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Biomedical subjects

L A Loeb

Publications and source records attributed to L A Loeb.

At least 55 records · Page 3Linked to original sources
PubMed1998

Random sequence mutagenesis and resistance to 5-fluorouridine in human thymidylate synthases.

Thymidylate synthase (TS) catalyzes the methylation of dUMP to dTMP and is the target for the widely used chemotherapeutic agent 5-fluorouracil. We used random sequence mutagenesis to replace 13 codons within the active site of TS and obtain variants that are resistant to 5-fluorodeoxyuridine (5-FdUR). The resulting random library was selected for its ability to complement a TS-deficient Escherichia coli strain, and sequence analysis of survivors found multiple substitutions to be tolerable within the targeted region. An independent selection of the library was carried out in the presence of 5-FdUR, resulting in a more limited spectrum of mutations. One specific mutation, C199L, was observed in more than 46% of 5-FdUR-resistant clones. A 5-FdUR-resistant triple mutant, A197V/L198I/C199F, was purified to apparent homogeneity. Kinetic studies with the substrate dUMP indicate that this mutant is similar to the wild type in regards to kcat and Km values for dUMP and the cosubstrate CH2H4-folate. In contrast, equilibrium binding studies with the inhibitor, FdUMP, demonstrate that the dissociation constant (Kd) for FdUMP binding into the ternary complex was 20-fold higher than values obtained for the wild-type enzyme. This 5-FdUMP-resistant mutant, or others similarly selected, is a candidate for use in gene therapy to render susceptible normal cells resistant to the toxic effects of systemic 5-fluorouracil.

Amino Acid Sequence↗
PubMed1998

Creation of RNA molecules that recognize the oxidative lesion 7,8-dihydro-8-hydroxy-2'-deoxyguanosine (8-oxodG) in DNA.

We used in vitro evolution to obtain RNA molecules that specifically recognize and bind with high affinity to the oxidative lesion 7, 8-dihydro-8-hydroxy-2'-deoxyguanosine (8-oxodG) in DNA. A pool of approximately 10(15) RNA molecules containing a random insert of 45 nucleotides in length was subject to 10 successive rounds of chromatographic enrichment using an 8-oxodG affinity matrix, reverse transcription, PCR amplification, and RNA synthesis. Selected RNA molecules bind to 8-oxodG located at the 3' terminus (Kd </= 270 nM) or in the center (Kd </= 2.8 microM) of a 19-nt strand of DNA, with no detectable affinity for the corresponding dG-containing DNA sequences. These 8-oxodG-binding RNAs will be used to monitor levels of 8-oxodG in DNA from biological sources and should provide a unique method for evaluating oxygen-mediated DNA damage. This approach should be applicable for the creation of RNA molecules that can bind to and identify the different modifications of DNA produced by a variety of environmental agents.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1998

Characterization of Werner syndrome protein DNA helicase activity: directionality, substrate dependence and stimulation by replication protein A.

Werner syndrome is an inherited disease characterized by premature aging, genetic instability and a high incidence of cancer. The wild type Werner syndrome protein (WRN) has been demonstrated to exhibit DNA helicase activity in vitro. Here we report further biochemical characterization of the WRN helicase. The enzyme unwinds double-stranded DNA, translocating 3'-->5' on the enzyme-bound strand. Hydrolysis of dATP or ATP, and to a lesser extent hydrolysis of dCTP or CTP, supports WRN-catalyzed strand-displacement. K m values for ATP and dATP are 51 and 119 microM, respectively, and 2.1 and 3.9 mM for CTP and dCTP, respectively. Strand-displacement activity of WRN is stimulated by single-stranded DNA-binding proteins (SSBs). Among the SSBs from Escherichia coli, bacteriophage T4 and human, stimulation by human SSB (human replication protein A, hRPA) is the most extensive and occurs with a stoichiometry which suggests direct interaction with WRN. A deficit in the interaction of WRN with hRPA may be associated with deletion mutations that occur at elevated frequency in Werner syndrome cells.

Adenine Nucleotides↗
PubMed1998

Fidelity of mutant HIV-1 reverse transcriptases: interaction with the single-stranded template influences the accuracy of DNA synthesis.

