Biomedical subjects

M F Goodman

Publications and source records attributed to M F Goodman.

At least 109 records · Page 6Linked to original sources

PubMed1989

Spontaneous and UV-induced mutations in Escherichia coli K-12 strains with altered or absent DNA polymerase I.

The induction of mutations to valine resistance and to rifampin resistance occurs after UV irradiation in bacteria carrying a deletion through the polA gene (delta polA), showing that DNA polymerase I (PolI) is not an essential enzyme for this process. The PolI deletion strain showed a 7- to 10-fold-higher spontaneous mutation frequency than the wild type. The presence in the deletion strain of the 5'----3' exonuclease fragment on an F' episome caused an additional 10-fold increase in spontaneous mutation frequency, resulting in mutation frequencies on the order of 50- to 100-fold greater than wild type. The mutator effect associated with the 5'----3' exonuclease gene fragment together with much of the effect attributable to the polA deletion was blocked in bacteria carrying a umuC mutation. The mutator activity therefore appears to reflect constitutive SOS induction. Excision-proficient polA deletion strains exhibited increased sensitivity to the lethal effect of UV light which was only partially ameliorated by the presence of polA+ on an F' episome. The UV-induced mutation rate to rifampin resistance was marginally lower in delta polA bacteria than in bacteria carrying the polA+ allele. This effect is unlikely to be caused by the existence of a PolI-dependent mutagenic pathway and is probably an indirect effect caused by an alteration in the pattern of excision repair, since it did not occur in excision-deficient (uvrA) bacteria. An excision-deficient polA deletion strain possessed UV sensitivity similar to that of an isogenic strain carrying polA+ on an F' episome, showing that none of the functions of PolI are needed for postreplication repair in the absence of excision repair. Our data provide no evidence for a pathway of UV mutagenesis dependent on PolI, although it remains an open question whether PolI is able to participate when it is present.

DNA Polymerase I↗

PubMed1988

Purification and characterization of an inducible Escherichia coli DNA polymerase capable of insertion and bypass at abasic lesions in DNA.

We have investigated the ability of DNA polymerases from SOS-induced and uninduced Escherichia coli to incorporate nucleotides at a well-defined abasic (apurinic/apyrimidinic) DNA template site and to extend these chains from this unpaired 3' terminus. A DNA polymerase activity has been purified from E. coli, deleted for DNA polymerase I, that appears to be induced 7-fold in cells following treatment with nalidixic acid. Induction of this polymerase (designated DNA polymerase X) appears to be part of the SOS response of E. coli since it cannot be induced in strains containing a noncleavable form of the LexA repressor (Ind-). The enzyme is able to incorporate nucleotides efficiently opposite the abasic template lesion and to continue DNA synthesis. Although we observe an approximate 2-fold induction of DNA polymerase III in cells treated with nalidixic acid, several lines of evidence argue that DNA polymerase X is unrelated to DNA polymerase III (pol III). In contrast to pol X, pol III shows almost no detectable ability to incorporate at or extend beyond the abasic site; incorporation efficiency at the abasic lesion is at least 100-fold larger for pol X compared to pol III holoenzyme, pol III core, or pol III* (the polymerase III holoenzyme subassembly lacking the beta subunit). Pol X does not cross-react with polyclonal antibody directed against pol III holoenzyme complex or with monoclonal antibody prepared to the alpha subunit of pol III. Despite these structural and biochemical differences, pol X appears to interact specifically with the beta subunit of the pol III holoenzyme in the presence of single-stranded binding protein. Pol X has a molecular mass of 84 kDa. Our results indicate that this novel activity is likely to be identical to DNA polymerase II of E. coli.

Base Sequence↗

PubMed1988

Equilibrium between a wobble and ionized base pair formed between fluorouracil and guanine in DNA as studied by proton and fluorine NMR.

A synthetic oligonucleotide duplex containing the chemotherapeutic and mutagenic agent 5-fluorouracil paired with guanine has been studied in solution by proton and fluorine NMR. The 7-mer duplex containing a central FU.G base pair adopts a normal right-handed configuration. At low pH, the predominant base-paired structure is wobble, whereas at higher pH an ionized structure in Watson-Crick geometry is observed. The two structures are in a pH-dependent equilibrium with one another with an apparent pK of 8.3 at 23 degrees C. This is the first demonstration of an equilibrium between two distinct base pairing schemes and the first demonstration of a negatively charged base pair in DNA.

