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PubMedDate not supplied

Interfacial reactions of cast titanium with mold materials.

PURPOSE: Dental casting requires replication of complex shapes with high fidelity. To achieve this objective, the problem of scale formation on titanium dental castings must be overcome. Scaling occurs readily at high temperatures because of the high reactivity of molten titanium with investment materials. The purpose of this study was to examine the effectiveness of using stable oxide coatings on the mold surface to reduce the interfacial reactions. MATERIALS AND METHODS: A traditional phosphate-bonded dental investment, a commercial titanium investment, and an experimental oxide coating were used for the molds. Pure titanium samples were cast, divested, and prepared for scanning electron microscopic and energy dispersive x-ray spectroscopic analyses. RESULTS: Layers of 7- to 10-micron-thick scale were formed on titanium castings during reaction with traditional mold materials. Interface reaction was reduced between the molten titanium and the investment material when yttrium oxide or zirconium oxide coatings were applied to the mold before casting. CONCLUSION: Less titanium-mold interfacial reactions occurred when protective coatings were used as diffusion barriers for titanium casting. Y2O3-coating oxide particles applied without binder were entrapped in the cast titanium surface layer. Further study of a binder system for Y2O3 coating is needed.

Dental Casting Investment↗
PubMed2004

DNA ligases ensure fidelity by interrogating minor groove contacts.

DNA ligases, found in both prokaryotes and eukaryotes, covalently link the 3'-hydroxyl and 5'-phosphate ends of duplex DNA segments. This reaction represents a completion step for DNA replication, repair and recombination. It is well established that ligases are sensitive to mispairs present on the 3' side of the ligase junction, but tolerant of mispairs on the 5' side. While such discrimination would increase the overall accuracy of DNA replication and repair, the mechanisms by which this fidelity is accomplished are as yet unknown. In this paper, we present the results of experiments with Tth ligase from Thermus thermophilus HB8 and a series of nucleoside analogs in which the mechanism of discrimination has been probed. Using a series of purine analogs substituted in the 2 and 6 positions, we establish that the apparent base pair geometry is much more important than relative base pair stability and that major groove contacts are of little importance. This result is further confirmed using 5-fluorouracil (FU) mispaired with guanine. At neutral pH, the FU:G mispair on the 3' side of a ligase junction is predominantly in a neutral wobble configuration and is poorly ligated. Increasing the solution pH increases the proportion of an ionized base pair approximating Watson-Crick geometry, substantially increasing the relative ligation efficiency. These results suggest that the ligase could distinguish Watson-Crick from mispaired geometry by probing the hydrogen bond acceptors present in the minor groove as has been proposed for DNA polymerases. The significance of minor groove hydrogen bonding interactions is confirmed with both Tth and T4 DNA ligases upon examination of base pairs containing the pyrimidine shape analog, difluorotoluene (DFT). Although DFT paired with adenine approximates Watson-Crick geometry, a minor groove hydrogen bond acceptor is lost. Consistent with this hypothesis, we observe that DFT-containing base pairs inhibit ligation when on the 3' side of the ligase junction. The NAD+-dependent ligase, Tth, is more sensitive to the DFT analog on the unligated strand whereas the ATP-dependent T4 ligase is more sensitive to substitutions in the template strand. Electrophoretic gel mobility-shift assays demonstrate that the Tth ligase binds poorly to oligonucleotide substrates containing analogs with altered minor groove contacts.

Base Pairing↗
PubMed2002

Fidelity of DNA polymerase epsilon holoenzyme from budding yeast Saccharomyces cerevisiae.

