Search PubMed⌕ Search

Biomedical subjects

L A Loeb

Publications and source records attributed to L A Loeb.

At least 19 recordsLinked to original sources
PubMed1992

Mutations induced by methylene blue plus light in single-stranded M13mp2.

Reactive oxygen species are generated by a variety of cellular processes. These endogenously generated, reactive intermediates produce a multiplicity of DNA alterations and mutations and have been implicated in the pathogenesis of several human diseases. We report here that treatment of single-stranded M13mp2 bacteriophage DNA with methylene blue and white light generates increased levels of 8-hydroxydeoxyguanosine and that mutagenesis is both highly specific and dependent on the SOS response. Lesions produced block the progression of DNA synthesis one base preceding template guanines. In SOS-induced Escherichia coli, 97% of all methylene blue-induced mutations in the lacZ alpha gene of M13mp2 DNA are single-base substitutions opposite template guanines. The most frequent mutations are G----C transversions. The G----T transversions expected from the presence of 8-hydroxydeoxyguanosine in the template strand occur, but at a lower frequency. Sequence data together with SOS dependency and the presence of replication blockage demonstrate that while 8-hydroxydeoxyguanosine may serve as an important marker to monitor oxygen-induced DNA damage in humans, it does not account for either the observed blockage to replication or the mutagenesis by methylene blue plus light in SOS-induced E. coli. Instead, an as yet unidentified lesion generated by active oxygen species is a more potent mutagenic event.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1992

Permissible amino acid substitutions within the putative nucleoside binding site of herpes simplex virus type 1 encoded thymidine kinase established by random sequence mutagenesis [corrected].

We determined the essentiality of all amino acid replacements within an 11-codon sequence in the putative nucleoside-binding site of thymidine kinase encoded by herpes simplex virus type 1. This involved partial randomization of 11 codons in the gene to create a degenerate library, followed by genetic complementation using a tk- Escherichia coli strain and selection of unnatural active enzymes. We produced and tested 53,000 variants; of which 190 were found to be biologically active. Sequence analyses of functional variants revealed a high degree of flexibility in accommodating different types of amino acid substitutions in this region. However, no replacement was tolerated at proline-173, whereas tyrosine-172 could be replaced by only phenylalanine. To further define permissible substitutions at specified positions, we constructed a library with randomization at only four test codons. We produced and tested 600,000 variants; of which only 5 were active. Again proline-173 was conserved, and only tyrosine and phenylalanine were found at position 172. The identification of these conserved amino acids should provide important insights into the understanding of the structural basis of catalysis by this enzyme.

Amino Acid Sequence↗
PubMed1992

Mutagenic specificity of oxygen radicals produced by human leukemia cells.

An important source of endogenous oxygen radicals are phagocytic cells such as neutrophils and macrophages. The human leukemia cell line HL-60 can be induced to differentiate into a neutrophil-like cell population. Among the properties of these differentiated cells is the ability to produce reactive oxygen species when stimulated by tumor promoters. Mutagenesis induced by HL-60-generated free radicals was assessed using the M13mp2 forward mutation assay. Single-stranded M13mp2 DNA was coincubated with phorbol ester-stimulated HL-60 cells, after which mutations were scored by transfecting the DNA into SOS-induced Escherichia coli. The mutation frequency was increased 6-fold above background in DNA incubated with HL-60 cells. The majority of the mutations were single-base substitutions. However, approximately 6% of the mutations were tandem double substitutions that occurred in runs of adjacent cytidines. Overall, the mutations were clustered at apparent "hot spots," many of which were similar to sites seen using iron to generate oxygen radicals. These results suggest that human cells able to produce oxygen radicals in response to tumor promoters might play a significant role in the generation of tumors.

DNA, Bacterial↗
PubMed1992

Fidelity of HIV-1 reverse transcriptase copying RNA in vitro.

