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PubMed1975

Physiological transition of a coliphage lambda DNA replication.

The "rolling-circle" replicative intermediate (sigma-type molecules) which is normally produced in the late stage of coliphage lambda DNA replication can be found during the first round of lambda DNA replication if cells infected with lambda replication mutant Ots28 are incubated at the nonpermissive temperature until the late stage of the latent period of lambda infection. After shifting to the permissive temperature, the vast majority of replicating forms are sigma-type rolling circle even during the first round of DNA replication. Concatemeric lambda DNA molecules, produced from these sigma-type intermediates, are efficiently packaged into progeny phage, indicating that in the first round of lambda DNA replication, double-branched theta-type molecules are not required for production of viable progeny phage.

Centrifugation, Density Gradient↗

PubMed1999

PriA: at the crossroads of DNA replication and recombination.

PriA is a single-stranded DNA-dependent ATPase, DNA translocase, and DNA helicase that was discovered originally because of its requirement in vitro for the conversion of bacteriophage phi X174 viral DNA to the duplex replicative form. Studies demonstrated that PriA catalyzes the assembly of a primosome, a multiprotein complex that primes DNA synthesis, on phi X174 DNA. The primosome was shown to be capable of providing both the DNA unwinding function and the Okazaki fragment priming function required for replication fork progression. However, whereas seven proteins, PriA, PriB, PriC, DnaT, DnaB, DnaC, and DnaG, were required for primosome assembly on phi X174 DNA, only DnaB, DnaC, and DnaG were required for replication from oriC, suggesting that the other proteins were not involved in chromosomal replication. Strains carrying priA null mutations, however, were constitutively induced for the SOS response, and were defective in homologous recombination, repair of UV-damaged DNA, and double-strand breaks, and both induced and constitutive stable DNA replication. The basis for this phenotype can now be explained by the ability of PriA to load replication forks at a D loop, an intermediate that forms during homologous recombination, double-strand break-repair, and stable DNA replication. Thus, a long-theorized connection between recombination and replication is demonstrated.

Base Sequence↗

PubMed1975

Ten proteins required for conversion of phiX174 single-stranded DNA to duplex form in vitro. Resolution and reconstitution.

Protein requirements for conversion of phiX174 single-stranded DNA to a double-stranded replicative form with a small gap (RF II) have been determined by resolution and reconstitution of the multienzyme system from extracts of gently lysed Escherichia coli. Assays depended on: (a) complementation of extracts of thermosensitive mutants and (b) fractionation of extracts of wild type cells to divide essential components into groups, each of which was further resolved. These procedures have yielded eight proteins: dnaB protein, dnaC protein, proteins i and n (two novel proteins without a defined genetic locus), dnaG protein, DNA polymerase III holoenzyme (polymerase III and copolymerase III), and DNA unwinding protein; purification procedures for the first four are presented here. (Closure of RF 22 requires as with phage M13, DNA polymerase I and ligase.)

Chromatography, Affinity↗

PubMed2005

Differentiation of somatic mitochondria and the structural changes in mtDNA during development of the dicyemid Dicyema japonicum (Mesozoa).

Dicyemids (Mesozoa) are extremely simple multicellular parasites found in the kidneys of cephalopods. Their mitochondria are known to contain single-gene minicircle DNAs. However, it is not known if the minicircles represent the sole form of mitochondrial genome in these organisms. Here we demonstrate that high-molecular-weight (HMW) mtDNA is present in dicyemids. This form of mtDNA is probably limited to germ cells, and has been analyzed by PCR and Southern hybridization. In situ hybridization revealed that mtDNA is initially amplified during early embryogenesis, and then gradually decreases in copy number as larval development proceeds. Furthermore, we demonstrated using BrdU as a tracer that many of the mitochondria in terminally differentiated somatic cells no longer support DNA synthesis. Taking these observations into account, we propose an "amplification-dilution" model for mesozoan mtDNA. "Stem" mitochondria in the germ cells (1) amplify the HMW form of mtDNA in early embryos, followed by minicircle formation via DNA rearrangement, or (2) selectively replicate minicircles from the HMW DNA, concomitantly with the differentiation of the soma. Minicircle formation may itself lead to the loss of replication origins. Thereafter, the minicircles are simply distributed to daughter mitochondria without replication, resulting in the "somatic" mitochondria, which have lost the replicative form of the HMW mtDNA. The change in mtDNA configuration is discussed in relation to mitochondrial differentiation.

