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B D Preston

Publications and source records attributed to B D Preston.

At least 37 records · Page 2Linked to original sources
PubMed1994

Marked infidelity of human immunodeficiency virus type 1 reverse transcriptase at RNA and DNA template ends.

Human immunodeficiency virus type 1 (HIV-1) is genetically highly variable. This is attributed to the error-prone nature of HIV-1 replication and its proclivity for recombination. During replication and recombination, reverse transcriptase (RT) must polymerize DNA to the 5' ends of multiple RNA and DNA template termini while converting HIV-1 RNA to double-stranded DNA. We have determined the fidelity of HIV-1 RT in vitro during polymerization to the 5' ends of HIV-1 long terminal repeat DNA template sequences and to the end of a partial HIV-1 genomic RNA template that mimics a recombination intermediate. HIV-1 RT readily extended recessed DNA primers to form full-length blunt-end DNA-DNA and DNA-RNA duplexes. In addition, HIV-1 RT catalyzed high yields of products with one to four extra nucleotides at the 3' ends of the nascent DNAs. These products were formed processively via a nontemplated mechanism that is highly specific for the addition of purine nucleotides (A > G >> T > or = C). Thus, HIV-1 RT is extremely unfaithful at both DNA and RNA template ends, introducing errors (extra nucleotides) in one out of every two or three nascent strands processively polymerized. This error rate is 1000 times higher than for HIV-1 RT-catalyzed errors at internal template positions. Blunt-end additions were also catalyzed by other retroviral RTs at relative rates of HIV-1 approximately Moloney murine leukemia virus > avian myeloblastosis virus. These data suggest a potentially important mechanism for retroviral mutation mediated by nontemplated blunt-end addition of purines prior to forced copy-choice recombination.

Base Sequence↗
PubMed1993

Template-directed pausing of DNA synthesis by HIV-1 reverse transcriptase during polymerization of HIV-1 sequences in vitro.

Replication of human immunodeficiency virus type 1 (HIV-1) requires reverse transcriptase (RT) to synthesize double-stranded proviral DNA (9.7 kilobases) through a complex mechanism utilizing both RNA and DNA templates. We have examined DNA synthesis by HIV-1 RT on RNA and DNA templates derived from the HIV-1 genome using a primer extension assay in vitro. Analysis of polymerization products on sequencing gels revealed strong pauses in synthesis, on both RNA and DNA templates, in homopolymeric nucleotide runs, and at regions of predicted secondary structure. Polymerization pauses occurred in runs of template rGs (> or = 4 bases) and rCs (> or = 3 bases) during minus-strand synthesis on RNA templates, and in most runs (> or = 4 bases) of template dTs and dAs during plus-strand synthesis on DNA templates. Pausing also occurred on both templates within the first few nucleotides of the predicted hairpin structures of the Rev response element. The locations of pauses were dependent on template sequence and were unaffected by primer positioning, RT concentration, and ionic strength. Recombinant and virion-derived HIV-1 RTs showed similar pausing patterns. DNA products that accumulated at HIV-1 RT pause sites on RNA templates were extended by continued incubation with excess RT from Moloney murine leukemia virus, showing that the RNA templates were not broken or otherwise unable to support polymerization. Polymerizations conducted in the presence of a poly(rA) oligo(dT) trap showed that pausing results from two mechanisms: 1) RT remaining bound to the primer-template and polymerizing at a greatly reduced rate, or 2) RT dissociating from the primer-template. These results demonstrate that specific HIV-1 RNA and DNA template sequences are capable of interrupting processive DNA synthesis by HIV-1 RT in vitro. Pausing may serve specific functions in HIV-1 replication and mutagenesis. Moreover, these data suggest that one or more accessory factors are required to complete proviral DNA synthesis in vivo and that efficient HIV-1 DNA synthesis may require multiple origins.

Base Sequence↗
PubMed1992

Comparison of Moloney murine leukemia virus mutation rate with the fidelity of its reverse transcriptase in vitro.