Base Composition↗

PubMed1988

The fidelity of base selection by the polymerase subunit of DNA polymerase III holoenzyme.

In common with other DNA polymerases, DNA polymerase III holoenzyme of E. coli selects the biologically correct base pair with remarkable accuracy. DNA polymerase III is particularly useful for mechanistic studies because the polymerase and editing activities reside on separate subunits. To investigate the biochemical mechanism for base insertion fidelity, we have used a gel electrophoresis assay to measure kinetic parameters for the incorporation of correct and incorrect nucleotides by the polymerase (alpha) subunit of DNA polymerase III. As judged by this assay, base selection contributes a factor of roughly 10(4)-10(5) to the overall fidelity of genome duplication. The accuracy of base selection is determined mainly by the differential KM of the enzyme for correct vs. incorrect deoxynucleoside triphosphate. The misinsertion of G opposite template A is relatively efficient, comparable to that found for G opposite T. Based on a variety of other work, the G:A pair may require a special correction mechanism, possibly because of a syn-anti pairing approximating Watson-Crick geometry. We suggest that precise recognition of the equivalent geometry of the Watson-Crick base pairs may be the most critical feature for base selection.

DNA Polymerase III↗

PubMed1988

Comparison between DNA melting thermodynamics and DNA polymerase fidelity.

The relation between DNA polymerase fidelity and base pairing stability is investigated by using DNA primer-template duplexes that contain a common 9-base template sequence but have either correct (A.T) or incorrect (G.T, C.T, T.T) base pairs at the primer 3' terminus. Thermal melting and enzyme kinetic measurements are compared for each kind of terminus. Analysis of melting temperatures finds that differences between the free energy changes upon dissociation (delta delta Go) are only 0.2, 0.3, and 0.4 kcal.mol-1 (1 cal = 4.18 J) for terminal A.T compared to G.T, C.T, and T.T mispairs, respectively, at 37 degrees C. We show that enthalpy changes are directly correlated with entropy changes for normal and abnormal base pairs in DNA in aqueous solution and that delta delta Go values are small because of near cancellation of corresponding enthalpy and entropy components. The kinetics of elongating primer termini are measured with purified Drosophila DNA polymerase alpha. The matched A.T terminus is found to be extended approximately 200 times faster than a G.T mismatch and 1400 and 2500 times faster than C.T and T.T mismatches, respectively. Enzymatic discrimination against elongating mismatched termini is based mainly on Km rather than Vmax differences. From Km at 37 degrees C, we find delta delta Go values of 2.6-3.7 kcal.mol-1, about an order of magnitude greater than indicated by melting data. A similar measurement of nucleotide insertion kinetics has previously found rates of forming A.T base pairs to be 500 times greater than G.T mispairs and 20,000 times greater than C.T and T.T mispairs. Here also, Km differences are mainly responsible for discrimination and indicate even larger delta delta Go values (4.3-4.9 kcal.mol-1). Thus, free energy differences between correct and incorrect base pairs in the active site cleft of polymerase appear to be greater than 10 times as large as in aqueous medium. We explore the idea that a binding cleft that snugly fits correct base pairs and excludes water at the active site may amplify base-pair free energy differences by reducing entropy differences and increasing enthalpy differences sufficiently to account for nucleotide insertion and extension fidelity.

Base Sequence↗

PubMed1987

Structural and dynamic properties of a fluorouracil-adenine base pair in DNA studied by proton NMR.

An oligodeoxynucleotide duplex containing the chemotherapeutic agent 5-fluorouracil (FU) has been constructed by solid phase phosphotriester synthesis and has been studied in solution by proton NMR. In this study, we provide the first structural characterization of a DNA complex containing a FU.A base pair. It has been determined that the 7-mer duplex containing a central FU.A base pair adopts a normal right-handed configuration and the A residue in the FU.A pair is oriented in the normal anticonfiguration giving a Watson-Crick base pair. The significant difference between T.A and FU.A base pairs is dynamic, not structural: the FU.A base pair opens faster than normal base pairs in the oligonucleotide studied. We provide evidence that the FU.A base pair has a significantly enhanced opening rate resulting form decreased stacking of the 5-fluorouracil residue and not from the enhanced acidity of the 5'-fluorouracil imino proton.

Adenine↗

PubMed1987

DNA polymerase insertion fidelity. Gel assay for site-specific kinetics.