Animals↗

PubMed1998

A 68-nucleotide sequence within the 3' noncoding region of simian hemorrhagic fever virus negative-strand RNA binds to four MA104 cell proteins.

The 3' noncoding region (NCR) of the negative-strand RNA [3'(-)NCR RNA] of the arterivirus simian hemorrhagic fever virus (SHFV) is 209 nucleotides (nt) in length. Since this 3' region, designated 3'(-)209, is the site of initiation of full-length positive-strand RNA and is the template for the synthesis of the 5' leader sequence, which is found on both full-length and subgenomic mRNAs, it is likely to contain cis-acting signals for RNA synthesis and to interact with cellular and viral proteins to form replication complexes. Gel mobility shift assays showed that cellular proteins in MA104 S100 cytoplasmic extracts formed two complexes with the SHFV 3'(-)209 RNA, and results from competition gel mobility shift assays demonstrated that these interactions were specific. Four proteins with molecular masses of 103, 86, 55, and 36 kDa were detected in UV-induced cross-linking assays, and three of these proteins (103, 55, and 36 kDa) were also detected by Northwestern blotting assays. Identical gel mobility shift and UV-induced cross-linking patterns were obtained with uninfected and SHFV-infected extracts, indicating that the four proteins detected are cellular, not viral, proteins. The binding sites for the four cellular proteins were mapped to the region between nt 117 and 184 (68-nt sequence) from the 3' end of the SHFV negative-strand RNA. This 68-nt sequence was predicted to form two stem-loops, SL4 and SL5. The 3'(-)NCR RNA of another arterivirus, lactate dehydrogenase-elevating virus C (LDV-C), competed with the SHFV 3'(-)209 RNA in competition gel mobility shift assays. UV-induced cross-linking assays showed that four MA104 cellular proteins with the same molecular masses as those that bind to the SHFV 3'(-)209 RNA also bind to the LDV-C 3'(-)NCR RNA and equine arteritis virus 3'(-)NCR RNA. However, each of these viral RNAs also bound to an additional MA104 protein. The binding sites for the MA104 cellular proteins were shown to be located in similar positions in the LDV-C 3'(-)NCR and SHFV 3'(-)209 RNAs. These data suggest that the binding sites for a set of the cellular proteins are conserved in all arterivirus RNAs and that these cell proteins may be utilized as components of viral replication complexes.

Animals↗

PubMed1977

Mapping and length measurements of restriction enzyme fragments by electron microscopy.

1. We have mapped by electron microscopy the DNA-fragments formed by the action of the restriction endonuclease from Arthrobacter luteus of phi X 174 replicative form DNA. These fragments were separated by polyacrylamide gel electrophoresis and hybridized to phiX 174 single stranded DNA. The partial duplex molecules were inspected in the electron microscope. In this way the relative order of eleven fragments ranging in size from approximately 100 to 1000 nucleotide pairs has been established and compared with that deduced from reciprocal digestion studies. 2. The measured lengths of the fragments agreed well with the lengths found by gel electrophoresis. 3. The purity of the isolated fragments was checked. Most of the contaminating fragments derive from nearest neighbours in the preparative polyacrylamide gels.

Arthrobacter↗

PubMed1988

Bacteriophage f1 DNA replication genes. II. The roles of gene V protein and gene II protein in complementary strand synthesis.

Filamentous phage gene V, which encodes a single-stranded DNA binding protein, has been cloned and placed under control of the lac promoter. Cells bearing the clone are refractory to filamentous phage infection if the expression of the gene is induced with isopropyl-1-thio-beta-D-galactoside. The inhibition of infection is shown to occur at an early stage, and can be reversed if the cells express gene II in addition to gene V protein. These observations support the hypothesis that gene II protein, in addition to its role in nicking and facilitating the synthesis of phage viral (+) strand DNA, functions to prevent the gene V-mediated inhibition of complementary (-) strand synthesis. We proposed a model in which the absolute and relative concentrations of the products of genes II, X and V determine whether a single strand is to be exported as phage or incorporated into double-stranded replicative form DNA.