The genomic hypervariation of human immunodeficiency virus 1 (HIV-1) could result from misincorporations by the viral reverse transcriptase. We developed an assay for reverse transcriptase fidelity during RNA-dependent as well as DNA-dependent DNA polymerization in vitro. A lacZ alpha RNA fragment transcribed by T3 RNA polymerase was used to mimic first-strand reverse transcription. The corresponding DNA template was used to examine errors by reverse transcriptase during second-strand DNA synthesis. With both templates, the mutations introduced by reverse transcriptase were identified by their mutant phenotypes in an M13 lacZ alpha-complementation assay. We found that the reverse transcriptase from human immunodeficiency virus 1 (HIV-1 RT) was less accurate than the reverse transcriptase from Moloney murine leukemia virus (MLV RT) or the Klenow fragment of Escherichia coli DNA polymerase I (Pol I) on either RNA or DNA templates. The frequency of misincorporation by HIV-1 RT was 1 in 6900 nucleotides polymerized on the RNA template and 1 in 5900 on the DNA template. The error rates of MLV RT and Pol I on the RNA template were less than 1 in 28,000 and 37,000, respectively. The most frequent mutations produced by HIV-1 RT copying the RNA template were C----T transitions and G----T transversions resulting from misincorporation of dAMP.

Animals↗
PubMed1992

Mammalian DNA polymerase beta can substitute for DNA polymerase I during DNA replication in Escherichia coli.

Mammalian DNA polymerase beta is the smallest known eukaryotic polymerase and is expressed as an active protein in Escherichia coli harboring a plasmid containing its cDNA. Since some catalytic functions of DNA polymerase beta and E. coli DNA polymerase I are similar, we wished to determine if DNA polymerase beta could substitute for DNA polymerase I in bacteria. We found that the expression of mammalian DNA polymerase beta in E. coli restored growth in a DNA polymerase I-defective bacterial mutant. Sucrose density gradient analysis revealed that DNA polymerase beta complements the replication defect in the mutant by increasing the rate of joining of Okazaki fragments. These findings demonstrate that DNA polymerase beta, believed to function in DNA repair in mammalian cells, can also function in DNA replication. Moreover, this complementation system will permit study of the in vivo function of altered species of DNA polymerase beta, an analysis currently precluded by the difficulty in isolating mutants in mammalian cells.

Animals↗
PubMed1992

A DNA polymerase alpha pause site is a hot spot for nucleotide misinsertion.

In this study we examined whether the arrest of DNA polymerase alpha (pol alpha)-catalyzed DNA synthesis at template pause sites entails terminal nucleotide misincorporation. An approach was developed to identify the 3'-terminal nucleotide in nascent DNA chains that accumulate at pause sites. A radioactive 5'-end-labeled primer was annealed to a bacteriophage M13mp2 single-stranded DNA template and elongated by pol alpha. Individual DNA chains that were accumulated at pause sites were resolved by sequencing gel electrophoresis, isolated, and purified. These DNA chains were elongated by pol alpha by using four annealed synthetic DNA templates, each of which contained a different nucleotide at the position opposite the 3' terminus of the arrested chain. Owing to the high preference of pol alpha for matched-over-mismatched primer termini, only those templates that contain a nucleotide that is complementary to the 3' terminus of the isolated pause-site chain are copied. Electrophoresis of product DNA showed the extent of copying of each template and thus identified the 3'-terminal nucleotide of the pause-site chains. We found that product DNA chains terminate with a noncomplementary 3'-terminal nucleotide opposite pause sites within the sequence 3'-d(AAAA)-5' at positions 6272-6269 of the M13mp2 genome. pol alpha catalyzed misincorporation of dG or dA into the 3' terminus of nascent chains opposite two of the M13mp2 template dA residues. A similar analysis of a different pause site did not reveal significant misincorporation opposite template dC. These results suggest that some but not all sites at which pol alpha pauses may constitute loci of mutagenesis.

