My personal thoughts on the recent joint U.S./Japan cancer meeting in Honolulu, Hawaii.
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Congresses as Topic↗
Biomedical subjects
L A Loeb
Publications and source records attributed to L A Loeb.
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↗
Selection of new biological activities from random nucleotide sequences: evolutionary and practical considerations.
Recent advances in the selection of biologically active DNA sequences from random populations are reviewed. Within the framework of evolution, forces are considered that have precluded the testing of all possible DNA sequences, purely with regard to their functionality as genetic regulatory elements or protein coding sequences. Examples are drawn from cassette mutagenesis of enzyme active sites, protein domain replacement by fusion with random genomic digests, and the selection of bacterial promoters from random DNA. Efforts to derive new activities are examined, and the likelihood of future success is evaluated.
Base Sequence↗
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↗
DNA sequences of random origin as probes of Escherichia coli promoter architecture.
In order to better understand the role of the -35 sequence motif in transcription initiation by Escherichia coli sigma 70 RNA polymerase holoenzyme, -35 promoter elements of limited nucleotide composition have been selected from random DNA sequences. Functional promoter elements have been identified that are composed of just two nucleotide species (A,T; G,C; T,C; and T,G) and one nucleotide species (poly(dT)). From this study of 81 promoter mutations, two conclusions can be made. First, given a population of random DNA sequences, there exist sequences that form stronger -35 promoter elements than the consensus sequence. These sequences lack some features of the consensus sequence and reveal unexpected homology in the ordinarily nonconserved spacer sequence. Second, the sequence requirements at the -35 site may suggest a possible role for DNA secondary structure in the recognition of promoters by RNA polymerase.
Base Sequence↗
Resistance of HeLa cell mitochondrial DNA to mutagenesis by chemical carcinogens.
The mutagenic potentials of ethylmethane sulfonate, N-methyl-N'-nitrosoguanidine, and benzo(a)pyrene diol-epoxide in human mitochondria were determined by cloning and nucleotide sequencing of mitochondrial (mt) DNA from HeLa cells treated with these mutagens. Mutagen concentrations that reduced cell survival to approximately 0.1% of untreated cultures were used. Mitochondrial DNA was prepared 2 to 3 weeks after mutagen treatment, at which time the treated cell population had regrown to 10 times the starting cell number. In one series of experiments, a portion of the D-loop region of mtDNA from treated or control HeLa cells was cloned into the bacteriophage vector M13mp19, and the nucleotide sequences of 102 independent clones were determined. Only a single G:C base pair deletion was observed in 1 of 12 clones derived from HeLa cells treated 6 times with ethylmethane sulfonate. From benzo(a)pyrene diol-epoxide-treated HeLa cells, G:C base pair deletions were found in 14 of 63 clones. All 14 of these G:C deletion mutations occurred at the same position in independent clones, however, and thus could be the progeny of a single mutational event. In a second series of experiments, a method for the selection of mtDNA mutants was utilized. Mutations in an "uncloneable" fragment of human mtDNA render the fragment cloneable and thus provide a selection for mutations in this region of human mtDNA. No enhancement in the cloning efficiency of this region of mtDNA was observed after exposure of cells to toxic concentrations of either MNNG or benzo(a)pyrene diol-epoxide. Moreover, the site and types of nucleotide sequence alterations observed after mutagen treatment were similar to those obtained in the absence of drug treatment. The results of both types of experiments suggest that mutagenesis of human mtDNA is an infrequent event, even after extensive treatment of HeLa cells with potent mutagens that can covalently modify mtDNA.
7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
An E. coli promoter that regulates transcription by DNA superhelix-induced cruciform extrusion.
DNA can form structures other than the Watson-Crick double helix. The potential contributions to gene regulation from one such structure have been investigated by assembling a promoter capable of adopting cruciform base-pairing. Transcription from this promoter by RNA polymerase in vitro was repressed as the cruciform was extruded by increasing negative DNA supercoiling. Transcription in vivo was induced as supercoiling was relaxed by growth in conditions that inhibit DNA gyrase. A DNA conformational change is therefore capable of regulating the initiation of transcription.
DNA Topoisomerases, Type II↗
Specificity of proofreading by the 3'----5' exonuclease of the DNA polymerase-primase of Drosophila melanogaster.