Cloning, Molecular↗

PubMed1987

Generation of free radical and hydrogen peroxide from 2-hydroxyemodin, a direct-acting mutagen, and DNA strand breaks by active oxygen.

Among several hydroxylated metabolites of emodin, a fungal anthraquinone and constituent of rhubarb, 2-hydroxyemodin was a direct-acting mutagen showing a large electron-spin resonance (ESR) signal in the presence of DNA, especially at alkaline pH. Coupled with generation of free radical, hydrogen peroxide but not superoxide was formed. The active oxygen produced from 2-hydroxyemodin induced strand breaks in phi X 174 replicative form I DNA (supercoiled covalently closed circular duplex DNA). These results suggest a possible role of active oxygen in the process of mutagenesis.

Anthraquinones↗

PubMed1974

Partial purification and characterization of four endodeoxyribonuclease activities from Escherichia coli K-12.

Four hitherto undescribed endodeoxyribonucleases, temporarily designated A(1), A(2), A(3), and B, have been isolated from E. coli K-12. Each requires Mg(++) and is not stimulated by ATP or S-adenosylmethionine. A(3) is strongly inhibited by Fe(+++) and weakly inhibited by ATP, S-adenosylmethionine, and DPN, whereas B is inhibited by caffeine. Each can be purified free of exonuclease or DNA-3'-phosphatase. A(1) (molecular weight approximately 72,000) cleaves single-stranded, circular fd DNA to form 3'-hydroxyl termini and introduces nicks and breaks in the closed, double-stranded replicative form DNA of fd (fd RFI). A(2) (molecular weight approximately 46,000) cleaves fd DNA and introduces nicks and breaks in RFI, forming 3'-hydroxyl- and 5'-phosphoryl termini. A(3) (molecular weight approximately 38,000) cleaves fd DNA to form 3'-hydroxyl termini and introduces only nicks in fd RFI. Irradiation of the RFI with ultraviolet light markedly increases the rate of hydrolysis by A(3). B appears to form 3'-phosphoryl termini with fd DNA, but its characterization is highly preliminary due to its instability.

Chemical Precipitation↗

PubMed1992

A single amino acid substitution reduces the superhelicity requirement of a replication initiator protein.

The origin of rolling circle replication in filamentous coliphage consists of a core origin that is absolutely required and an adjacent replication enhancer sequence that increases in vivo replication 30 to 100-fold. The core origin binds the initiator protein (gpII) which either nicks or relaxes negatively superhelical replicative form DNA (RFI). Nicking at the origin, but not relaxation, leads to initiation of DNA replication. Our results indicate that the ratio of nicking to relaxation (nicking-closing) in vitro depends on the superhelical density of the substrate. We have studied the effect of a single amino acid substitution in gpII, which allows wild-type levels of replication in the absence of the enhancer, on origin nicking and binding. The enhancer-independent mutation yields more nicking and less relaxation of RFI, compared to the wild-type protein. The mutant gpII also shows a reduced requirement for superhelicity of the substrate in the nicking reaction. At the same time, the mutant gpII increases the cooperativity of protein-protein interactions in origin binding. We propose that the relaxation activity of gpII negatively regulates replication initiation, and that both increase in the negative superhelicity of the substrate and action of the replication enhancer may antagonize the relaxation activity.

Amino Acid Sequence↗

PubMed1998

Molecular and functional analysis of pTAV320, a repABC-type replicon of the Paracoccus versutus composite plasmid pTAV1.

