Host DNA Polymerase Research Articles

Replication of ColE2 and ColE3 plasmids: in vitro replication dependent on plasmid-coded proteins.

We developed an in vitro replication system for ColE2 and ColE3 plasmids using cell extracts prepared from bacteria with or without these plasmids. DNA synthesis depended on host DNA polymerase I and was sensitive to rifampicin and chloramphenicol. Preincubation of the extracts with plasmid DNA, however, allowed replication of template DNA added subsequently in a plasmid-specific manner in the presence of rifampicin and chloramphenicol. The plasmid-specified trans-acting factor(s) was detected in cell extracts from bacteria carrying a recombinant plasmid with the region of ColE2 or ColE3 encoding the Rep protein. The plasmid-specified factor(s) consisted at least in part of protein, probably the Rep protein. In vitro replication started within a region of ColE2 or ColE3 containing the smallest cis-acting segment essential for in vivo replication and proceeded in a fixed direction.

Reverse transcriptase, transposase and phage DNA polymerase genes are selfish genes evolved from duplicated host DNA polymerase gene(s)

Reverse transcriptase, transposase and phage DNA polymerase genes are selfish genes evolved from duplicated host DNA polymerase gene(s)

The location, sequence, transcription, and regulation of a baculovirus DNA polymerase gene

The Autographa californica nuclear polyhedrosis virus (AcMNPV) DNA polymerase gene was identified with the aid of an oligonucleotide probe corresponding to an amino acid sequence conserved among viral DNA polymerases of other virus families. A 3.6-kb pair region of the AcMNPV DNA, from 39.5 to 42.5 map units (m.u.), was sequenced and an open reading frame (ORF) of 2994 by was observed. From the first ATG of this ORF, a translation product of 984 amino acids ( Mr 114,310) was predicted. Amino acid sequence similarities to other viral DNA polymerases were found. Transcription was analyzed by Northern RNA blot analysis and nuclease protection studies of RNA:DNA hybrids. The ORF is transcribed in the counterclockwise direction as a 3-kb RNA. Transcripts appear to initiate at two differently regulated sites (ca. −120 and −212 bp) upstream of the initiating ATG (+1,+2,+3) and to be polyadenylated at a single site near a signal (A 2UA 3) which overlaps the translational termination signal (UAA) at +2952. Transcripts were observed only during a narrow window between 2 to 8 hr postinfection (p.i.) with maximum expression between 4 and 6 hr p.i. Polymerase gene transcripts were observed in the presence of the protein synthesis inhibitor cycloheximide which also blocked the shut-off of these early transcripts. Aphidicolin, an inhibitor of both viral and host DNA polymerases, inhibited polymerase gene transcription suggesting a unique regulation involving DNA replication.

Regulation of Host RNA Levels during Baculovirus Infection

During infection of the permissive host insect cell line Spodoptera frugiperda IPLB-SF-21 by the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV), the levels of host actin, histone, and heat shock 70 (hsp70) RNAs are reduced substantially. Reduction of the host RNA levels occurs primarily during a narrow window of the replication process, from approximately 12 to 18 hr postinfection (p.i.), corresponding to the phase in which the extracellular form of the virus buds into the media. A late viral protein appears to be required for this reduction since cycloheximide, an inhibitor of cytosolic protein synthesis, and aphidicolin, an inhibitor of host and viral DNA polymerases, inhibit the reduction of actin and histone RNA levels. A cDNA corresponding to the car☐yl half of the S. frugiperda mitochondrial cytochrome c oxidase subunit III (COIII) gene was isolated, sequenced, and characterized. Two differentially regulated mitochondrial transcripts of this gene are observed. The level of the larger of these transcripts, which is dependent on active cytosolic protein synthesis, is reduced during virus infection in a fashion similar to that of the nuclear host genes. The smaller COIII transcript is stable until at least 24 hr p.i. but the level of this RNA eventually declines by 48 hr p.i.