The DNA polymerase-primase from Drosophila melanogaster contains a cryptic 3'----5' exonuclease that can be detected after separation of the 182-kDa polymerase subunit from the four-subunit enzyme. To determine the specificity of excision of mispaired nucleotides by the exonuclease, we have utilized primed phi X174am3 single-stranded DNA containing a noncomplementary nucleotide at the 3'-primer terminus, opposite deoxyadenosine at position 587 in the amber3 codon of the template strand. In the absence of polymerization, the preference for excision of the mispaired nucleotide from the primer is C greater than A much greater than G. Excision under these conditions is inhibited by the addition of deoxyguanosine monophosphate. Under conditions of concomitant DNA synthesis, the preference for excision at this site becomes A = G much greater than C, and excision is insensitive to deoxyguanosine monophosphate. The high fidelity of DNA synthesis exhibited by the isolated 182-kDa polymerase subunit is not reduced by concentrations of deoxyguanosine monophosphate or adenosine monophosphate that inhibit proofreading by prokaryotic DNA polymerases. Thus, the 3'----5' exonuclease of the Drosophila DNA polymerase-primase participates in exonucleolytic proofreading by excising noncomplementary nucleotides prior to extension of the primer by polymerase action. The deoxynucleoside triphosphate analogs N2-(p-butylphenyl)deoxyguanosine triphosphate and N2-(p-butylphenyl)deoxyadenosine triphosphate are potent inhibitors of DNA polymerase alpha. Like calf thymus DNA polymerase delta, recently determined to have proofreading capability, DNA synthesis by the isolated Drosophila 182-kDa polymerase subunit was not inhibited by the two analogs. In contrast, DNA synthesis by the intact Drosophila polymerase-primase complex was inhibited greater than 95% by these analogs.
Animals↗
Factor D is a selective single-stranded oligodeoxythymidine binding protein.
Factor D, a protein purified from rabbit liver that selectively enhances traversal of template oligodeoxythymidine tracts by diverse DNA polymerases, was examined for the sequence specificity of its binding to DNA. Terminally [32P]-labeled oligomers with the sequence 5'-d[AATTC(N)16G]-3', N being dT, dA, dG, or dC, were interacted with purified factor D and examined for the formation of protein-DNA complexes that exhibit retarded electrophoretic mobility under nondenaturing conditions. Whereas significant binding of factor D to 5'-d[AATTC(T)16G]-3' is detected, there is no discernable association between this protein and oligomers that contain 16 contiguous moieties of dG, dA, or dC. Furthermore, factor D does not form detectable complexes with the duplexes oligo(dA).oligo(dT) or poly(dA).poly(dT). The preferential interaction of factor D with single-stranded poly(dT) is confirmed by experiments in which the polymerase-enhancing activity of this protein is protected by poly(dT) against heat inactivation two- and four-fold more efficiently than by poly(dA) or poly(dA).poly(dT), respectively.
Animals↗
Direct selection of mutations in the human mitochondrial tRNAThr gene: reversion of an 'uncloneable' phenotype.
Several regions of the human mitochondrial genome are refractory to cloning in plasmid and bacteriophage DNA vectors. For example, recovery of recombinant M13 clones containing a 462 basepair MboI-Kpn I restriction fragment that spans nucleotide positions 15591 to 16053 of HeLa cell mitochondrial DNA was as much as 100-fold lower than the recovery of M13 clones containing other regions of the human mitochondrial genome. All of 50 recombinant M13 clones containing this 'uncloneable' fragment had one or more changes in nucleotide sequence. Each clone contained at least one alteration in two nucleotide positions within the tRNAThr gene that encode portions of the anticodon loop and D-stem of the HeLa mitochondrial tRNAThr. These results imply that the HeLa mitochondrial tRNAThr gene is responsible for the 'uncloneable' phenotype of this region of human mitochondrial (mt) DNA. A total of 61 nucleotide sequence alterations were identified in 50 independent clones containing the HeLa mt tRNAThr gene. 56 mutations were single-base substitutions; 5 were deletions. Approximately 80% of the base substitution mutations were A:T----G:C transitions. A preference for A:T----G:C transition mutations also characterizes polymorphic base substitution variants in the mitochondrial DNA of unrelated individuals. This similarity suggests that human mitochondrial DNA sequence variation within and between individuals may have a common origin.
Base Sequence↗
Mutagenesis by the autoxidation of iron with isolated DNA.