The second replicator region of the native plasmid pTAV1 of Paracoccus versutus has been identified thus proving the composite nature of this replicon. The minimal replicon designated pTAV320 (4.3 kb) was cloned and sequenced. pTAV320 encodes three putative proteins--RepA, RepB and RepC. This replicator region shows strong structural and functional similarity to repABC-type replicons found in several Agrobacterium and Rhizobium plasmids. The origin of replication appears to be localized within the coding sequence of the repC gene. RepC was shown to be essential for replication. RepA and RepB were necessary for stable maintenance of the plasmid, which implies a role in active partitioning. The presence of the complete sequence of pTAV320 (in its non-replicative form) could stabilize in cis pTAV202, a mini-replicon derived from the other pTAV1 replicator region. Deletions introduced into the repC gene abolished the 'stabilizing' activity of pTAV320, suggesting that the centromere-like sequence, necessary for partitioning, might be localized within this gene. The two replicator regions of pTAV1 (pTAV320 and pTAV202) expressed incompatibility towards the parental plasmid but were compatible in trans in P. versutus cells. The pTAV320 replicon can be maintained in several Paracoccus, Agrobacterium, Rhizobium and Rhodobacter strains in addition to P. versutus.

Base Sequence↗

PubMed1982

DNA polymerase requirements for parvovirus H-1 DNA replication in vitro.

An in vitro system using nuclei from parvovirus H-1-infected cells was used to characterize the influence of inhibitors of mammalian DNA polymerases on viral DNA synthesis. The experiments tested the effects of aphidicolin, which is highly specific for DNA polymerase alpha, and 2',3'-dideoxythymidine-5'-triphosphate (ddTTP), which inhibits cellular DNA polymerases in the order gamma greater than beta greater than alpha. Both aphidicolin and ddTTP were inhibitory, indicating that both polymerase alpha and a ddttp-sensitive enzyme are required for viral DNA synthesis. This was seen more clearly in kinetic measurements, which indicated an initial period of rapid DNA synthesis with the participation of polymerase alpha, followed by a period of less rapid, but more sustained, rate of DNA synthesis carried out by a ddTTP-sensitive enzyme, probably polymerase gamma. One interpretation of the results is that polymerase alpha functions in a strand displacement stage of the viral DNA replication mechanism, whereas polymerase gamma serves to convert the displaced single strands back to double-strand replicative form.

Aphidicolin↗

PubMed1985

Feline panleukopenia virus replicates in cells in which cellular DNA synthesis is blocked.

The components of the cell cycle for a feline embryo cell line were defined. Thymidine (6mM)-supplemented medium reversibly arrested cells 1 h into the S phase of the cell cycle and was used in a double blocking procedure to synchronize cells to the early S phase. The kinetics of feline panleukopenia virus replication in synchronized cells was studied by using (i) inclusion body formation, (ii) a plaque assay for cell-associated and cell-free virus under one-step growth conditions, (iii) an enzyme immunoassay for viral protein, (iv) electron microscopy of infected cells, and (v) the detection and identification of viral replicative form DNA by restriction endonuclease analysis. Parallel studies by each of these procedures of the replication of feline panleukopenia virus in cells in which a 6 mM thymidine block was maintained indicated that parvovirus replicated with essentially similar kinetics in both unblocked, synchronized cells and in cells in which the block was maintained. Accordingly, a 6 mM thymidine-supplemented medium, although it effectively blocks cellular DNA synthesis, does not block the replication of parvovirus.

Animals↗

PubMed1981

Replication of phase fd RF with fd gene 2 protein and phage T4 enzymes.

Bacteriophage fd replicative form DNA with a nick in the viral strand serves as a template for DNa replication with purified bacteriophage T4 enzymes. As anticipated from previous in vitro studies carried out with this system (Morris, C. F., Sinha, N. K., and Alberts, B. M. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 4800-4804), DNA is synthesized by a rolling circle mechanism. We show here that the DNA strands synthesized are processed by the phage fd gene 2 protein into unit length products, providing that the gene 2 protein is present at the moment when this DNA is made. The products are mostly unit length linear single strands, indicating that the circularization step normally catalyzed by gene 2 protein subsequent to its site-specific cleavage of an fd DNA strand occurs only inefficiently in this system. The gene 2 protein reduces the level of DNA synthesis by 2-fold at low concentrations, even though it only cleaves the DNA products efficiently at higher levels of the enzyme. This indicates that there are at least two different effects of the fd gene 2 protein in processing of viral fd DNA.