Interaction of Epstein-Barr virus DNA polymerase and 5'-triphosphates of several antiviral nucleoside analogs

The 5'-triphosphates of 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-methyluracil, 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-iodocytosine, 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-methylcytosine, 9-[(2-hydroxyethoxy)methyl]guanine, and 9-(1,3-dihydroxy-2-propoxymethyl)guanine had lower Ki values for Epstein-Barr virus DNA polymerase than has been reported elsewhere for host DNA polymerase. Inhibition of DNA elongation by these analogs ranged from moderate to strong, suggesting that preferential incorporation of these analogs into DNA by virus DNA polymerase may contribute to antiviral selectivity.

Interaction of DNA polymerase and nucleotide analog triphosphates

The properties of virus and host DNA polymerases are important factors in determining the selectivity of deoxynucleotide analogs used in antiviral chemotherapy. The high affinity of herpes DNA polymerase for nucleotide analogs may be particularly important in CMV and EBV-infected cells, since these viruses do not induce the synthesis of a virus-specified thymidine kinase. In general, the effect of nucleotide analog incorporated into DNA may be summarized as follows: analogs with modifications at the base moiety do not affect the rate of DNA chain elongation whereas those modified at the sugar moiety will inhibit the rate of chain elongation. ACGTP and DHPGTP competitively inhibit incorporation of dGTP into DNA; however, steric freedom of the acyclic phosphate may allow these nucleotides to bind virus enzyme in a conformation similar to that assumed by dGTP only at the transitional stage of the enzyme reaction. This may explain the high affinity of virus enzyme for these inhibitors. The interaction of aphidicolin with virus enzyme differs from that with host enzyme. These differences suggest new strategies for antiviral chemotherapy using aphidicolin derivatives.

Properties of the adenovirus DNA polymerase.

The 140,000-Da adenovirus-encoded DNA polymerase (Ad Pol) is required for viral DNA replication both in vitro and in vivo. The polymerase co-purifies in a complex with the 80,000-Da precursor (pTP) of the terminal protein (TP) found covalently attached to the 5' ends of adenovirus DNA. To better understand their function in DNA replication, we have examined the properties of the Ad Pol and the pTP X Ad Pol complex on natural and synthetic DNA templates. The pTP X Ad Pol complex utilizes a variety of homopolymer template-primer combinations including poly(dC) X oligo(dG), poly(dA) X oligo(dT), poly(dT) X oligo(dA), and poly(dT) X oligo(rA). With poly(dT) as template and oligo(rA) or oligo(dA) as primer, DNA synthesis by the pTP X Ad Pol complex is stimulated as much as 100-fold by the 59,000-Da adenovirus DNA-binding protein (Ad DBP). ATP (4 mM) can further increase the rate of DNA synthesis 3- to 10-fold. The Ad DBP does not stimulate the activity of host (HeLa cell) DNA polymerase alpha with poly(dT) X oligo(dA) (or oligo(rA)) as the template-primer, and Escherichia coli single-stranded DNA binding protein cannot substitute for the Ad DBP in the stimulation of the Ad Pol activity. Under optimal conditions, poly(dA) chains 30,000 nucleotides in length are formed indicating that the Ad Pol can be a highly processive enzyme. An exonuclease activity co-sediments with the pTP X Ad Pol complex during glycerol gradient centrifugation, and co-purifies with the 140,000-Da Ad Pol after dissociation of the pTP X Ad Pol complex with urea. The Ad Pol-associated nuclease hydrolyzes single-stranded DNA in a 3'----5' direction and is at least 10-fold more active on single-stranded DNA than on duplex DNA. The Ad Pol has no detectable endonuclease activity on single-stranded DNA or duplex circular DNA. Analysis of the products of the nuclease activity showed that 5'-deoxynucleoside monophosphates were released during the hydrolysis of single-stranded DNA. The Ad DBP inhibits the hydrolysis of DNA by the polymerase-associated nuclease activity.

Acyclovir triphosphate is a suicide inactivator of the herpes simplex virus DNA polymerase.