The role of Moloney murine leukemia virus (MoMLV) reverse transcriptase (RT) in the generation of base substitution mutations during retroviral replication was analyzed. To that effect, the in vitro fidelity of the MoMLV RT was compared to the rate of base substitution mutations occurring during the replication of an MoMLV-based retroviral vector. Using the vector in an amber reversion assay, the base substitution mutation rate at a single locus was found to be 2 x 10(-6)/base pair in one cycle of vector virus replication. Analysis of the fidelity of the purified RT using the same template sequence revealed that, of the two mispairs (A.C and T.G) that would lead to reversion of the amber codon during replication, A.C occurs at a rate of 4.0 x 10(-6), and T.G occurs at a rate of 0.7 x 10(-4). While the rate of formation of A.C is very similar to the vector mutation rate, the rate of formation of T.G is more than 30 times higher. This discrepancy in rates suggests that there are other elements in the infected cells that contribute to the fidelity of viral replication.

3T3 Cells↗
PubMed1992

Inhibition of reverse transcriptase from feline immunodeficiency virus by analogs of 2'-deoxyadenosine-5'-triphosphate.

The replication of feline immunodeficiency virus (FIV) in cultured cells was inhibited by 2',3'-dideoxyadenosine (ddA) and by 9-(2-phosphonylmethoxyethyl)adenine (PMEA) with IC50 values of 0.98 and 0.95 microM, respectively. The effects of the presumed active forms of these inhibitors, ddATP and PMEA-diphosphate (PMEApp), upon the FIV reverse transcriptase (RT) were examined with two different template-primer systems. Both of these compounds were potent inhibitors of the FIV RT in reactions with primed phi X-174 DNA, yielding Ki values of 8.8 nM for ddATP and 5.0 nM for PMEApp. However, they were both poor inhibitors of the reaction with poly(rU)-oligo(dA); concentrations of ddATP or PMEApp greater than 10 microM were required to inhibit this reaction by 50%. Further analysis of the reaction with poly(rU)-oligo(dA) revealed that even in the absence of inhibitors the primers were extended by less than 20 nucleotides. In contrast, high molecular weight products were obtained in reactions with phi X-174 DNA. These results suggest that the reaction of FIV RT with poly(rU)-oligo(dA) is not highly processive. The high degree of termination encountered during this reaction with poly(rU)-oligo(dA) may be responsible for the low inhibitory potential of ddATP and PMEApp.

Adenine↗
PubMed1991

Dose-dependent differences in the profile of mutations induced by an ultimate carcinogen from benzo[a]pyrene.

Mutations in the coding region of the hypoxanthine (guanine) phosphoribosyltransferase (HPRT) gene of Chinese hamster V-79 cells were examined after exposure of the cells to a high cytotoxic dose (0.48 microM; 35% survival) and a low noncytotoxic dose (0.04 microM; 100% survival) of the ultimate carcinogen (+)-7R,8S-dihydroxy-9S,10R-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-BPDE]. Independent 8-azaguanine-resistant colonies were isolated and cDNAs were prepared by reverse transcription. The coding region of the cDNA of the HPRT gene was amplified by the polymerase chain reaction and sequenced. An examination of the DNA base sequence changes induced by different doses of (+)-BPDE demonstrated that the high dose of (+)-BPDE caused base substitution mutations almost exclusively at G.C base pairs whereas the low dose of (+)-BPDE caused mutations at both G.C and A.T base pairs. Thus, use of a low dose of (+)-BPDE allowed the detection of mutations (at A.T base pairs) that were not readily observed with a high dose of (+)-BPDE. The data also suggest that the low dose of (+)-BPDE may have caused a different profile of base substitutions at G.C base pairs and exon deletions than the high dose. The results indicate dose-dependent differences in the profile of mutations for an ultimate carcinogen.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1991

The vinyl chloride DNA derivative N2,3-ethenoguanine produces G----A transitions in Escherichia coli.