We have used random sequence mutagenesis and complementation in a bacterial selection system to establish a large library of immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) mutants with amino acid substitutions in the beta3-beta4 region of the fingers subdomain [Kim, B., Hathaway, T. R., and Loeb, L. A. (1996) J. Biol. Chem. 271, 4872-4878]. We show here that one of these mutants, D76V, exhibits increased accuracy of copying both DNA and RNA templates in a primer extension assay with biased dNTP pools. More detailed analysis of DNA-dependent polymerization showed that the D76V mutation conferred an up to 14-fold increase in fidelity of nucleotide insertion and a 9-fold reduced mutation rate in an M13mp2 lacZalpha forward mutation assay. Substitution at D76 with positively charged (D76R) and nonpolar (D76V and D76I) residues increased replicational accuracy, while substitutions with negatively charged (D76E) and polar residues (D76S and D76C) had little effect on fidelity. We propose that D76 affects replicational accuracy by mediating interaction between the fingers subdomain and the single-stranded template. Our work shows that the Escherichia coli complementation system can yield HIV RT mutants with increased fidelity that have not been isolated from the natural host and that are valuable in understanding the molecular bases of replicational accuracy.

Amino Acid Substitution↗
PubMed1998

Engineering human DNA alkyltransferases for gene therapy using random sequence mutagenesis.

O6-Alkylguanine-DNA alkyltransferase (AGT) is a suicide enzyme that repairs alkylation damage at the O6 position of guanine in DNA. The essentiality of a limited number of amino acid residues at the active site has been determined by site-directed mutagenesis. We used random mutagenesis techniques to create a plasmid library of > 10(6) human AGT mutants with substitutions at residues 150-172 and selected for clones with mutations rendering Escherichia coli resistant to both the alkylating agent, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and the AGT inhibitor, O6-benzylguanine (BG). On sequencing surviving clones resistant to MNNG in the presence and absence of BG, we found that a majority of the clones contained multiple substitutions at mostly nonconserved positions. We selected nine mutants resistant to a combination of MNNG and BG, and the survival of these mutants was as much as 341-fold higher than that of cells harboring wild-type AGT under the same conditions. Each of the mutants contained at least three amino acid substitutions and as many as eight, suggesting that maximum resistance to MNNG in the presence of BG requires even more substitutions than resistance to MNNG alone. BG is being tested clinically as a way to sensitize tumors to chemotherapeutic alkylating agents. Therefore, our BG-resistant mutants hold strong potential as gene therapy candidates for protecting normal tissue in patients receiving BG in combination with alkylating agents for the treatment of cancer.

Alkyl and Aryl Transferases↗
PubMed1998

On the origin of multiple mutations in human cancers.

Tumor progression is a multi-step process, proceeding by multiple alterations from a normal cell to a localized tumor, and finally to one that acquires the ability to invade and metastasize. Tumors are characterized by many mutations in the form of base substitutions, deletions, chromosomal translocations, and gene amplifications, and these mutations are found to accumulate as tumors progress. In contrast, spontaneous mutations are very rare events. Considering the high fidelity of DNA replication in normal cells, it seems improbable that spontaneous mutations could be the source of the large numbers of genetic alterations that are observable in cancer cells. The question of how multiple mutations accumulate in tumor cells is one of considerable interest, since understanding the source of these mutations may facilitate the detection of tumors and may provide new approaches to cancer prevention. We have proposed that the multiple mutations detectable in cancer cells result from a mutator phenotype, in which loss of a genome stability function occurs early during tumor development and predisposes the tumor cell to the accumulation of further mutations. We will first consider the evidence that cancer cells manifest a mutator phenotype, and subsequently discuss the possibility that a mutator phenotype can be selected and can be transient as tumors progress.

Clone Cells↗
PubMed1998

Cancer cells exhibit a mutator phenotype.