The triphosphate form of 9-[(2-hydroxyethoxy)-methyl]guanine (acyclovir), ACVTP, inactivates the herpes simplex virus type 1 DNA polymerase. ACVTP does not innately inactivate resting polymerase, but becomes an inactivator only while being processed as an alternative substrate. Pseudo first-order rates of inactivation were measured at varying concentrations of ACVTP and fixed concentrations of the natural substrate, deoxyguanosine triphosphate. These studies indicated that a reversible enzyme-ACVTP (Michaelis-type) complex is formed at the active site prior to inactivation. The formation of this complex was competitively retarded by deoxyguanosine triphosphate. An apparent dissociation constant (KD) of 3.6 +/- 0.2 (S.D.) nM was determined for ACVTP from this reversible complex. A second method for the estimation of the KD which used the extrapolated initial velocities produced a value of 5.9 +/- 0.4 (S.D.) nM. The rate of conversion of the reversible complex to the inactivated complex, at saturating ACVTP, was calculated to be 0.24 min-1. No reactivation of enzyme activity was detected following isolation of the inactivated complex by rapid desalting on Sephadex G-25. Under these conditions, an overall reactivation rate of 1.5 X 10(-5) min-1 could have been easily detected. Therefore, the overall inhibition constant must have been less than 3 pM. In contrast, when host DNA polymerase alpha was incubated with 14 microM ACVTP, only 60% inhibition of enzyme activity was observed, but inactivation was not detected. These data indicate that ACVTP functions as a suicide inactivator of the herpes simplex virus type 1 DNA polymerase, and is only a weak reversible inhibitor of DNA polymerase alpha.

Factors involved in the initiation of phage o29 DNA replication in vitro: requirement of the gene 2 product for the formation of the protein p3-dAMP complex

To study the requirements for the in vitro formation of the protein p3-dAMP complex, the first step in phi29 DNA replication, extracts from B. subtilis infected with phi29 mutants in genes 2, 3, 5, 6 and 17, involved in DNA synthesis, have been used. The formation of the initiation complex is completely dependent on the presence of a functional gene 2 product, in addition to protein p3 and phi29 DNA-protein p3 as template. ATP is also required, although it can be replaced by other nucleotides. The products of genes 5, 6 and 17 do not seem to be needed in the formation of the initiation complex. Inhibitors of the host DNA polymerase III, DNA gyrase or RNA polymerase had no effect on the formation of the protein p3-dAMP complex, suggesting that these proteins are not involved in the initiation of phi29 DNA replication. ddATP or aphidicolin, inhibitors of DNA chain elongation, had also no effect on the formation of the initiation complex.

A novel DNA polymerase induced byBacillus subtilisphage φ29

A novel DNA polymerase induced by Bacillus subtilis bacteriophage phi 29 has been identified. This polymerase can be separated from host DNA polymerase, by fractionation of extracts prepared from phage infected cells, using phosphocellulose chromatography. The isolated polymerase prefers poly(dA)oligo(dT) as template. The DNA polymerase isolated from the cells infected with a gene 2 temperature sensitive mutant (ts2) showed greater heat-lability than that induced by wild type phi 29. The ts2 DNA polymerase was also thermolabile for its activity in the formation of a covalent complex between phi 29 terminal protein and dAMP, the initiation step of phi 29 DNA replication. These findings indicate that gene 2 is the structural gene for a phi 29 DNA polymerase required for the complex formation step of DNA initiation.

Separation of the adenovirus terminal protein precursor from its associated DNA polymerase: role of both proteins in the initiation of adenovirus DNA replication.

A complex containing the 80,000-dalton precursor to the adenovirus (Ad)-encoded terminal protein (pTP) and a 140,000-dalton protein is required for Ad DNA replication in vitro. This complex has been separated into subunits by glycerol gradient centrifugation in the presence of urea. The isolated 140,000-dalton subunit contains a DNA polymerase activity which can be differentiated from all host DNA polymerases. No enzyme activity was detected with the isolated pTP. The requirements for reactions involved in the initiation of Ad DNA replication were determined by using the isolated subunits. The covalent addition of dCMP, the first nucleotide in the DNA chain, to the pTP, which serves as the primer for replication, required the DNA polymerase subunit as well as the pTP. Synthesis of viral DNA in vitro also required both subunits. The properties of the DNA polymerase suggest that it may be a viral gene product.

Studies with aphidicolin on the Fv-1 host restriction of Friend murine leukemia virus.