Vinyl chloride is a known human and rodent carcinogen that forms several cyclic base derivatives in DNA. The mutagenic potential of these derivatives has been examined in vitro but not in vivo. One of these derivatives, N2,3-ethenoguanine (epsilon G), is known to base pair with both cytosine and thymine during in vitro DNA synthesis, which would result in G----A transitions. To determine the base pairing specificity of this labile guanine derivative in Escherichia coli, we have developed a genetic reversion assay for guanine derivatives. The assay utilizes DNA polymerase-mediated analogue insertion into a bacteriophage vector, M13G*1, which detects all single-base substitutions at position 141 of the lacZ alpha gene by change in plaque color. After the insertion of a single epsilon G opposite the template cytosine at position 141 by use of epsilon dGTP and DNA polymerase and further extension with all four normal dNTPs, the DNA was transfected into E. coli. Transfection of M13G*1 containing epsilon G at the target site yielded 135 mutants among 26,500 plaques, 134 of which represented G----A transitions. The uncorrected mutation frequency was 0.5%, as compared with the control value, approximately 0.02%; when corrected for epsilon G content and penetrance, the calculated mutagenic potential of epsilon G (mutations/analogue) was about 13%. We thus conclude that epsilon G specifically induces G----A transitions during DNA replication in E. coli. The M13G*1 assay may permit the testing of other labile guanine derivatives not otherwise amenable to mutagenesis studies.

Adenine↗
PubMed1991

Mutagenic spectrum resulting from DNA damage by oxygen radicals.

Oxygen free radicals are highly reactive species that damage DNA and cause mutations. We determined the mutagenic spectrum of oxygen free radicals produced by the aerobic incubation of single-stranded M13mp2 DNA with Fe2+. The Fe2(+)-treated DNA was transfected into component Escherichia coli, and mutants within the nonessential lac Z alpha gene for beta-galactosidase were identified by decreased alpha-complementation. The frequency of mutants obtained with 10 microM Fe2+ was 20- to 80-fold greater than that obtained with untreated DNA. Mutagenesis was greater after the host cells were exposed to UV irradiation to induce the SOS "error-prone" response. The ability of catalase, mannitol, and superoxide dismutase to diminish mutagenesis indicates the involvement of oxygen free radicals. The sequence data on 94 of the mutants establish that mutagenesis results primarily from an increase in single-base substitutions. Ninety-four percent of the mutants with detectable changes in nucleotide sequence were single-base substitutions, the most frequent being G----C transversions, followed by C----T transitions and G----T transversions. The clustering of mutations at distinct gene positions suggests that Fe2+/oxygen damage to DNA is nonrandom. This mutational spectrum provides evidence that a multiplicity of DNA lesions produced by oxygen free radicals in vitro are promutagenic and could be a source of spontaneous mutations.

Base Sequence↗
PubMed1989

Extension of mismatched 3' termini of DNA is a major determinant of the infidelity of human immunodeficiency virus type 1 reverse transcriptase.

The unusually high error rate of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) suggests that polymerization errors by this enzyme contribute to the genetic variability of the AIDS virus. We have analyzed the mechanism for HIV-1 RT infidelity by studying two distinct steps that might lead to base substitution mutations: nucleotide misinsertions and elongation from 3'-terminal DNA mispairs. Our results indicate that the capacity of HIV-1 RT to polymerize nucleotides onto mispaired termini is a major factor in the production of mutations by this enzyme. When a noncomplementary dAMP was inserted opposite a template adenine by HIV-1 RT, the nascent 3'-terminal A.A mispair was readily extended by subsequent incorporation of the next complementary nucleotide. The frequencies of nucleotide addition onto 3'-terminal A-A, A-C, and A-G mispairs were determined by quantitating the amount of extended primers with a gel electrophoresis assay and by measuring mutagenesis after hybridization of mismatched primers opposite an amber mutation in bacteriophage phi X174 DNA. The mispair extension frequencies are approximately 50-fold higher by HIV-1 RT than by the mammalian replicative enzyme DNA polymerase alpha.