Coliphages↗

PubMed1982

The beta subunit of the DNA polymerase III holoenzyme becomes inaccessible to antibody after formation of an initiation complex with primed DNA.

The initiation of the DNA polymerase III holoenzyme-catalyzed reaction is blocked by antibody directed against the beta subunit; elongation is unaffected (Johanson, K., and McHenry, C. (1980) J. Biol. Chem. 255, 10984-10990). We have developed an immunological method for quantitating nanogram quantities of beta in reaction complexes. Using this method, we have demonstrated that beta is present in all stages of the DNA polymerase III holoenzyme reaction. Upon initiation complex formation, the antigenic determinants of beta become inaccessible to anti-beta immunoglobulin G. The methods described herein should be generally applicable to the study of a variety of multienzyme complexes. Even after conversion of a primed G4 single strand to the duplex replicative form, beta does not readily dissociate. This creates a kinetic barrier to the overall holoenzyme replicative reaction.

Antibodies↗

PubMed1976

A compact form of double-stranded RNA in solutions containing poly(ethyleneglycol).

Molecules of single-stranded ribosomal RNA and double-stranded replicative form of phage f2 RNA (dsRNA) adopt a compact form in solutions, containing sufficiently high concentrations of salt (NaCl) and polymer (PEG). However, only in the cases of native dsRNA molecules the compact particles are characterized by a regular internal structure, which accounts for the appearance of an intense positive band in CD spectra. Heating or acidification of PEG-containing solutions of dsRNA leads to the disappearance of the intense positive CD band, which results from the "destruction" of the regular internal structure of compact particles. Comparison of properties of DNA and dsRNA compact particles formed in PEG-containing water-salt solutions suggests the existence of similar mechanisms of compactization of double-stranded polynucleotides.

Circular Dichroism↗

PubMedDate not supplied

[Compact form of DNA in solution. XII. Double-stranded polyribonucleotide compacting in the presence of polyethylene glycol].

Double-stranded polyribonucleotides (a replicative form of phage f2 RNA--dsRNA and poly(A) poly(U), can adopt a compact from in solutions, containing NaCl and poly(ethylene glycol) (PEG). According to electron-microscopic observations dsRNA compact particles have the form of disks or doughnuts 200--400 A in diameter. X-ray diffraction patterns from dense slurries of dsRNA compact particles show a reflection at a spacing of 35 A, which is indicative of the existance of ordered regions in compact particles. The intense positive CD band, which is characteristic of dsRNA and poly(A) poly(U) compact particles, presumably results from the ordered regions in the particles. Heating of the solution leads to the disappearance of the intense positive CD band, probably as a result of the destruction of the ordered structure of compact particles. Heat or acid denatured dsRNA molecules as well as single-stranded molecules of ribosomal RNA also form large particles in PEG-containing solutions. However, X-ray diffraction patterns from these particles do not show the 35 A reflection and the specific positive band is not present in their CD spectra, which indicates that such particles lack ordered internal structure. It is suggested that similar mechanism of compactization of double-stranded polynucleotides (DNA and RNA) exist, and compact particles may be divided into two families (psi+ and psi-), differing by the secondary structure of double-stranded polynucleotides, which form the particles.

Bacteriophages↗

PubMed2002

A positive-strand RNA virus replication complex parallels form and function of retrovirus capsids.

We show that brome mosaic virus (BMV) RNA replication protein 1a, 2a polymerase, and a cis-acting replication signal recapitulate the functions of Gag, Pol, and RNA packaging signals in conventional retrovirus and foamy virus cores. Prior to RNA replication, 1a forms spherules budding into the endoplasmic reticulum membrane, sequestering viral positive-strand RNA templates in a nuclease-resistant, detergent-susceptible state. When expressed, 2a polymerase colocalizes in these spherules, which become the sites of viral RNA synthesis and retain negative-strand templates for positive-strand RNA synthesis. These results explain many features of replication by numerous positive strand RNA viruses and reveal that these viruses, reverse transcribing viruses, and dsRNA viruses share fundamental similarities in replication and may have common evolutionary origins.

Bromovirus↗