This review analyzes the concept and evidence in support of a mutator phenotype in human cancer. The large number of mutations reported in tumor cells cannot be accounted for by the low mutation rates observed in normal somatic cells; rather, it must be a manifestation of a mutator phenotype present early during the tumorigenic process. The interaction between increased mutagenesis and clonal selection provides a mechanism for the selection of cells with increased proliferative advantage. The concept of a mutator phenotype in cancer has gained considerable support from the findings of enormous numbers of somatic mutations in repetitive sequences in human tumors. Moreover, cell lines exhibiting microsatellite instability demonstrate an increased mutation frequency in expressed genes. A knowledge of mechanisms that generate multiple mutations in cancer cells has important implications for prevention. For many tumors, a delay in the rate of accumulation of mutations by a factor of two could drastically reduce the death rates from these tumors.

Animals↗
PubMed1998

The mutation rate and cancer.

The stability of the human genome requires that mutations in the germ line be exceptionally rare events. While most mutations are neutral or have deleterious effects, a limited number of mutations are required for adaptation to environmental changes. Drake has provided evidence that DNA-based microbes have evolved a mechanism to yield a common spontaneous mutation rate of approximately 0.003 mutations per genome per replication (Drake 1991). In contrast, mutation rates of RNA viruses are much larger (Holland et al. 1982) and can approach the maximum tolerable deleterious mutation rate of one per genome (Eigen and Schuster 1977; Eigen 1993). Drake calculates that lytic RNA viruses display spontaneous mutation rates of approximately one per genome while most have mutation rates that are approximately 0.1 per genome (Drake 1993). This constancy of germline mutation rates among microbial species need not necessarily mean constancy of the somatic mutation rates. Furthermore, there need not be a constant rate for somatic mutations during development. In this review, we consider mutations in cancer, a pathology in which there appears to be an increase in the rate of somatic mutations throughout the genome. Moreover, within the eukaryotic genome, as in microbes, there are "hot-spots" that exhibit unusually high mutation frequencies. It seems conceivable to us that many tumors contain thousands of changes in DNA sequence. The major question is: how do these mutations arise, and how many are rate-limiting for tumor progression?

Animals↗
PubMed1997

Inefficient repair of RNA x DNA hybrids.

RNA x DNA hybrids are commonly observed during normal biological processes. We tested the ability of three DNA-repair enzymes to remove lesions from the DNA strand of RNA x DNA heteroduplexes. Three nucleotide analogs, 5-hydroxy-2'-deoxycytidine triphosphate, 8-oxo-2'-deoxyguanosine triphosphate, and O6-methyl-2'-deoxyguanosine triphosphate, representative of lesions generated by oxygen damage and methylating agents, were incorporated into the DNA strand synthesized using either a DNA or RNA template. The extended DNA x DNA and RNA x DNA hybrids were used as substrates for bacterial formamidopyrimidine-DNA glycosylase, Nth protein (endonuclease III) and O6-methylguanine-DNA methyltransferase. We show that all three lesions are readily cleaved from the DNA strand of a DNA x DNA duplex but are relatively resistant to cleavage when present in the DNA strand of an RNA x DNA hybrid. Our in vitro studies suggest that damaged DNA in RNA x DNA hybrids is less likely to be repaired in vivo.

DNA Damage↗
PubMed1997

Creation of human alkyltransferases resistant to O6-benzylguanine.

O6-benzylguanine (BG), an inhibitor of O6-alkylguanine-DNA alkyltransferase, is being tested clinically for its ability to chemosensitize tumors to alkylating agents. Although this drug may increase the killing of tumors that express high levels of alkyltransferase, it would also be expected to reduce the already low alkyltransferase levels of hematopoietic stem cells and, thus, exacerbate the dose-limiting side effect of myelosuppression. One way to overcome this problem would be to transduce hematopoietic stem cells with a gene encoding a BG-resistant alkyltransferase prior to BG/alkylation treatment. We used the technique of random mutagenesis followed by positive genetic selection to create such a mutant gene. A pool of 6.5 x 10(6) human alkyltransferases that were randomly mutated at six amino acids near the alkyl-accepting cysteine was transformed into alkyltransferase-deficient Escherichia coli. Five mutants were selected based on their ability to provide the bacteria with resistance to both N-methyl-N'-nitro-N-nitrosoguanidine and BG. One mutant, V139F/P140R/L142M, not only had the highest BG resistance (50% inhibitory concentration, >500 microM) but also offered E. coli the best protection from N-methyl-N'-nitro-N-nitrosoguanidine and, thus, is a promising gene therapy candidate.