Base Composition↗
PubMed1989

Fidelity of two retroviral reverse transcriptases during DNA-dependent DNA synthesis in vitro.

We determined the fidelity of avian myeloblastosis virus and Moloney murine leukemia virus reverse transcriptases (RTs) during DNA synthesis in vitro using the M13mp2 lacZ alpha gene as a mutational target. Both RTs commit an error approximately once for every 30,000 nucleotides polymerized. DNA sequence analysis of mutants generated in a forward mutation assay capable of detecting many types of errors demonstrated that avian myeloblastosis virus RT produced a variety of different mutations. The majority (58%) were single-base substitutions; all of which resulted from the misincorporation of either dAMP or dGMP. Minus-one frameshifts were also common, composing about 30% of the mutations. In addition to single-base events, eight mutants contained sequence changes involving from 2 to 59 bases. The frequency of these mutants suggests that, at least during DNA synthesis in vitro, RTs also commit errors by mechanisms other than classical base miscoding and misalignment. We examined the ability of RTs to synthesize DNA from a mismatched primer terminus at a sequence where the mismatched base was complementary to the next base in the template. Unlike cellular DNA polymerases which polymerize from the mismatched template-primer, RTs preferred to polymerize from a rearranged template-primer containing a matched terminal base pair and an unpaired base in the template strand. The unusual preference for this substrate suggests that the interactions between RTs and the template-primer are different from those of cellular DNA polymerases. The overall error rate of RT in vitro is sufficient to account for the estimated mutation rate of these viruses.

Avian Myeloblastosis Virus↗
PubMed1988

Fidelity of HIV-1 reverse transcriptase.

The human immunodeficiency virus type 1 (HIV-1) shows extensive genetic variation and undergoes rapid evolution. The fidelity of purified HIV-1 reverse transcriptase was measured during DNA polymerization in vitro by means of three different assays. Reverse transcriptase from HIV-1 introduced base-substitution errors in DNA from the bacteriophage phi X174 amber3 at estimated frequencies of 1/2000 to 1/4000. Analyses of misincorporation rates opposite a single template adenine residue showed that HIV-1 reverse transcriptase catalyzed nucleotide mismatches with a specificity of A:C much greater than A:G greater than A:A. The high error rate of HIV-1 reverse transcriptase in vitro translates to approximately five to ten errors per HIV-1 genome per round of replication in vivo. This high error rate suggests that misincorporation by HIV-1 reverse transcriptase is, at least in part, responsible for the hypermutability of the AIDS virus. The specificity of misincorporation may provide a basis for the systematic construction of antiviral nucleosides.

Avian Myeloblastosis Virus↗
PubMed1987

Comparison of the relative mutagenicities of O-alkylthymines site-specifically incorporated into phi X174 DNA.

The relative mutagenicities of O-alkylthymine-DNA adducts were analyzed in vivo by site-specific mutagenesis. Purified DNA polymerases were used to incorporate O4-methyl (Me)-, O4-ethyl (Et)-, O4-isopropyl (iPr)-, or O2-Me-dTTP onto the 3' terminus of a synthetic oligonucleotide (15-mer) hybridized to phi X174 am3 DNA. The product oligonucleotides were further extended in the presence of unmodified dNTPs to yield 21-mers containing single O-alkylthymine adducts opposite the adenine residue of the bacteriophage amber codon. Polyacrylamide gel electrophoresis and nearest-neighbor analyses confirmed the identities and nucleotide positions of the adducts. Transfection and replication of the site-specifically alkylated DNAs in ada- Escherichia coli (defective in the alkyltransferase capable of repairing O4-alkylthymine-DNA adducts) yielded mutant progeny phage with reversion frequencies of: O4-Me-dThd (19.5 X 10(-6) ) greater than O4-Et-dThd (7.5 X 10(-6) ) greater than O4-iPr-dThd (3.0 X 10(-6) ) greater than or equal to O2-Me-dThd (1.0 X 10(-6) ) approximately equal to dThd (2.0 X 10(-6) ). None of the adducts produced mutations above background following replication in ada+ E. coli. DNA sequence analyses of 40 independently isolated mutant phage derived from the O4-Me- or O4-Et-dThd-containing DNAs showed that all mutants contained guanine residues opposite the original site of the alkylthymines. These data are consistent with a mechanism of mutagenesis involving the formation of O4-alkyl-T.G base pairs during DNA replication in E. coli and suggest that the formation of A.T----G.C transition mutations is characteristic of mutagenesis by O4-Me- and O4-Et-dThds in vivo.

