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PubMed1980

The role of gene A protein and E. coli rep protein in the replication of phi X 174 replicative form DNA.

The gene A protein of bacteriophage phi X 174 initiates replication of super-twisted RFI DNA by cleaving the viral (+) strand at the origin of replication and binding to the 5' end. Upon addition of E. coli rep protein (single-stranded DNA dependent ATPase), E. coli single-stranded DNA binding protein and ATP, complete unwinding of the two strands occurs. Electron microscopic analyses of intermediates in the reaction reveal that the unwinding occurs by movement of the 5' end into the duplex, displacing the viral strand in the form of a single-stranded loop. Since unwinding will not occur in the absence of either gene A protein or rep protein, it is presumed that the rep protein interacts to form a complex with the bound gene A protein. Single-stranded DNA binding protein facilitates the unwinding by binding to the exposed single-stranded DNA. Further addition of the four deoxyribotriphosphates and DNA polymerase III holoenzyme to the reaction results in synthesis of viral (+) single-stranded circles in amounts exceeding that of the input template. A model describing the role of gene A protein and rep protein in duplex DNA replication is presented and other properties of gene A protein discussed.

Bacterial Proteins↗

PubMed1974

Replication process of the parvovirus H-1. II. Isolation and characterization of H-1 replicative form DNA.

The Parvovirus H-1 replicates autonomously in hamster embryo cells. A DNA synthetic event, called HA-DNA synthesis, upon which subsequent viral RNA and viral hemagglutinin synthesis is dependent, is initiated in late S phase of the infected cell (18). It was postulated that HA-DNA represents parental viral replicative form DNA (RF DNA). This study describes the isolation and characterization of H-1 RF DNA as part of the continuing study of the mechanisms and control of DNA replication in the eukaryotic cell. The H-1 RF DNA is a linear duplex molecule containing the viral strand and its complement. The complementary strands of the RF DNA have been separated by equilibrium density gradient centrifugation. The RF DNA has a buoyant density of 1.705 in neutral CsCl and an estimated guanine plus cytosine (GC) content of 45.9%. It has a sedimentation coefficient of 17S. The calculated molecular weight of 3.7 x 10(6) is twice that of the single-stranded virion DNA. H-1 virions contain DNA that is homogeneous and free of complementary strands.

Animals↗

PubMed1982

Replication of mycoplasma virus L51. VII. Effect of chloramphenicol on the synthesis of DNA replicative intermediates.

Chloramphenicol affects several steps in the DNA replication of mycoplasma virus L51, a noncytocidal, naked, bullet-shaped virion containing circular single-stranded (SS) DNA of 1.5 X 10(6) daltons (4.5 kilobases). In the presence of chloramphenicol, adsorption was normal and parental SS DNA was converted to double-stranded replicative forms (RF), but subsequent RF leads to RF replication was inhibited. Chloramphenicol added late in infection, when most viral nascent DNA is in progeny SS molecules, inhibited SS synthesis, but nascent RF molecules were formed. However, a chase experiment showed that these RF molecules could not be converted to SS DNA. Therefore, viral RF molecules made in the presence of chloramphenicol are not functional as SS DNA precursors.

Acholeplasma laidlawii↗

PubMed1998

The replication protein A binding site in simian virus 40 (SV40) T antigen and its role in the initial steps of SV40 DNA replication.

Physical interactions of simian virus 40 (SV40) large tumor (T) antigen with cellular DNA polymerase alpha-primase (Pol/Prim) and replication protein A (RPA) appear to be responsible for multiple functional interactions among these proteins that are required for initiation of viral DNA replication at the origin, as well as during lagging-strand synthesis. In this study, we mapped an RPA binding site in T antigen (residues 164 to 249) that is embedded within the DNA binding domain of T antigen. Two monoclonal antibodies whose epitopes map within this region specifically interfered with RPA binding to T antigen but did not affect T-antigen binding to origin DNA or Pol/Prim, ATPase, or DNA helicase activity and had only a modest effect on origin DNA unwinding, suggesting that they could be used to test the functional importance of this RPA binding site in the initiation of viral DNA replication. To rule out a possible effect of these antibodies on origin DNA unwinding, we used a two-step initiation reaction in which an underwound template was first generated in the absence of primer synthesis. In the second step, primer synthesis was monitored with or without the antibodies. Alternatively, an underwound primed template was formed in the first step, and primer elongation was tested with or without antibodies in the second step. The results show that the antibodies specifically inhibited both primer synthesis and primer elongation, demonstrating that this RPA binding site in T antigen plays an essential role in both events.