DNA polymerases delta and epsilon (pol delta and epsilon) are the major replicative polymerases and possess 3'-5' proofreading exonuclease activities that correct errors arising during DNA replication in the yeast Saccharomyces cerevisiae. This study measures the fidelity of the holoenzyme of wild-type pol epsilon, the 3'-5' exonuclease-deficient pol2-4, a +1 frameshift mutator for homonucleotide runs, pol2C1089Y, and pol2C1089Y pol2-4 enzymes using a synthetic 30-mer primer/100-mer template. The nucleotide substitution rate for wild-type pol epsilon was 0.47 x 10(-5) for G:G mismatches, 0.15 x 10(-5) for T:G mismatches, and less than 0.01 x 10(-5) for A:G mismatches. The accuracy for A opposite G was not altered in the exonuclease-deficient pol2-4 pol epsilon; however, G:G and T:G misincorporation rates increased 40- and 73-fold, respectively. The pol2C1089Y pol epsilon mutant also exhibited increased G:G and T:G misincorporation rates, 22- and 10-fold, respectively, whereas A:G misincorporation did not differ from that of wild type. Since the fidelity of the double mutant pol2-4 pol2C1089Y was not greatly decreased, these results suggest that the proofreading 3'-5' exonuclease activity of pol2C1089Y pol epsilon is impaired even though it retains nuclease activity and the mutation is not in the known exonuclease domain.

Base Sequence↗
PubMed1997

The fidelity of misinsertion and mispair extension throughout DNA synthesis exhibited by mutants of the reverse transcriptase of human immunodeficiency virus type 2 resistant to nucleoside analogs.

The AIDS-causing retroviruses, human immunodeficiency virus types 1 and type 2 (HIV-1 and HIV-2, respectively) undergo extensive genetic variations, which effect their pathogenesis and resistance to drug therapy. It was postulated that this genetic hypervariability results from high rates of viral replication in conjugation with a relatively low fidelity of DNA synthesis [typical to the reverse transcriptases (RT) of these retroviruses]. As part of studying structure/function relationship in HIV RT, mutational analyses were conducted to identify amino acid residues which are involved in affecting the fidelity of DNA synthesis. The formation of 3'-mispaired DNA due to nucleotide misinsertions, and the subsequent elongation of this mismatched DNA were shown to be major determinants in affecting those substitutions during DNA synthesis (exhibited in vitro by HIV RT). It was interesting to find a correlation between sensitivity to nucleoside analogs (due to the ability to incorporate or reject an incoming analog) and the fidelity of DNA synthesis (which depends on the capacity to incorporate and extend a wrong nucleotide). Such a connection has already been found for several drug-resistant mutants of HIV-1 RT, with an increased fidelity of DNA synthesis relative to the wild-type RT. In the present study we have examined the fidelity of DNA synthesis using the same parameters of misinsertion and mispair extension for five novel drug-resistant mutants of HIV-2 RT; i.e. the single mutants [Val74]RT, [Gly89]RT and [Tyr215]RT and the double mutants [Val74,Tyr215]RT and [Gly89, Tyr215]RT. This comparative study suggests that unlike the Val74 mutant of HIV-1 RT, which was shown earlier to display a substantially enhanced fidelity, the comparable mutant of HIV-2 RT has fidelity similar to that of the wild-type RT. Depending on the assay employed and the DNA sequences extended, most other mutants of HIV-2 RT display moderate effects on the enzyme, leading to mild increases in fidelity of DNA synthesis. This implies a more complex and less distinctive correlation between drug-resistance, misinsertion and mispair extension in HIV-2 RT in contrast to HIV-1 RT, providing evidence for potential biochemical differences between these two related RT.

Anti-HIV Agents↗
PubMed2001

Promotion of evolution by intracellular coexistence of mutator and normal DNA polymerases.

The efficient evolution of a population requires both genetic diversity and stable reproduction of advantageous genotypes. The accuracy of DNA replication guarantees the stable reproduction, while errors during DNA replication produce the genetic diversity. Thus, one key to the promotion of evolution is inherent in DNA replication. In bacteria, replication forks progress bidirectionally from the single origin of replication on a genome. One replication fork contains two DNA polymerase molecules so that four DNA polymerases simultaneously carry out the replication of a genome. It is generally believed that the fidelity of the intracellular DNA polymerases is identical (parity strategy). To test this, we examined the effects of the intracellular coexistence of a mutator polymerase with low fidelity and a normal polymerase with high fidelity on adaptive evolution (disparity strategy). From the analysis using genetic algorithms based on the bacterial replication, it was found that the population using the disparity strategy could further expand its genetic diversity and preserve the advantageous genotypes more profoundly than the parity population. This strongly suggests that bacteria replicating with a disparity strategy may undergo rapid evolution, particularly during severe environmental changes. The implications of the conspicuous adaptability of Escherichia coli mutator strains are discussed in this context.

