter or a host-derived promoter, suggesting that enhancement is promoter specific during infection. To investigate its role during AcMNPV replication, hr5 was deleted from its normal position within the viral genome. The resulting hr5 mutants exhibited no apparent defects in replication, as judged by production of budded virus and levels of very late gene expression, even though steady-state levels of p35 RNA were reduced. These results indicated for the first time that hr5 functions as a transcriptional enhancer within the viral genome. However, the element is not required for AcMNPV replication in cultured cells. Thus, loss of one of five possible origins of DNA replication is not deleterious to viral growth. Since p26 was removed from the hr5 deletion mutants, this gene is also nonessential for viral replication.

Animals↗

PubMed2005

Involvement of human MCM8 in prereplication complex assembly by recruiting hcdc6 to chromatin.

The MCM2-MCM7 complex is an essential component of the prereplication complex (pre-RC), which is recruited by the cdc6 and cdt1 proteins to origins of DNA replication during G(1) phase. Here, we report that the accumulation on chromatin of another member of the MCM protein family, human MCM8 (hMCM8), occurs during early G(1) phase, before the hMCM2-hMCM7 complex binds. hMCM8 interacts in vivo with two components of the pre-RC, namely, hcdc6 and hORC2. Depletion of endogenous hMCM8 protein by RNA interference leads to a delay of entry into S phase, suggesting a role for hMCM8 in G(1) progression. Furthermore, down-regulation of hMCM8 also leads to a reduced loading of hcdc6 and the hMCM2-hMCM7 complex on chromatin. These results suggest that hMCM8 is a crucial component of the pre-RC and that the interaction between hMCM8 and hcdc6 is required for pre-RC assembly.

Cell Cycle Proteins↗

PubMed1987

Purification and characterization of the gene 1.2 protein of bacteriophage T7.

Gene 1.2 of bacteriophage T7, located near the primary origin of DNA replication at position 15.37 on the T7 chromosome, encodes a 10,059-dalton protein that is essential for growth on Escherichia coli optA1 strains (Saito, H., and Richardson, C. C. (1981) J. Virol. 37, 343-351). In the absence of the T7 1.2 and E. coli optA gene products, the degradation of E. coli DNA proceeds normally, and T7 DNA synthesis is initiated at the primary origin. However, T7 DNA synthesis ceases prematurely and the newly synthesized DNA is degraded; no viable phage particles are released. The gene 1.2 protein has been purified to apparent homogeneity from cells in which the cloned 1.2 gene is overexpressed. Purification of the [35S] methionine-labeled protein was followed by monitoring the radioactivity of the protein and by gel electrophoresis. The purified protein has been identified as the product of gene 1.2 on the basis of molecular weight and partial amino acid sequence. We have found that extracts of E. coli optA1 cells infected with T7 gene 1.2 mutants are defective in packaging exogenous T7 DNA when such extracts are prepared late in infection. Purified gene 1.2 protein restores packaging activity to these defective extracts, thus providing a biological assay for gene 1.2 protein. No specific enzymatic activity has been found associated with the purified gene 1.2 protein.

Amino Acid Sequence↗