The murine gene Fv-1 exerts a major control over the replication of Friend murine leukemia virus (F-MuLV). An effect of the gene product has been determined to be at the level of accumulation and integration of viral DNA. Aphidicolin, an inhibitor of eucaryotic DNA polymerase alpha, was studied in murine cells infected either permissively or nonpermissively with regard to the Fv-1 genotype. Results indicated that inhibition of DNA polymerase alpha did not affect the accumulation of form III viral DNA in either permissive or nonpermissive cells. However, the normal accumulation of circular form I DNA in permissive cells was inhibited. The block in the accumulation of form I DNA resembled that occurring in some F-MuLV Fv-1-nonpermissive infections. Additionally, aphidicolin treatment resulted in the accumulation of novel low-molecular-weight viral DNA species, normally detectable in a nonpermissive infection of NIH cells with B-tropic F-MuLV. These data suggest that the Fv-1 gene product may interact with host DNA polymerase alpha to prevent viral replication.

Plasmid R46-mediated protection against bleomycin is poLA+-dependent.

Strains of Escherichia coli deficient in post-replication recombination repair were more sensitive to bleomycin than wild-type, repair-proficient strains. Mutants lacking excision repair functions were no more sensitive to bleomycin than the wild-type strains, indicating that this pathway is not involved in the repair of bleomycin-damaged DNA. Plasmid R46 not only protected repair-proficient strains but also those with recB, recC, uvrA or lig genotypes, suggesting that R46 protection against bleomycin is independent of these host repair functions. However, R46 protection was abolished in recA or polA strains, indicating that these gene functions are necessary for plasmid-mediated protection. It is suggested that protection may be due to a recA+-dependent interaction of a plasmid-encoded product with host DNA polymerase I, resulting in an increase in the DNA repair capacity of cells.

Induction of host DNA synthesis and DNA polymerase by DNA-negative temperature-sensitive mutants of human cytomegalovirus

Four DNA-negative temperature-sensitive ( ts) mutants of human cytomegalovirus, belonging to different complementation groups, were studied for their ability to induce cell DNA synthesis and DNA polymerase in permissive human embryo lung (HEL) and nonpermissive rabbit lung (RL) cells. These is mutants stimulated host cell DNA synthesis in HEL and RL cells and DNA polymerase activity in HEL and RL cells at permissive (33.5°) and nonpermissive temperatures (39.5°). Salt stimulation of induced DNA polymerase activity was used to distinguish between virus and cell DNA polymerase from HEL cells. DNA polymerase activity was stimulated by 100 m M (NH 4) 2SO 4 at either 33.5 or 39.5° in cultures infected with three of the mutants ( ts 9, is 153, and ts 155). However, DNA polymerase activity was not stimulated by 100 m M (NH 4) 2SO 4, in cultures infected with one of the ts mutants ( ts 13). These data suggest that at least four cistrons control the synthesis of virus DNA and that virus DNA synthesis is not required for the induction of cell DNA synthesis.

Prevalence of Pseudomonas aeruginosa FP plasmids which enhance spontaneous and UV-induced mutagenesis

From work reported here and from previous studies 16 out of 53 (30%) FP plasmids (i.e. those plasmids that promote host chromosome transfer) of Pseudomonas aeruginosa are found to protect host cells against UV irradiation. 13 of these UV-protecting FP plasmids were tested to determine their mode of DNA repair and were found to contribute to error-prone repair because of their enhancement of UV-induced mutagenesis and in most instances spontaneous mutagenesis as well. Some of these plasmids were tested for their behaviour in a DNA polymerase I deficient (Pol −) mutant of P. aeruginosa; the remainder could not be tested due to plasmid instability in the Pol − mutant. 11 of these FP plasmids provided wild-type level of UV protection to the mutant. 4 of the plasmids tested (FP18, FP103, FP109 and FP111) were able to enhance the mutant's ability to host cell reactivate UV irradiated phage, though not to the level of the Pol + parent. The presence of FP18 or FP111 in the Pol − mutant did not increase polymerase I-like enzymatic activity. It is concluded that the plasmids do not confer a polymerase activity functionally equivalent to host DNA polymerase I. It is possible however, that the plasmids code for another polymerase or for a cofactor which interacts with a host polymerase, as seen by the partial restoration by FP plasmids of host-cell reactivation of UV-irradiated phage in the polymerase I deficient mutant. The mutagenic properties of those FP plasmids tested appears to be nonspecific because of their ability to mutate two host chromosomal genes, trpB1 and leu38 and an R plasmid gene, bla. The implications of the prevalence of FP plasmids in P. aeruginosa which enhance mutagenesis are discussed.