Base Sequence↗
PubMed1992

8-Hydroxyguanine, an abundant form of oxidative DNA damage, causes G----T and A----C substitutions.

Mutations caused by oxidative DNA damage may contribute to human disease. A major product of that damage is 8-hydroxyguanine (oh8Gua). Because of differences in experimental design, the base pairing specificity of oh8G in vivo is not completely resolved. Here, oh8dGTP and DNA polymerase were used in two complementary bacteriophage plaque color assays to examine the mutagenic specificity of oh8Gua in vivo. The first is a reversion assay that detects all three single-base substitutions caused by misreading of guanine analogues inserted at a specific site. oh8Gua at that site gave a mutation frequency of 0.7%. Twenty-two of the 23 mutations were G----T substitutions. The second assay, a forward mutation assay, tests the mispairing potential of any altered nucleotide 1) during incorporation as substrate nucleotide, and 2) after multiple incorporations into a single-stranded DNA gap region of M13mp2. Substituting oh8dGTP for dGTP during polymerization produced 16% mutants; two classes of mutations were observed, both caused by pairing of oh8Gua with A. Seventy-six of 78 mutations were A----C substitutions, and two were G----T substitutions. These assays thus illustrate mutagenic replication of oh8Gua as template causing G----T substitutions and misincorporation of oh8Gua as substrate causing A----C substitutions, both caused by oh8Gua.A mispairs.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1992

DNA damage and repair in brain: relationship to aging.

The usefulness of conducting DNA damage and repair studies in a postmitotic tissue like brain is emphasized. We review studies that use brain as a tissue to test the validity of the DNA damage and repair hypothesis of aging. As far as the accumulation of age dependent DNA damage is concerned, the data appear to overwhelmingly support the hypothesis. However, attempts to demonstrate a decline in DNA repair capacity as a function of age are conflicting and equally divided. Possible reasons for this discrepancy are discussed. It is suggested that assessment of the repair capacity of neurons with respect to a specific type of damage in a specific gene might yield more definitive answers regarding the role of DNA repair potential in the aging process and as a longevity assurance system.

Aging↗
PubMed1991

Artificial mutants generated by the insertion of random oligonucleotides into the putative nucleoside binding site of the HSV-1 thymidine kinase gene.

We have obtained 42 active artificial mutants of HSV-1 thymidine kinase (ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21) by replacing codons 166 and 167 with random nucleotide sequences. Codons 166 and 167 are within the putative nucleoside binding site in the HSV-1 tk gene. The spectrum of active mutations indicates that neither Ile166 nor Ala167 is absolutely required for thymidine kinase activity. Each of these amino acids can be replaced by some but not all of the 19 other amino acids. The active mutants can be classified as high activity or low activity on two bases: (1) growth of Escherichia coli KY895 (a strain lacking thymidine kinase activity) in the presence of thymidine and (2) uptake of thymidine by this strain, when harboring plasmids with the random insertions. E. coli KY895 harboring high-activity plasmids or wild-type plasmids can grow in the presence of low amounts of thymidine (less than 1 microgram/mL), but are unable to grow in the presence of high amounts of thymidine. On the other hand, E. coli KY895 harboring low-activity plasmids can grow at a high concentration of thymidine (greater than 50 microgram/mL) in the media. The high-activity plasmids also have an enhanced [3H]dT uptake. The amounts of thymidine kinase activity in vitro in unfractionated extracts do not correlate with either growth at low thymidine concentration or the rate of thymidine uptake. Heat inactivation studies indicate that the mutant enzymes are without exception more temperature-sensitive than the wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
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

Isolation of temperature-sensitive DNA polymerase III from Saccharomyces cerevisiae cdc2-2.