Binding Sites↗
PubMed1997

Low fidelity mutants in the O-helix of Thermus aquaticus DNA polymerase I.

We screened 67 mutants in the O-helix of Thermus aquaticus (Taq) DNA polymerase I (pol I) for altered fidelity of DNA synthesis. These mutants were obtained (Suzuki, M., Baskin, D., Hood, L., and Loeb, L. A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 9670-9675) by substituting an oligonucleotide containing random sequences for codons 659-671, and selecting for complementation of a growth defect in Escherichia coli caused by temperature-sensitive host pol I. Thirteen mutants decreased fidelity in a screen that employed primer extension reactions lacking one of four complementary deoxynucleoside triphosphates (dNTPs). Three mutants were purified and exhibited 29-68% of wild-type specific activity. Homogeneous polymerases A661E, A661P, and T664R extended primers further than the wild-type, synthesizing past template nucleotides for which the complementary dNTP was absent. The data indicate that both misinsertion of incorrect nucleotides and extension of mispaired primer termini were increased. In a lacZalpha forward mutation assay, A661E and T664R yielded mutation frequencies at least 7- and 25-fold greater, respectively, than that of the wild-type polymerase. These findings emphasize the importance of the O-helix in substrate recognition and are compatible with a role for pyrophosphate release in enhancing fidelity of DNA synthesis.

Base Sequence↗
PubMed1997

Mutagenesis by human immunodeficiency virus reverse transcriptase: incorporation of O6-methyldeoxyguanosine triphosphate.

The high frequency of incorporation of non-complementary nucleotides by HIV-1 reverse transcriptase is likely to be a major factor in the exceptionally rapid accumulation of viral mutations during the course of AIDS infections. To investigate whether this high level of infidelity is also associated with the incorporation of nucleotide analogs, we analyzed O6-methyldeoxyguanosine triphosphate and compared the incorporation of this analog by HIV-1 reverse transcriptase to that catalyzed by other DNA synthesizing enzymes. Our results indicate that O6-methyldeoxyguanosine triphosphate serves as a substrate for DNA synthesized in vitro by HIV-1 RT on both DNA and RNA templates. The product DNA contains the modified purine; it is sensitive to the repair enzyme, O6-methylguanine methyltransferase, which specifically reacts with DNA containing methylated guanines at the O6 position. Using a forward mutation assay we demonstrated that the nucleotide analog incorporated by HIV-1 RT is mutagenic. The mutations produced are single-base substitutions opposite template thymidines and result in A:T --> G:C transitions. The incorporation of a mutagenic nucleotide by HIV-1 RT highlights the possibility of increasing the rate of mutagenesis of HIV by the use of nucleotides that form non-complementary base pairs at high frequency.

Base Sequence↗
PubMed1997

Incorporation of the guanosine triphosphate analogs 8-oxo-dGTP and 8-NH2-dGTP by reverse transcriptases and mammalian DNA polymerases.

We have measured the efficiencies of utilization of 8-oxo-dGTP and 8-NH2-dGTP by human immunodeficiency virus type 1 and murine leukemia virus reverse transcriptases and compared them to those of DNA polymerases alpha and beta. Initially, we carried out primer extension reactions in the presence of dGTP or a dGTP analog and the remaining three dNTPs using synthetic DNA and RNA templates. These assays revealed that, in general, 8-NH2-dGTP is incorporated and extended more efficiently than 8-oxo-dGTP by all enzymes tested. Second, we determined rate constants for the incorporation of each analog opposite a template cytidine residue using steady state single nucleotide extension kinetics. Our results demonstrated the following. 1) Both reverse transcriptases incorporate the nucleotide analogs; discrimination against their incorporation is a function primarily of Km or Vmax depending on the analog and the enzyme. 2) Discrimination against the analogs is more stringent with the DNA template than with a homologous RNA template. 3) Polymerase alpha exhibits a mixed kinetic phenotype, with a large discrimination against 8-oxo-dGTP but a comparatively higher preference for 8-NH2-dGTP. 4) Polymerase beta incorporates both analogs efficiently; there is no discrimination with respect to Km and a significantly lower discrimination with respect to Vmax when compared with the other polymerases.

DNA Polymerase II↗