SPP1 DNA replicative forms: growth of phage SPP1 in Bacillus subtilis mutants temperature-sensitive in DNA synthesis.

The development of bacteriophages SPP1 and phi 29 has been studied in several B. sutilis mutants defective in host DNA replication, under non permissive conditions. Several gene products, involved in the synthesis of host DNA, are required for phi 29 replication, while SPP1 seems to require only the host DNA polymerase III. In addition both phages are unable to grow in a dna A mutant (ribonucleotide reductase). Taking advantage of the fact that SPP1 DNA is actively replicated in several dna mutants at non-permissive temperature, we have studied the structure of the replicative intermediates of this phage in the absence of interfering host DNA synthesis. Fast sedimenting forms of SPP1 DNA can be isolated from phage infected cells and evidence of covalently joined concatemers has been obtained, suggesting the presence of terminally repeated sequences.

In vivo effects of recBC DNase, exonuclease I, and DNA polymerases of Escherichia coli on the infectivity of native and single-stranded DNA of bacteriophage T7

The effect of several enzymes of the DNA metabolism of Escherichia coli on the biological activity of native and single-stranded T7 DNA was studied by transfection of lysozyme-EDTA spheroplasts prepared from various E. coli mutants. It is shown that the presence of the recBC DNase in the recipient cells decreases the infectivity of native and denatured DNA by about 100- and 10-fold, respectively. Lack of exonuclease I did not stimulate transfection by single-stranded DNA. Separated light (l) and heavy (r) strands of T7 DNA are fully infective, with a linear dependence on DNA concentrations, whereas heat-denatured DNA shows a two-hit kinetics. Single-stranded DNA was observed to depend on a functional DNA polymerase III for infectivity in polAB cells, whereas transfection with native T7 DNA was independent of the host DNA polymerases. The results are discussed with respect to the mode of T7 DNA replication.

Lysogenization of Escherichia coli by bacteriophage Lambda: complementary activity of the host's DNA polymerase I and ligase and bacteriophage replication proteins Q and P.

When bacteriophage lambda DNA replication is blocked by mutation in phage genes O or P, the efficiency of lysogenization drops to a very low value unless high multiplicities of infecting phage are used. Our results show that even at high multiplicity, lambda O or P mutants cannot efficiently lysogenize some hosts that are defective in either DNA polymerase I or DNA ligase. Covalent closure of infecting DNA molecules, a preliminary step for insertion according to Campbell's model and an obvious candidate for this lysogenization defect, appears to occur normally under our conditions. In addition, prophage excision as measured by the frequency of curing O- and P- lysogens seemed normal when tested in the poll- strain. These results suggest that the Escherichia coli enzymes DNA polymerase I and ligase, and phage proteins O and P, are able to provide some complementary activity whose function is required specifically for prophage integration.

Effect of Phosphonoacetate on Marek's Disease Virus Replication

Phosphonoacetate (PA), but not any of its analogues tested, effectively inhibited avian herpesvirus replication and viral DNA synthesis in cell cultures. At 100 mug/ml culture medium, PA completely inhibited the replication of Marek's disease virus (MDV), herpesvirus of turkeys, and owl herpesvirus, but had no measurable effect on normal cell growth. PA also inhibited DNA polymerases induced by these avian viruses. Enzyme inhibition was 50% at a PA concentration of 0.2 mug/ml. At a concentration of 3-6 mug/ml, the compound also effected a 50% inhibition of alpha (maxi) enzyme of the host DNA polymerase. It had no effect on the host beta (mini) enzyme. When administered to chickens, PA did not inhibit the replication of MDV, nor did it prevent the development of lymphoma.

Induction of mutations in B. subtilis phage SPP1 by growth on host cells carrying a mutator DNA polymerase III.

Phage SPP1 infecting a mutator strain of B. subtilis (BD337) which carries a defective DNA polymerase III is mutagenized. This effect is absent in phages SPO2, SP82G and øe. The results confirm previous observations that SPP1 uses host DNA polymerase III for its DNA synthesis.