Animals↗

PubMed2004

Low level of lytic replication in a recombinant Epstein-Barr virus carrying an origin of replication devoid of BZLF1-binding sites.

Binding of the BZLF1 viral transactivator to Epstein-Barr virus (EBV) oriLyt has been reported to be essential for viral DNA replication. We have constructed a recombinant virus (E2-oriLyt-R) in which the oriLyt BZLF1-binding sites (ZRE) were exchanged against papilloma E2-binding sites. A fusion protein between the BZLF1 protein-transactivating domain and the E2 protein-binding domain was able to reactivate lytic replication in E2-oriLyt-R. However, BZLF1 alone could also induce E2-oriLyt-R, albeit with much lower efficiency. ZRE are therefore important but not absolutely essential cis elements for lytic replication. This shows the importance of recombinants to evaluate viral functions.

Binding Sites↗

PubMed1997

An origin of replication and a centromere are both needed to establish a replicative plasmid in the yeast Yarrowia lipolytica.

Two DNA fragments displaying ARS activity on plasmids in the yeast Yarrowia lipolytica have previously been cloned and shown to harbor centromeric sequences (P. Fournier, A. Abbas, M. Chasles, B. Kudla, D. M. Ogrydziak, D. Yaver, J.-W. Xuan, A. Peito, A.-M. Ribet, C. Feynerol, F. He, and C. Gaillardin, Proc. Natl. Acad. Sci. USA 90:4912-4916, 1993; and P. Fournier, L. Guyaneux, M. Chasles, and C. Gaillardin, Yeast 7:25-36, 1991). We have used the integration properties of centromeric sequences to show that all Y. lipolytica ARS elements so far isolated are composed of both a replication origin and a centromere. The sequence and the distance between the origin and centromere do not seem to play a critical role, and many origins can function in association with one given centromere. A centromeric plasmid can therefore be used to clone putative chromosomal origins coming from several genomic locations, which confer the replicative property on the plasmid. The DNA sequences responsible for initiation in plasmids are short (several hundred base pairs) stretches which map close to or at replication initiation sites in the chromosome. Their chromosomal deletion abolishes initiation, but changing their chromosomal environment does not.

Base Sequence↗

PubMed1997

Denaturation of the simian virus 40 origin of replication mediated by human replication protein A.

The initiation of simian virus 40 (SV40) replication requires recognition of the viral origin of replication (ori) by SV40 T antigen, followed by denaturation of ori in a reaction dependent upon human replication protein A (hRPA). To understand how origin denaturation is achieved, we constructed a 48-bp SV40 "pseudo-origin" with a central 8-nucleotide (nt) bubble flanked by viral sequences, mimicking a DNA structure found within the SV40 T antigen-ori complex. hRPA bound the pseudo-origin with similar stoichiometry and an approximately fivefold reduced affinity compared to the binding of a 48-nt single-stranded DNA molecule. The presence of hRPA not only distorted the duplex DNA flanking the bubble but also resulted in denaturation of the pseudo-origin substrate in an ATP-independent reaction. Pseudo-origin denaturation occurred in 7 mM MgCl2, distinguishing this reaction from Mg2+-independent DNA-unwinding activities previously reported for hRPA. Tests of other single-stranded DNA-binding proteins (SSBs) revealed that pseudo-origin binding correlates with the known ability of these SSBs to support the T-antigen-dependent origin unwinding activity. Our results suggest that hRPA binding to the T antigen-ori complex induces the denaturation of ori including T-antigen recognition sequences, thus releasing T antigen from ori to unwind the viral DNA. The denaturation activity of hRPA has the potential to play a significant role in other aspects of DNA metabolism, including DNA repair.

Base Sequence↗

PubMed2000

DNA replication but not nucleotide excision repair is required for UVC-induced replication protein A phosphorylation in mammalian cells.