Animals↗
PubMed1999

Toward the experimental codon reassignment in vivo: protein building with an expanded amino acid repertoire.

The high precision and fidelity of the genetic message transmission are ensured by numerous proofreading steps, from DNA replication and transcription to protein translation. The key event for translational fidelity is the proper codon assignment for 20 canonical amino acids. An experimental codon reassignment is possible for noncanonical amino acids in vivo using artificially constructed expression hosts under efficient selective pressure. However, such amino acids may interfere with the cellular metabolism and thus do not belong to the 'first' or 'restricted' part of the universal code, but rather to a second or 'relaxed' part, which is limited mainly by the downstream proofreading in the natural translational machinery. Correspondingly, not all possible alpha-amino acids can be introduced into proteins. The aim of this study is to discuss biological and evolutionary constraints on possible candidates for this second coding level of the universal code. Engineering of such a 'second' code is expected to have great academic as well as practical impact, ranging from protein folding studies to biomedicine.

Acylation↗
PubMed1991

Exonucleolytic proofreading of leading and lagging strand DNA replication errors.

We have asked whether exonucleolytic proofreading occurs during simian virus 40 origin-dependent, bidirectional DNA replication in extracts of human HeLa cells. In addition, we have compared the fidelity of leading and lagging strand DNA synthesis. In a fidelity assay that scores single-base substitution errors that revert a TGA codon in the lacZ alpha gene in an M13mp vector, providing an excess of a single dNTP substrate over the other three dNTP substrates in a replication reaction generates defined, strand-specific errors. Fidelity measurements with two vectors having the origin of replication on opposite sides of the opal codon demonstrate that error rates for two different A.dCTP and T.dGTP mispairs increase when deoxyguanosine monophosphate is added to replication reaction mixtures or when the concentration of deoxynucleoside triphosphates is increased. The data suggest that exonucleolytic proofreading occurs on both strands during bidirectional replication. Measurements using the two simian virus 40 origin-containing vectors suggest that base substitution error rates are similar for replication of the leading and lagging strands.

Antigens, Polyomavirus Transforming↗
PubMed1984

Critical spatial requirement within the origin of simian virus 40 DNA replication.

We inserted a single base pair into the center of a 27-base-pair palindrome within the replication origin of simian virus 40. The mutation did not directly alter the symmetry of the palindrome or the protein-binding sequences within the palindrome. DNA binding studies showed that subunits of the simian virus 40 A protein (T antigen) bound to each of the four recognition pentanucleotides in the origin palindrome but did so with reduced affinity in comparison with wild-type origins. The mutant origin cloned in a plasmid DNA failed to replicate in COS cells. Thus, precise spatial interactions among subunits of A protein are necessary for stable origin binding and are crucial for subsequent steps in the initiation of DNA replication. Furthermore, any possible functional interactions of the simian virus 40 A protein with cellular DNA would require a great fidelity of protein binding arrangements to initiate cellular DNA replication.

Base Sequence↗
PubMed2001

Study of the impact of HIV genotypic drug resistance testing on therapy efficacy.