Bacteriophage phi X 174↗
PubMed1987

O-Alkyl deoxythymidines are recognized by DNA polymerase I as deoxythymidine or deoxycytidine.

The O2- and O4-methyldeoxythymidine triphosphates (O-alkyl dTTP) can be used to substitute for dTTP in Escherichia coli DNA polymerase I (Pol I)-catalysed synthesis of poly[deoxyadenosine-deoxythymidine] (dA-dT). When incorporated into the polynucleotide, no detectable perturbation of structure occurred with even 20% O-methyldeoxythymidine in place of dT. However, on replication of such polymers with Pol I, significant amounts of deoxyguanosine triphosphate (dGTP) were incorporated, as well as high levels of deoxyadenosine triphosphate (dATP), indicating tautomer-like behaviour. Higher homologues, such as O4-ethyl (e4) dTTP or O4-isopropyl (ip4) dTTP, could also replace dTTP, but with lower efficiency. Nevertheless, their presence, like O4-methyl (m4) dT substitutions, caused transitions as well as inhibiting enzyme digestion with a variety of 3' nucleases, particularly to the 3'----5' exonuclease activity (proofreading) of polymerases. Further proof of mutagenicity comes from site-directed experiments placing m4dT or e4dT in place of dT at position 587 in am3 of phi X174, in which all revertants sequenced had A----G transitions. This implies that, since m4dT and e4dT are poorly repaired in eukaryotes, it is likely that they will remain in the DNA and lead to effects on enzyme activity, as well as mutations which contribute to the carcinogenicity of N-nitroso compounds.

Alkylation↗
PubMed1986

Mutagenic potential of O4-methylthymine in vivo determined by an enzymatic approach to site-specific mutagenesis.

O4-Alkylthymine-DNA adducts have been implicated as causative lesions in chemical mutagenesis and carcinogenesis. To directly assess the mutagenic potential of these adducts in vivo, we have designed an enzymatic technique for introducing nucleotide analogues at predetermined sites of biologically active DNA. Escherichia coli DNA polymerase I was used in vitro to incorporate a single O4-methylthymine residue at the 3' terminus of an oligonucleotide primer opposite the adenine residue of the amber codon in bacteriophage phi X174 am3 DNA. After further extension of the primer with unmodified nucleotides, the partial-duplex product was transfected into E. coli spheroplasts. Replication of the site-specifically methylated DNA in E. coli deficient in O4-methylthymine-DNA methyltransferase (ada-) yielded 10-fold more mutant progeny phage than replication of nonmethylated DNA; no increase in mutation frequency was observed after replication in repair-proficient (ada+) E. coli. The DNA from 20 independently isolated mutant plaques all contained A.T----G.C transitions at the original site of O4-methylthymine incorporation. These data demonstrate that O4-methylthymine induces base-substitution mutations in E. coli and suggest that this adduct may be involved in mutagenesis by N-nitroso methylating agents. This enzymatic technique for site-specific mutagenesis provides an alternative to the chemical synthesis of oligonucleotides containing altered bases.

Base Sequence↗
PubMed1985

The activities of 2,2',5,5'-tetrachlorobiphenyl, its 3,4-oxide metabolite, and 2,2',4,4'-tetrachlorobiphenyl in tumor induction and promotion assays.