Oxygen free radicals are highly reactive species generated by many cellular oxidation-reduction processes. These radicals damage cellular constituents and have been causally implicated in the pathogenesis of many human diseases. We report here that oxygen free radicals generated by Fe2+ in aqueous solution are mutagenic. Aerobic incubation of luminal diameter X174 am3 (amber 3 mutation) DNA with Fe2+ results in decreased phage survival when the treated DNA is transfected into Escherichia coli spheroplasts. Transfection of the treated DNA into SOS-induced spheroplasts results in an increase in mutagenesis as great as 50-fold. Both killing and mutagenesis can be prevented by binding of Fe2+ with deferoxamine or by the addition of catalase or mannitol. These results suggest that DNA damage and mutagenesis brought about by Fe2+ are likely to occur by a Fenton-type mechanism that involves the generation of (i) hydrogen peroxide by the autoxidation of iron and (ii) hydroxyl radicals by the interaction of the hydrogen peroxide with Fe2+. DNA sequence analysis of the Fe2+-induced mutants indicates that reversion of the phage phenotype to wild type occurs largely by a transversion type of mutation involving substitution of deoxyadenosine for thymidine opposite a template deoxyadenosine. Mutagenesis is not abolished by incubation of Fe2+-treated luminal diameter X174 am3 DNA with an apurinic endonuclease and only partially abolished by incubation with alkali, suggesting that a large fraction of the mutagenesis by oxygen free radicals is not caused by formation of apurinic sites but instead involves an as-yet-to-be-defined alteration in deoxyadenosine. These findings raise the possibility that free iron localized in cellular DNA may cause mutations by the generation of oxygen free radicals.
Bacteriophage phi X 174↗
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↗
On the fidelity of DNA replication. Isolation of high fidelity DNA polymerase-primase complexes by immunoaffinity chromatography.
Error rates for conventionally purified DNA polymerase-alpha from calf thymus, chicken, and human sources have been reported to be one in 10,000 to one in 40,000 nucleotides incorporated. Isolation of polymerase-alpha by immunoaffinity chromatography yields a multiprotein high molecular weight replication complex that contains an associated DNA primase (Wong, S. W., Paborsky, L. R., Fisher, P. A., Wang, T. S-F., and Korn, D. (1986) J. Biol. Chem. 261, 7958-7968). We have isolated DNA polymerase-primase complexes from calf thymus, from a human lymphoblast cell line (TK-6), and from Chinese hamster lung cells (V-79) using two different methods of immunoaffinity chromatography. These enzyme complexes are 12- to 20-fold more accurate than conventionally purified calf thymus DNA polymerase-alpha when assayed using the phi X174am3 fidelity assay; estimated error rates are one in 460,000 to one in 830,000 nucleotides incorporated when the enzyme complex is freshly isolated. The polymerase-primase complex from calf thymus exhibited no detectable 3'----5' exonuclease activity using a heteroduplex substrate containing a single 3'-terminal mismatched nucleotide. Upon prolonged storage at -70 degrees C, the error rate of the immunoaffinity-purified calf thymus DNA polymerase-primase complex increases to about one in 50,000 nucleotides incorporated, an error rate similar to that exhibited by conventional isolates of DNA polymerase-alpha.
Animals↗
The DNA sequence specificity of stimulation of DNA polymerases by factor D.
The mechanism of enhancement of DNA polymerase activity by the murine DNA-binding protein factor D was investigated. Extension by Escherichia coli DNA polymerase I and calf thymus DNA polymerase-alpha of 5'-32P-labeled oligodeoxynucleotide primers that are complementary to poly(dT) or to bacteriophage M13 DNA was measured in the absence or presence of factor D. With 5'-[32P](dA)9.poly(dT), factor D enables E. coli polymerase I to fill approximately 15-nucleotide gaps between adjacent primers; whereas in the absence of the stimulatory protein, poly(dT) is not copied significantly. In order to study the nucleotide specificity of synthesis enhancement, we used M13mp10 DNA containing 4 consecutive thymidine residues downstream from the 3-hydroxyl terminus of an oligonucleotide primer. Upon addition of factor D, both polymerase I and polymerase-alpha can traverse this sequence more efficiently and thus generate longer DNA products. Densitometric analysis of nonextended and elongated 5'-32P-labeled M13 primer indicates that, without changing the frequency of primer utilization, factor D enhances the activity of these DNA polymerases by increasing their apparent processivity. By positioning oligonucleotide primers 4, 8, and 12 bases upstream from the (dT)4 template sequence, we show that the enhancement of synthesis by factor D is independent of the position of the oligothymidine cluster. We hypothesize that factor D interacts with oligo(dT).oligo(dA) domains in DNA to alter their conformation, which may normally obstruct the progression of DNA polymerases.
Animals↗
On the fidelity of DNA replication: herpes DNA polymerase and its associated exonuclease.