Exposure of mammalian cells to short-wavelength light (UVC) triggers a global response which can either counteract the deleterious effect of DNA damage by enabling DNA repair or lead to apoptosis. Several stress-activated protein kinases participate in this response, making phosphorylation a strong candidate for being involved in regulating the cellular damage response. One factor that is phosphorylated in a UVC-dependent manner is the 32-kDa subunit of the single-stranded DNA-binding replication protein A (RPA32). RPA is required for major cellular processes like DNA replication, and removal of DNA damage by nucleotide excision repair (NER). In this study we examined the signal which triggers RPA32 hyperphosphorylation following UVC irradiation in human cells. Hyperphosphorylation of RPA was observed in cells from patients with either NER or transcription-coupled repair (TCR) deficiency (A, C, and G complementation groups of xeroderma pigmentosum and A and B groups of Cockayne syndrome, respectively). This exclude both NER intermediates and TCR as essential signals for RPA hyperphosphorylation. However, we have observed that UV-sensitive cells deficient in NER and TCR require lower doses of UV irradiation to induce RPA32 hyperphosphorylation than normal cells, indicating that persistent unrepaired lesions contribute to RPA phosphorylation. Finally, the results of UVC irradiation experiments on nonreplicating cells and S-phase-synchronized cells emphasize a major role for DNA replication arrest in the presence of UVC lesions in RPA UVC-induced hyperphosphorylation in mammalian cells.

Cell Line, Transformed↗

PubMed1996

DnaA- and PriA-dependent primosomes: two distinct replication complexes for replication of Escherichia coli chromosome.

Enzymatic analyses of primosome assembly at chromosomal and plasmid origins as well as that at single-stranded replication origins revealed the presence of two distinct primosomes in Escherichia coli for primer RNA synthesis and duplex unwinding. A DnaA-dependent primosome is assembled at oriC, the chromosomal origin of Escherichia coli, as well as at the A site, a single-stranded DNA hairpin containing a dnaA box sequence within its stem. In contrast, PriA protein recognizes a hairpin, called n'-pas (primosome assembly site), and initiates assembly of the phiX174-type PriA-dependent primosome in conjunction with other prepriming proteins. Genetic analyses of the prepriming proteins required specifically for the latter primosome strongly suggested that it is responsible for RecA-dependent, DnaA/oriC-independent replication of the Escherichia coli chromosome. Furthermore, primosome assembly in replication of various plasmids may also be classified into either DnaA-dependent or PriA-dependent type. We propose that Escherichia coli possesses two distinct, mutually exclusive primosomes which are differentially utilized by the chromosome as well as by the plasmids. PriA protein appears to be conserved in a wide range of prokaryotic species, and we will also discuss possible biological function of the PriA-dependent primosome in the process of responses to DNA damages.

Bacterial Proteins↗

PubMed1991

Characterization of a cDNA encoding the 70-kDa single-stranded DNA-binding subunit of human replication protein A and the role of the protein in DNA replication.

Replication protein A (RP-A) is a three-subunit single-stranded DNA-binding protein that has been isolated from human cells. RP-A is essential for SV40 DNA replication and may also be important in genetic recombination. The sequence of a cDNA encoding the 70-kDa subunit of human RP-A is reported. The 616-amino acid predicted open reading frame of the human protein is 31% identical with the 621-amino acid open reading frame of the 70-kDa subunit of RP-A from the yeast Saccharomyces cerevisiae. Both proteins share a highly conserved putative metal binding domain of the 4-cysteine type. The human cDNA directs production in Escherichia coli of a 70-kDa protein that reacts with a monoclonal antibody directed against the 70-kDa subunit of human RP-A. The recombinant 70-kDa subunit, purified from bacteria, exhibits single-stranded DNA binding activity comparable to that of the complete RP-A complex. The 70-kDa subunit is able to substitute for the complete human RP-A complex in stimulating the activity of DNA polymerase alpha-primase on a poly(dA).oligo(dT) template. However, the 70-kDa subunit alone cannot substitute for the complete RP-A complex in SV40 DNA replication in vitro, suggesting an important functional role for the other subunits.

Amino Acid Sequence↗

PubMed1995

The possible involvement of replication-related proteins with a DEAD-box-like motif in cell-free DNA replication of Xenopus eggs.