During recent years significant progress has been made in the treatment of HIV-1, at least in part due to the availability of potent antiretroviral drugs. The goal of the current treatment strategies is to inhibit the viral replication as completely as possible by using a combination of 3 or more antiretroviral drugs. This Highly Active Antiretroviral Therapy (HAART) has radically changed the clinical outcome of HIV, leading to decreased mortality and morbidity, at least in developed countries. Additionally to the advent of new and potent drugs, demonstrations of the prognostic value of the CD4 cell count and the plasma viral load were of major importance in the development of therapeutic strategies. Especially the ability of viral load assays to assess accurately the true level of viral replication, led to a better understanding of the pathogenesis of the disease. HIV proved to be a highly dynamic infection even during the period of clinical latency. The initial enthusiasm that HAART could radically change the outcome of HIV was cooled off in face of the difficulties in real life associated with the complex treatment strategies. Besides long-term side effects and suboptimal drug potency, the emergence of resistant virus and the necessity of perfect therapy adherence are major concerns for obtaining a sustained control of viral replication. In this study we focused on HIV resistance, which remains one of the major threats for a sustained response to antiretroviral therapy. HIV proved to be able to develop resistance to all currently used antiretroviral drugs. The high replication rate of the virus together with the low fidelity of the viral reverse transcriptase, from the basis for the presence of enormous amounts of viral variants. Whenever viral replication is ongoing in the presence of antiretroviral drugs, these variants that escape the inhibitory effects of the drugs will be selected. Although the knowledge in the field of HIV resistance has expanded enormously, many issues need to be answered. Genotypic and phenotypic resistance patterns are evolving continuously, due to changes in the treatment strategies. Moreover the relation between drug resistance and therapy failure needs further investigation, in order to prove the relevance of performing resistance testing in the follow-up of HIV-infected patients. The wide availability of antiretroviral drugs has led to the transmission of resistant HIV. Infection with HIV resistant to one or more antiretroviral drugs has been observed to occur through the different transmission routes. In a first study we assessed the prevalence of genotypic resistance to antiretroviral drugs in Belgian antiretroviral-naïve HIV-infected patients. We observed that HIV strains with resistance-related mutations to one or more classes of antiretroviral drugs are not uncommon in the Belgian naïve patients. Furthermore the inclusion of samples from patients visiting the Belgian hospitals for the first time in 1995, 1997 and 1998, showed that the overall prevalence of baseline genotypic resistance remains rather constant (26-30%). The increasing trend in genotypic baseline resistance to 3TC (2% to 6.3%) and PIs (4.4% to 9.9%) as well as the decrease in ZDV-resistance (13.3% to 5.4%), reflect the change in treatment strategies, and resistance to these drugs is most probably caused by transmission of variants with resistance mutations selected during therapy. The presence of NNRTI-related mutations (around 16%) is likely to reflect the occurrence of baseline polymorphisms, since NNRTI-related mutations can occur without a replication deficit for the virus. Moreover, despite the rather recent introduction of NNRTIs into the clinic from 1997 onwards, no clear trend in NNRTI baseline resistance over time is observed. The best current therapeutic strategy for HIV-infected patients is to start antiretroviral therapy with HAART in order to avoid the accumulation of resistance towards drugs in less suppressive regimens. In a second study, we showed that the start of HAART in antiretroviral-naive HIV-patients in daily clinical practice could prevent viral breakthrough for up to 44 months in 60% of patients (n = 25). Six of 10 patients with virologic failure developed resistance to the drugs included in their treatment regimens. In comparison to patients with a sustained virologic response, patients with virologic failure were in a later disease stage when starting therapy and showed lower PI drug-levels, what can be an indication of poor adherence. Despite a poor virologic response for some of them, a rise in CD4 cell count was observed for all patients during the study period. A large number of HIV-infected patients started treatment in the pre-HAART period. The use of NRTIs is mono- or bitherapy was not able to prevent the development of resistant virus. In a third study we studied the prevalence and characteristics of 2 patterns of multinucleoside resistance (MNR) in European patients (n = 755). In patients without NRTI-exposure or with exposure to only one NRTI, no MNR was observed. MNR was present in low prevalence (each pattern < 2%) in patients pretreated with multiple NRTIs. Despite this low prevalence, MNR should be closely monitored, since it results in broad cross-resistance to NRTIs in vitro and a poor therapy response in vivo. We also assessed the predictive value of baseline resistance on the virologic response to later added drugs. The genotype of patients starting or changing a therapy consisting solely of NRTIs was analyzed at baseline and 6 months later. In patients without genotypic mutations towards the added drug the virologic response was significantly better compared to patients with baseline resistance. At 6 months however, both patient groups showed a rise in viral load due to the accumulation of NRTI-related mutations under the presence of poorly suppressive regimens, although the difference between the two groups remained significant. In this study, and also in studies reported by others, the presence of baseline resistance has a high predictive value for therapy failure, while the absence of resistance is not predictive for therapy response. Suboptimal adherence may be one of the reasons of the poor predictive value of the absence of baseline resistance for a good therapy response. In a last observational study, we investigated the relation between adherence, the presence and development of genotypic resistance and the virologic response in patients during HAART therapy. Adherence to 1 protease inhibitor was monitored using Electronic Event Monitoring. Patients with perfect therapy adherence and in particular without drug holidays, can control viral replication provided that the activity of the drugs included in the combination is not entirely compromised by the presence of baseline resistance mutations. In our patient population, reduced adherence resulted in therapy failure, mostly associated with a subsequent accumulation of resistance mutations. In conclusion, the outcome of the HIV disease has been revolutionarily changed with the advent of HAART. Both resistance and treatment adherence are crucial factors in determining the therapy response. Retrospective studies, such as ours, and a limited number of prospective trials already proved the short-term benefit of therapy switch based on the results of resistance tests in addition to standard of care. To ultimately define the role of tools as resistance testing and adherence monitoring with eventual adherence interventions, more prospective trials are needed as well in treatment-naïve as in experienced patients.