Procaryotic DNA polymerases contain an associated 3'----5' exonuclease activity which provides a proofreading function and contributes substantially to replication fidelity. DNA polymerases of the eucaryotic herpes-type viruses contain similar associated exonuclease activities. We have investigated the fidelity of polymerases purified from wild type herpes simplex virus, as well as from mutator and antimutator strains. On synthetic templates, the herpes enzymes show greater relative exonuclease activities, and greater ability to excise a terminal mismatched base, than procaryotic DNA polymerases which proofread. On a phi X174 natural DNA template, the herpes enzymes are more accurate than purified eucaryotic DNA polymerases; the error rate is similar to E. coli polymerase I. However, conditions which abnegate proofreading by E. coli polymerase I have little effect on the herpes enzymes. We conclude that either these viral polymerases are accurate in the absence of proofreading, or the conditions examined have little effect on proofreading by the herpes DNA polymerases.
DNA Replication↗
Aphidicolin-resistant mutator strains of mouse teratocarcinoma.
From among a series of stable, aphidicolin-resistant mutant strains of mouse teratocarcinoma, derived from a multipotent parental line (PSA-1-80), three were selected for further study on the basis of their comparatively high degrees of resistance and elevated frequencies of spontaneous forward mutation to 6-thioguanine and ouabain resistance. Fluctuation tests confirmed that they were mutator strains. Since each of the three mutants was isolated after multiple rounds of selection, and since a variety of biochemical abnormalities were observed, it is likely that a number of mechanisms, probably consisting of overlapping subsets, determine the phenotypes. Abnormalities in the metabolism of the nucleotide substrates for polymerization are likely to be of major importance in mutants designated Aph-2 and Aph-3, as there were marked alterations in the dCTP and dATP pool sizes. The specific activity of DNA polymerase alpha was also increased. For the case of Aph-3, which exhibited the greatest (400-fold) increase in resistance to aphidicolin, a mutation in the structural gene for DNA polymerase alpha may be an additional important component, since in vitro assays revealed that the isolated enzyme was resistant to aphidicolin. For the case of Aph-1 however, only minor alterations in dNTP pools were observed, and there was no increase in the specific activity of DNA polymerase alpha or in the aphidicolin resistance of the isolated DNA polymerase alpha, suggesting yet another mechanism(s) underlying the aphidicolin resistance/mutator phenotype. All three mutants formed subcutaneous tumors in syngeneic mice; both Aph-1 and Aph-2 were multipotent; whereas Aph-3 was nullipotent.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Metal-induced lethality and mutagenesis: possible role of apurinic intermediates.
The possible mechanisms by which various metals exert their mutagenic effects were investigated using both chemical and biochemical techniques. Ions of Cu, Ni and Cr enhanced the release of either adenine [Cu(II) and Ni(II)] or guanine [Cr(VI)] from DNA as measured in a chromatography assay, suggesting the possible importance of depurination in metal-induced mutagenesis. Transfection experiments with single-stranded bacteriophage phi X174 DNA indicated that micromolar levels of Cu(II), Cr(III), Cr(VI) and Pt(IV) are capable of causing extensive lethal damage to the phage DNA. In case of Cu(II) and Pt(IV) this damage proved mutagenic for phi X174 am3 after transfection of DNA into SOS-induced spheroplasts. For Cu(II) mutagenesis is likely due to the release of adenine residues from the phage DNA based on the abolishment of mutagenesis by alkali and the observed specificity of the phage revertants. The enhancement of the adenine depurination rate by Cu(II) was estimated to be as high as 10,000-fold.
Adenine↗
Base substitution mutagenesis by terminal transferase: its role in somatic mutagenesis.
We have addressed the possibility of terminal transferase involvement in somatic mutagenesis and the creation of N-region diversity, by measuring the ability of TdT to enhance single-base substitution mutagenesis during in vitro DNA synthesis. Using 3 independent assays we find that terminal transferase produces only a small increase in base-substitution mutagenesis when assayed in the presence of DNA polymerase-beta. In the presence of either polymerase-alpha or E. coli polymerase-I, however, no detectable increase in TdT-induced mutagenesis is seen. Furthermore, in an assay capable of detecting a variety of mutational events, terminal transferase primarily produces complex addition/deletion mutations, as well as a few multiple, tightly-clustered, single-base mutations. We conclude that the majority of the scattered single-base changes that occur during antibody gene differentiation are not catalyzed by terminal transferase, but instead result from another error-prone DNA synthetic process (possibly utilizing DNA polymerase-beta).
Bacteriophage phi X 174↗