Sensitive assays for the induction of lung adenomas in A/J mice or skin papillomas in SENCAR mice failed to show activity for either 2,2',5,5'-tetrachlorobiphenyl or 2,2',5,5'-tetrachlorobiphenyl 3,4-oxide. Injections of the 3,4-oxide into preweanling A/J mice caused considerable mortality, whereas the parent hydrocarbon did not. Both 2,2',5,5'- and 2,2',4,4'-tetrachlorobiphenyl showed promoting activity for hepatic gamma-glutamyl transpeptidase-positive foci initiated in rat liver by treatment with diethylnitrosamine. The promoting activity of 2,2',4,4'-tetrachlorobiphenyl was approximately 10-fold greater than that of the 2,2',5,5'-isomer.

Adenoma↗
PubMed1984

Reactions of 2,2',5,5'-tetrachlorobiphenyl 3,4-oxide with methionine, cysteine and glutathione in relation to the formation of methylthio-metabolites of 2,2',5,5'-tetrachlorobiphenyl in the rat and mouse.

Non-enzymatic reactions of the 3,4-oxide of 2,2'5,5'-tetrachlorobiphenyl (TCB) with methionine or N-acetylmethionine in ethanol/neutral buffer at 37 degrees C proceeded very slowly to yield an approx. 1:1 ratio of 3- and 4-methylthio-TCB. Under similar conditions reaction of TCB 3,4-oxide with cysteine proceeded about 100 times more rapidly to yield an approx. 1:1 ratio of 3- and 4-(cystein-S-yl)-TCB as the major products. Cystein-S-yl-3,4-dihydro-hydroxy-TCB(s) was also formed as a minor product from reaction of TCB 3,4-oxide with cysteine in dimethyl sulfoxide/neutral buffer. TCB 3,4-oxide did not react detectably with glutathione in ethanol/neutral buffer at 37 degrees C or 70 degrees C, but reaction in ethanol/pH 8.7 buffer at 37 degrees C proceeded very rapidly to yield about a 1:1 ratio of 3- and 4-(glutathion-S-yl)-TCB and of two glutathion-S-yl-TCB precursors. Glutathion-S-yl-TCB(s) and its precursor(s) were also formed rapidly in a rat liver cytosol-catalyzed reaction of TCB 3,4-oxide with glutathione at neutral pH. The glutathion-S-yl-TCBs readily degraded upon concentration in aqueous alcohol solutions under mild conditions to yield compounds tentatively identified as [N-(5-carboxy-1-pyrrolin-2-yl)-1-glycinocystein-S-yl]-TCBs, (1-glycinocystein-S-yl)-TCBs and 2-oxopyrrolidine-5-carboxylic acid. Rats given a single dose of TCB excreted about 0.07% of the dose in the feces during the first 4 days as 3-methylthio-TCB, 4-methylthio-TCB, 4-methylsulfonyl-TCB, methylthio-hydroxy-TCBs (tentatively identified) and mercapto-TCB(s) (tentatively identified) in about a 1:5:0.1:0.1:0.05 ratio, respectively. Rats given an equimolar dose of TCB 3,4-oxide excreted similar ratios of these fecal metabolites in approx. 10-fold greater quantities. Mice given TCB excreted about 0.1% of the dose in the feces during the first 4 days as 3-methylthio-TCB, 4-methylthio-TCB and 3-methylsulfonyl-TCB in about a 1.5:1:0.05 ratio, respectively. Methylthio-TCBs were not detected (less than 0.0004% of the dose) in the bile of a cannulated rat given a single dose of TCB. About 1.5% of the TCB dose was excreted in the bile as glutathion-S-yl-TCB(s) and its precursor(s). Collectively, the data indicate that TCB 3,4-oxide is a primary metabolic intermediate in the formation of methylthio-metabolites of TCB.

Animals↗