Anti-HIV Agents↗
PubMedDate not supplied

Intervention research: establishing fidelity of the independent variable in nursing clinical trials.

BACKGROUND: Internal validity of a randomized clinical trial of a nursing intervention is dependent on intervention fidelity. Although several methods have been developed, evaluating audio or audiovisual tapes for prescribed and proscribed interventionist behaviors is considered the gold standard test of treatment fidelity. This approach requires development of a psychometrically sound instrument to meaningfully categorize and quantify interventionist behaviors. OBJECTIVE: To outline critical steps necessary to develop a treatment fidelity instrument. METHODS: A comprehensive literature review was conducted to determine procedures used by other researchers. The literature review produced five quantitative studies of treatment fidelity, all in the field of psychotherapy, and two replication studies. A synthesis of methodologies across studies combined with researchers' experiences resulted in identification of the steps necessary to develop a treatment fidelity measure. RESULTS: Seven sequential steps were identified as essential to the development of a valid and reliable measure of treatment fidelity. These steps include (a) identification of the essential elements of the experimental and control treatment modalities; (b) construction of scale items; (c) development of item scaling; (d) identification of the units for coding; (e) item testing and revision; (f) specification of rater qualifications and development of rater training program; and (g) development and completion of pilot testing to test psychometric properties. Development of the Possibilities Project Psychotherapy Coding Questionnaire is described as an illustration of the seven-step process. DISCUSSION: The results show the essential steps that are unique to the development of treatment fidelity measures and show the feasibility of using these steps to construct a psychometrically sound treatment-specific fidelity measure.

Adolescent↗
PubMed1986

Studies of DNA polymerases alpha and beta from cultured human cells in various replicative states.

DNA polymerase activities from HeLa cells and from cultured diploid human fibroblasts in various growth states were compared. alpha-Polymerase activities from log phase fibroblasts treated with sodium butyrate and from stationary phase HeLa cells had DEAE-cellulose elution patterns that differed from those of polymerases from dividing cells. Moreover, alpha- and beta-polymerases from nondividing cells replicated synthetic polymers less faithfully. Although similar changes were observed previously for polymerases from late-passage and postconfluent early passage fibroblasts, amounts of alpha-polymerase activity recovered from nondividing cells in this study did not dramatically decline as they had in the former cases. The alpha-polymerase activities from HeLa cells and fibroblasts in various growth states sedimented near 7.5S in 0.4 M KCI and could be inhibited by a monoclonal IgG fraction prepared against KB cell alpha-polymerase. By several criteria, there was no significant differences in levels of UV-stimulated repair synthesis observed in early or late-passage postconfluent fibroblasts or in log phase fibroblasts treated with sodium butyrate. In summary, levels of alpha-polymerase do not necessarily correlate either with replicative activity or with apparent levels of repair synthesis. However, cells with decreased replicative activity always yielded enzyme with decreased fidelity in vitro and altered chromatographic behavior. It appears, therefore, that the alterations observed for alpha-polymerase from late-passage cells may be attributed more generally to the nondividing nature of these cells.