DNA polymerase III of the yeast Saccharomyces cerevisiae has been reported to be encoded at the CDC2 locus based on two observations. First, the CDC2 gene has homology to known DNA polymerase genes [Boulet et al. (1989) EMBO J. 8, 1849-1854], and second, the mutants cdc2-1 and cdc2-2 yield little or no DNA polymerase III activity in vitro [Boulet et al. (1989); Sitney et al. (1989) Cell 56, 599-605]. We describe here the isolation of temperature-sensitive DNA polymerase III from cdc2-2 strains. Our results provide direct experimental confirmation of the previously inferred gene/enzyme relationship and verify the conclusion that DNA polymerase III is required to replicate the genome. We isolated DNA polymerase III from two cdc2-2 strains, one containing the wild-type allele for DNA polymerase I (CDC17) and the other a mutant DNA polymerase I allele (cdc17-1). Yields from cdc2-2 cells of both DNA polymerase III activity and an associated 3'-5'-exonuclease activity [exonuclease III; Bauer et al. (1988) J. Biol. Chem. 263, 917-924] were decreased relative to yields from CDC2 cells. DNA polymerase III activity from cdc2-2 strains is thermolabile, displaying at least a 4-fold reduction in half-life at 44 degrees C. The activity is also labile at 37 degrees C, a temperature which is restrictive for growth of cdc2-2 but not CDC2 strains. At 23 degrees C, a temperature which is permissive for growth of both cdc2-2 and CDC2 strains, the mutant and wild-type DNA polymerase III activities display equal stability. These observations provide a demonstrable biochemical basis for the thermosensitive phenotype of cdc2-2 cells.

Alleles↗
PubMed1991

Mutation spectrum of copper-induced DNA damage.

The ability of metal ions to damage DNA and cause mutagenesis has been analyzed with reversion and forward mutation assays using single-stranded DNA templates. We previously reported that incubation of phi X174 am3 DNA with Fe2+ in vitro results in mutagenesis when the treated DNA is transfected into Escherichia coli spheroplasts (Loeb, L. A., James, E. A., Waltersdorph, A. M., and Klebanoff, S. J. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 3918-3922, 1988). We now extend these studies to other metal ions. Of the metal ions tested, copper ions were the most mutagenic; the frequency of mutants produced was equal to or greater than that produced by Fe2+. Mutagenesis by Cu+ was diminished by catalase, mannitol, and superoxide dismutase suggesting the involvement of H2O2, hydroxyl ions, and superoxide, respectively. However, the findings that Cu+ and Cu2+ are nearly equally mutagenic and that the mutagenic activities are not completely inhibited by oxygen free radical scavengers make it unlikely that the mechanism for mutagenesis is simply the production of hydroxyl free radicals. The spectra of mutations produced by either copper ion using the lacZ gene as a target are very similar and differ from those reported with other agents. The predominant mutagenic sequence changes are single-base substitutions, the most frequent being replacement of a template C by a T. This transition presumably results from mispairing of an altered C with deoxyadenosine. Copper-induced mutations are not randomly distributed. Instead, they are found predominantly in clusters suggesting direct interaction of copper ions with specific nucleotide sequences in DNA. Evidence is considered that the high frequency of C----T transitions may be a common manifestation of DNA damage by oxygen radicals.

Bacteriophage phi X 174↗
PubMed1991

The association of thymidine kinase activity and thymidine transport in Escherichia coli.

We have constructed a series of mutants within the putative nucleoside-binding site of the herpes simplex type-1 virus (HSV-1) thymidine kinase (TK)-encoding gene (tk), contained within an expression vector. While most mutations within this sequence produce an inactive protein, we find no absolute requirement for the wild-type Ile166 and Ala167. The uptake of thymidine (dT) into Escherichia coli tdk-, lacking functional endogenous TK activity, is proportional to the amount of TK activity expressed from the heterologous HSV-1 tk gene. In contrast, there is no enhancement in deoxycytidine uptake into E. coli producing (HSV-1) TK. These results imply a specific role for TK in the active transport of dT into E. coli.

Amino Acid Sequence↗
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↗