Two types of antibodies were prepared: one directed against an oligopeptide specific to P1 protein, a mammalian homologue of yeast MCM3, and the other against an oligopeptide with a DEAD box motif, which is a highly conserved sequence in the P1 protein family. Immunoprecipitation of the eluate from anti-P1 family IgG-bound beads, which had been incubated in Xenopus egg extracts, with anti-P1 IgG-bound beads revealed that three proteins were coprecipitated. Two proteins remained in the supernatant after the immunoprecipitation of the eluate from anti-P1 family IgG-bound beads with anti-P1 IgG-bound beads. The immunodepleted extracts with anti-P1 family IgG-bound beads showed much lower DNA replication activity than did mock-treated extracts. Recovery of replication was achieved by supplementing the depleted extracts with both the eluate from anti-P1 IgG-bound beads and the supernatant obtained after the immunoprecipitation of the eluate with anti-P1 IgG-bound beads but not by supplementing the extracts with only the proteins eluted from anti-P1 IgG-bound beads. These findings suggest that some proteins containing a DEAD-box-like motif as well as mammalian homologues of yeast MCM2, MCM3 and CDC46 play an important role in cell-free DNA replication of Xenopus eggs.

Amino Acid Sequence↗

PubMed2000

Mutations in domain V of the Sendai virus L polymerase protein uncouple transcription and replication and differentially affect replication in vitro and in vivo.

The Sendai virus L and P proteins comprise the viral RNA-dependent RNA polymerase. The L subunit is thought to be responsible for all the catalytic activities necessary for viral RNA synthesis. Sequence alignment of the L proteins of negative-stranded RNA viruses revealed six regions of good conservation, domains I-VI, which are thought to correspond to functional domains of the protein. Domain V, amino acids 1129-1378, has no recognizable motifs, and to date its function is unknown. Site-directed mutagenesis was used to construct mutations across domain V. The mutant L proteins were all stably expressed and were tested for activity in several aspects of RNA synthesis. One set of mutants could synthesize more le+ RNA than mRNA, while two mutants showed the opposite phenotype, synthesizing more mRNA than le+ RNA. The majority of the mutants could synthesize mRNA, but not genome RNA in vitro, thus uncoupling transcription and replication. Several mutants could replicate in vivo, but not in vitro, at nearly wildtype L levels, suggesting the importance of the intact host cell for replication in some instances. One L mutant, SS24, was virtually inactive in all viral RNA synthesis. SS24 L was able to form a polymerase complex that recognized the nucleocapsid template, and thus these amino acids are essential for the initiation of RNA synthesis.

DNA-Directed RNA Polymerases↗

PubMed1979

Cloning of the replication gene P of bacteriophage lambda: effects of increased P-protein synthesis on cellular and phage DNA replication.

A restriction fragment of lambdaDNA carrying the P gene was cloned in the high copy number plasmid RSF2124. Cells harbouring this new plasmid RSF2124/lambdaE complement lambdaPam80 phage. A lac promoter-operator region (lacP), produced by EcoRI digestion of plasmid pKB252, was inserted into RSF2124/lambdaE such that induction of the lac promoter by IPTG or lactose leads to increased production of the P gene product. A high amount of P protein in E. coli cells results in a slow inhibition of bacterial DNA synthesis, suggesting that the initiation reaction is blocked by P protein. Synthesis of lambdaDNA proceeds normally under these conditions. Nonsuppressing groPA15 mutant bacteria which are unable to support the replication of wild-type lambda (lambdawt), acquire the ability to replicate lambdaPam80 phage but not lambdawt when they are transformed with a plasmid carrying the lambdaP gene. When harbouring a plasmid containing the mutant Pamber 80 gene, groPA15 mutants are able to support the replication of lambdawt phage when infected at a high multiplicity. lambdaPam80 phage does not multiply in these cells.

Coliphages↗

PubMed1981

Structure and function of the region of the replication origin of the Bacillus subtilis chromosome. II. Identification of the essential regions for inhibitory functions shown by the DNA segment containing the replication origin.

A BamHI restriction endonuclease fragment B7, which contains the replication origin of the Bacillus subtilis chromosome, showed inhibitory effects on cell growth and plasmid replication in Escherichia coli and Bacillus subtilis, when B7 was inserted into a composite plasmid pMS102' and introduced into these cells. In order to localize these properties in more limited regions within the B7 fragment, we developed a new and widely applicable method for deletion of DNA segments of various lengths from one or other end of a given region of the plasmid molecule. Using a set of deletions in the B7 fragments of pMS102'-B7, we determined the loci responsible for the inhibitory effects of B7 as described below. (1) Stickiness appearing in E. coli cells was caused by a segment residing in a region of approximately 2.2 kilobase pairs (kbp) overlapping the E19 and E22 fragments. (2) instability of the plasmid in E. coli was due to a segment localized in the 440 bp region of the E19-side terminal portion of the 2.2 kbp region. (3) The same 440 bp were also responsible for inhibition of the replication of the plasmid in B. subtilis. Hybridization of the cloned DNA fragments containing the 2.2 kbp region with the whole B. subtilis chromosome revealed that several regions of the chromosome are homologous to this characteristic sequence.