Cell Line↗
PubMed2006

The structural basis for the mutagenicity of O(6)-methyl-guanine lesions.

Methylating agents are widespread environmental carcinogens that generate a broad spectrum of DNA damage. Methylation at the guanine O(6) position confers the greatest mutagenic and carcinogenic potential. DNA polymerases insert cytosine and thymine with similar efficiency opposite O(6)-methyl-guanine (O6MeG). We combined pre-steady-state kinetic analysis and a series of nine x-ray crystal structures to contrast the reaction pathways of accurate and mutagenic replication of O6MeG in a high-fidelity DNA polymerase from Bacillus stearothermophilus. Polymerases achieve substrate specificity by selecting for nucleotides with shape and hydrogen-bonding patterns that complement a canonical DNA template. Our structures reveal that both thymine and cytosine O6MeG base pairs evade proofreading by mimicking the essential molecular features of canonical substrates. The steric mimicry depends on stabilization of a rare cytosine tautomer in C.O6MeG-polymerase complexes. An unusual electrostatic interaction between O-methyl protons and a thymine carbonyl oxygen helps stabilize T.O6MeG pairs bound to DNA polymerase. Because DNA methylators constitute an important class of chemotherapeutic agents, the molecular mechanisms of replication of these DNA lesions are important for our understanding of both the genesis and treatment of cancer.

Binding Sites↗
PubMed1996

Strand specificity of mutagenic bypass replication of DNA containing psoralen monoadducts in a human cell extract.

Psoralens are mutagenic compounds of vegetable origin that are used as photosensitizing agents in the treatment of various skin diseases, blood cell cancer, and autoimmune disorders. To study the mechanism of mutagenicity of psoralens in humans, we examined the efficiency and fidelity of simian virus 40 origin-dependent replication in a human cell extract of M13mp2 DNA randomly treated with the psoralen derivative 4'-hydroxymethyl-4,5',8-trimethyl psoralen plus UVA irradiation. Replication of DNA treated with variable amounts of 4'-hydroxymethyl-4,5',8-trimethyl psoralen and a fixed UVA fluence was inhibited in a concentration-dependent manner. However, covalently closed monomer-length circular replication products were observed. Product analysis by renaturing agarose gel electrophoresis after cross-linking with 250- to 280-nm UV light indicated that approximately 1 of 9 psoralen monoadducts was bypassed during in vitro replication. Introduction of product DNA into Escherichia coli to score replication errors in the lacZalpha reporter gene demonstrated that replication of the damaged DNA was more mutagenic than was replication of undamaged DNA. Sequence analysis of lacZ mutants revealed that damage-dependent replication errors were predominantly T.A-->C.G transitions, transversions at C.G base pairs, and deletions of single A.T base pairs, the last occurring most frequently in homopolymeric runs. A comparison of error specificities with two substrates having the replication origin asymmetrically placed on opposite sides of the mutational target suggests that the lagging-strand replication apparatus is less accurate than the leading-strand replication apparatus for psoralen monoadduct-dependent deletion errors. A model is proposed based on the preferential loopout of the monoadducted base from the strand that templates retrograde discontinuous synthesis.

Bacteriophage M13↗
PubMed1997

Sequence context is an important determinant in the mutagenic potential of 1,N6-ethenodeoxyadenosine (epsilonA): formation of epsilonA basepairs and elongation in defined templates.

Many laboratories have obtained data on mutagenicity of modified bases in naturally occurring DNA sequences. It has often been noted that mutation is favored in certain sequence contexts, sometimes termed 'hot spots'. This approach to the contribution of neighboring sequences does not permit a systematic study of both the qualitative and quantitative mutational frequencies. In the present experiments we have chosen to use the exocyclic adduct, 1,N6-etheno A (epsilonA), site-specifically placed in a defined 25-mer oligonucleotides in which epsilonA is flanked by differing 5' and 3' tandem bases. Mutation was assessed using an in vitro replication assay and five polymerases of varying fidelity. The relevant central sequences were 3' --> 5' -CC-epsilonA-CC-, -GG-epsilonA-GG-, -TT-epsilonA-TT-, -AA-epsilonA-AA-, -GG-epsilonA-TT-, -TT-epsilonA-AA-, -AT-epsilonA-TT- and -TA-epsilonA-TA-. Using the Klenow fragment (Kf) (exo+ or exo-) of E. coli Pol I, it was found the epsilonA is an ambiguous base and, with varying efficiencies, all four dNTPs could be inserted opposite epsilonA in all sequences. However, only 3' --> 5' -TT-epsilonA-TT-, -GG-epsilonA-TT- and -AT-epsilonA-TT- were fully extended to a significant extent. The only sequences essentially blocked at the position of epsilonA were -AA-epsilonA-AA- and -TT-epsilonA-AA-. The others were intermediate. When replication was performed with Sequenase, MMLV RT or HIV RT, different patterns were observed, in which replication terminated one base prior to epsilonA, at epsilonA, or one base after epsilonA without further extension. In favored sequences, using the Klenow fragment, an epsilonA x N pair could be extended to form normal basepairs. No extension could be demonstrated in sequences in which tandem adenines were 5' to epsilonA. Kinetic data showed that two of the epsilonA x N pairs, epsilonA x A and epsilonA x C, could form at 10 microM or less dNTP. Which bases were preferentially inserted opposite epsilonA was a function of the flanking bases. Under the kinetic conditions used, epsilonA x T did not form even at 1 mM dTTP. These results indicate that the chemical structure of an adduct is not the only determinant of mutagenic efficiency. It is likely that the effect of the adduct on replication is due to the changes in the structural environment conferred by the flanking bases.

DNA Adducts↗
PubMed2004

Chemical synthesis and translesion replication of a cis-syn cyclobutane thymine-uracil dimer.

The cytosine base in DNA undergoes hydrolytic deamination at a considerable rate when UV radiation induces formation of a cyclobutane pyrimidine dimer (CPD) with an adjacent pyrimidine base. We have synthesized a phosphoramidite building block of a cis-syn cyclobutane thymine-uracil dimer (T[]U), which is the deaminated form of the CPD at a TC site, and incorporated it into oligodeoxyribonucleotides. The previously reported method for synthesis of the thymine dimer (T[]T) was applied, using partially protected thymidylyl-(3'-5')-2'-deoxyuridine as the starting material, and after triplet- sensitized irradiation, the configuration of the base moiety in the major product was determined by NMR spectroscopy. Presence of the cis-syn cyclobutane dimer in the obtained oligonucleotides was confirmed by UV photoreversal and reaction with T4 endonuclease V. Using a 30mer containing T[]U, translesion synthesis by human DNA polymerase eta was analyzed. There was no difference in the results between the templates containing T[]T and T[]U and pol eta bypassed both lesions with the same efficiency, incorporating two adenylates. This enzyme showed fidelity to base pair formation, but this replication causes a C-->T transition because the original sequence is TC.

DNA Replication↗
PubMed1985

DNA modification by chemical carcinogens.

The chemistry and molecular biology of DNA adducts is only one part of the carcinogenic process. Many other factors will determine whether a particular chemical will exert a carcinogenic effect. For example, the size of particles upon which a carcinogenic may be adsorbed will influence whether or not, and if so where, deposition within the lung will occur. The simultaneous exposure to several different agents may enhance or inhibit the metabolism of a chemical to its ultimate carcinogenic form (Rice et al., 1984; Smolarek and Baird, 1984). The ultimate carcinogenic metabolites may be influenced in their ability to react with DNA by a number of factors such as internal levels of detoxifying enzymes, the presence of other metabolic intermediates such as glutathione with which they could react either enzymatically or non-enzymatically, and the state of DNA which is probably most heavily influenced by whether or not the cell is undergoing replication or particular sequences being expressed. Replicating forks have been shown to be more extensively modified than other areas of DNA. Another critical factor which can influence the final outcome of the DNA damage is whether or not the modifications can be repaired. If this occurs with high fidelity and the cell has not previously undergone replication then the effect of the damage by the carcinogen is likely to be minimal. The major area in which progress is needed is an understanding of what this damage really does to the cell such that after an additional period of time, which may be as long as twenty or more years, these prior events are expressed and cell proliferation occurs. Clearly additional stimulatory factors, for example tumor promoting agents such as the phorbol esters or phenobarbital, are often needed. After such prolonged periods it seems likely that the DNA adducts would no longer be present. However, the way in which their earlier presence is remembered is not clear. Simple mutations do not explain all the characteristics of tumor progression and, when it occurs, regression. Even if a specific site mutation does occur then its expression must be under other types of control. Any explanation of the action of DNA modification at the molecular level also requires that account be taken of the diverse nature of the DNA adducts from simple modifications such as methylation to bulkier adducts such as benzo[a]pyrene, aflatoxin or aromatic amines.(ABSTRACT TRUNCATED AT 400 WORDS)