Bacillus subtilis↗

PubMed1978

Regional replication of the bacterial chromosome induced by derepression of prophage lambda. III. Role of the replication in escape synthesis of gal operon.

Recent evidence suggests that the escape synthesis of gal operon following derepression of the prophage lambda in Escherichia coli K12 involves transcription originating at the lambda promoter (PL) to extend through gal under the conditions in which lambda DNA replication is prevented. Whether the observed expression of gal is due to transcription initiating at PL or at the bacterial promoter for gal (Pgal) was examined in the case of lambda DNA replication being normal. The experiments are based on that two types of transcription are distinguished from each other by the following properties: 1. Pgal-promoted transcription is inhibited by chloramphenicol, while PL-promoted transcription is not. 2. PL-promoted transcription suppresses the polar effect caused by nonsense mutation in a bacterial gene, while Pgal-promoted transcription does not do so. -he results have suggested that gal escape synthesis in lambda-induced lysogen results from transcription which initiates not only at PL but also at Pgal. The Pgal-promoted transcription may be a consequence, direct or indirect, of the concomitant replication of gal DNA.

Chloramphenicol↗

PubMed1992

Transcription in vivo within the replication origin of the Escherichia coli chromosome: a mechanism for activating initiation of replication.

Within the replication origin, oriC, of the Escherichia coli chromosome, novel in vivo transcripts were detected which proceeded rightward and whose production was activated by DnaA protein. In contrast, DnaA protein repressed the previously described ori-L leftward transcription. The former should introduce negative supercoiling, and the latter positive supercoiling, into the 13-mers. The effects of transcription on the initiation of replication were also investigated by making constructs with promoters placed near oriC. Transcription was found to enhance the origin activity only when it was oriented in such a way as to introduce negative supercoiling into the 13-mers. From these results, we propose that transcription within oriC regulates replication initiation by altering the topology of the 13-mer region.

Base Sequence↗

PubMed1978

Analysis of DNA replication patterns of human fibroblast chromosomes: the replication map.

A replication map of human fibroblast chromosomes from two diploid human female fibroblast lines, 46,XX and 46,X, del (X)(q13), was determined using the 'fluorescent plus Giemsa' (FPG) technique. Each chromosome was found to stain homogeneously dark when thymidine was incorporated for the entire S phase of that particular cell. As the duration of exposure to thymidine progressively decreased by increasing the incubation time in bromodeoxyuridine, the staining intensity of chromosomes decreased and, concurrently, gaps in the staining began to appear. These gaps coincide with R bands and represent the earliest areas to complete DNA synthesis. As these areas widen and increase in frequency, first Q and G bands appear, and finally C bands. Homologous X chromosomes were easily differentiated by either a comparison of the bands present or their staining intensity. The replication kinetics of the structurally abnormal heterocyclic X chromosome were very similar to those of the normal heterocyclic X chromosome. The X chromosome with deletion of a portion of the long arm was consistently late in replication.

Cell Line↗

PubMed1976

Comparative study of human chromosome replication in primary cultures of embryonic fibroblasts and in cultures of peripheral blood leucocytes. II. Replication of centromeric regions of chromosomes at the termination of the S period.

Replication of regions of chromosomes 1, 2, 3, 16, and group 4-5 was studied at the termination of the S period in primary cultures of embryonic fibroblasts (two embryos) and in cultures of peripheral blood (two women). Distinct differences were established in the pattern of late replication of the studied chromosomes in the cultures of the two types of cells. These differences consern first of all the centromeric and neighbouring regions of the chromosome. The content of late label in this region is 1.5-3 times higher in the cultures of fibroblasts than in the corresponding regions of leucocyte cultures. The difference is most pronounced in chromosomes 1, 3 and 16. It is suggested that the difference between cultures of these two types of cells in chromosome replication may be connected with the different genetic functioning of the centromeric and neighbouring regions in them. It is also possible that this difference is due to underreplication (or partial loss in an other way) of heterochromatin DNA of centromeric and neighbouring regions in leucocytes functioning for a long period without division.

Adult↗