4-Nitroquinoline-1-oxide↗
PubMed1996

Fidelity and error specificity of the alpha catalytic subunit of Escherichia coli DNA polymerase III.

Escherichia coli DNA polymerase III holoenzyme is the replicative enzyme primarily responsible for the duplication of the E. coli chromosome. This process occurs with high accuracy, less than 10(-9) to 10(-10) errors being committed per base pair per round of replication. As a first step in understanding the mechanisms responsible for the high fidelity of this process, we have purified the polymerase III alpha catalytic subunit, free of exonuclease activity, and analyzed its fidelity in vitro. We employed a newly developed gap-filling assay using the N-terminal 250 bases of the lacI gene as a forward mutational target. When synthesizing across this target, alpha subunit produced mutations at a frequency of 0.6%. DNA sequencing revealed that the mutants created in vitro consisted mostly of frameshift mutations, although some base substitutions were also observed. The frameshifts, occurring at more than 120-fold above the background, consisted largely of -1 deletions. Among them, about 80% were the deletion of a purine template base with a pyrimidine 5'-neighbor. These results suggest that the alpha subunit (i) has a relatively low ability to extend from misincorporated bases, accounting for the low level of observed base substitutions, and (ii) has a relatively high capability of extension after misalignment of a misincorporated base on the next (complementary) template base, accounting for the high level of frameshift mutations. This model is supported by an experiment in which alpha subunit was required to initiate DNA synthesis from a terminal mispair in a sequence context that allowed slippage on the next template base. Among the products of this reaction, frameshifts outnumbered base pair substitutions by greater than 70-fold. A comparison to in vivo mutational spectra suggests that the pol III accessory factors may play a major role in modulating the fidelity of DNA synthesis.

Bacterial Proteins↗
PubMed1992

Unequal human immunodeficiency virus type 1 reverse transcriptase error rates with RNA and DNA templates.

Sequence variation in the type 1 human immunodeficiency virus (HIV-1) results, in part, from inaccurate replication by reverse transcriptase. Although this enzyme is error-prone during synthesis in vitro with DNA templates, the fidelity of RNA-dependent DNA synthesis relevant to minus-strand replication in the virus life cycle has not been examined extensively. In the present study, we have developed a system to determine the fidelity of transcription and reverse transcription and have used it to compare the fidelity of DNA synthesis by the HIV-1 reverse transcriptase with RNA and DNA templates of the same sequence. Overall, fidelity was several-fold higher with RNA than with DNA. Sequence analysis of mutants generated with the two substrates revealed that differences in error rates were substantial for specific errors. Fidelity with RNA was greater than 10-fold higher for substitution and minus-one nucleotide errors at five different homopolymeric positions. Because such errors likely result from template-primer slippage, this result suggests that misaligned intermediates are formed and/or used less frequently with an RNA template-DNA primer than with a DNA template-DNA primer. The results also suggest that HIV-1 reverse transcriptase synthesis with an RNA template-DNA primer was error-prone during incorporation of the first two nucleotides, perhaps due to aberrant enzyme-substrate interactions as synthesis initiates. The unequal error rates with RNA and DNA templates suggest that mistakes during minus- and plus-strand DNA synthesis may not contribute equally to the mutation rate of HIV-1. The data also provide estimates of substitution and frameshift error rates during transcription by T7 RNA polymerase.

Avian Myeloblastosis Virus↗