A quantitative assay based on gel electrophoresis is described to measure nucleotide insertion kinetics at an arbitrary DNA template site. The assay is used to investigate kinetic mechanisms governing the fidelity of DNA synthesis using highly purified Drosophila DNA polymerase alpha holoenzyme complex and M13 primer-template DNA. Km and Vmax values are reported for correct insertion of A and misinsertion of G, C, and T opposite a single template T site. The misinsertion frequencies are 2 X 10(-4) for G-T and 5 X 10(-5) for both C-T and T-T relative to normal A-T base pairs. The dissociation constant of the polymerase-DNA-dNTP complex, as measured by Km, plays a dominant role in determining the rates of forming right and wrong base pairs. Compared with Km for insertion of A opposite T (3.7 +/- 0.7 microM), the Km value is 1100-fold greater for misinsertion of G opposite T (4.2 +/- 0.4 mM), and 2600-fold greater for misinsertion of either C or T opposite T (9.8 +/- 4.2 mM). These Km differences indicate that in the enzyme binding site the stability of A-T base pairs is 4.3 kcal/mol greater than G-T pairs and 4.9 kcal/mol greater than C-T or T-T pairs. In contrast to the large differences in Km, differences in Vmax are relatively small. There is only a 4-fold reduction in Vmax for insertion of G opposite T and an 8-fold reduction for C or T opposite T, compared with the correct insertion of A. For the specific template T site investigated, the nucleotide insertion fidelity for Drosophila polymerase alpha seems to be governed primarily by a Km discrimination mechanism.

Base Composition↗

PubMed1987

An abasic site in DNA. Solution conformation determined by proton NMR and molecular mechanics calculations.

We have determined the three-dimensional structure of a non-selfcomplementary nonanucleotide duplex which contains an abasic (apyrimidinic) site in the centre, i.e. a deoxyribose residue opposite an adenosine. The majority of the base and sugar proton resonances were assigned by NOESY, COSY and 2DQF spectra in D2O and H2O. We have measured the initial slope of buildup of NOEs in NOESY spectra at very short mixing times (25 to 50 ms), and from these were able to establish interproton distances for the central part of the duplex. We propose a different strategy for proton-proton distance determinations which takes into account the observed variations in correlation times for particular proton-proton vectors. A set of 31 measured interproton distances was incorporated into the refinement of the oligonucleotide structure by molecular mechanics calculations. Two structures were obtained which retain all aspects of a classical B DNA in which the unpaired adenine and the abasic deoxyribose lie inside the helix. We observe that the non-hydrogen bonded adenine is held well in the helix, the Tm of this base being the same as that of the A.T base pairs in the same duplex.

Algorithms↗

PubMed1987

NMR studies on an oligodeoxynucleotide containing 2-aminopurine opposite adenine.

A heteroduplex containing the mismatch 2-aminopurine (AP)-adenine has been synthesized and studied by proton NMR. The mismatch was incorporated into the sequence d[CGG(AP)GGC].d-(GCCACCG). One-dimensional nuclear Overhauser effect measurements in H2O and two-dimensional nuclear Overhauser effect spectra in D2O show AP.A base pairs in a wobble structure in which both bases are in the anti conformation. The adenine is stacked well in the helix, but the helix twist between the adenine and neighboring cytosine in the 3' direction is unusually small. As a result, the aminopurine on the opposite strand is somewhat pushed out of the helix. From the measurements of the imino proton line widths, the two adjacent G.C base pairs are not found to be significantly destabilized by the presence of the purine-purine wobble pair.

2-Aminopurine↗

PubMed1987

Size of the directing moiety at carbon 5 of cytosine and the activity of human DNA(cytosine-5) methyltransferase.

M13 DNAs in which carbon 5 of each deoxycytidine residue in one strand is replaced with a bulky group are very good substrates for human DNA (cytosine-5) methyltransferase. Rate enhancements of up to 35 fold are obtained depending on the size of the moiety at C-5. The enzyme appears optimally suited to sense a methyl group in one strand at this position. Alkaline density gradient analyses of the distribution of methyl groups applied to 5-BrdCyd or 5-IdCyd substituted DNA reveal that these groups serve to direct the enzyme to methylate the unsubstituted strand.

Cytosine↗

PubMed1987

Nucleotide insertion kinetics opposite abasic lesions in DNA.

A gel assay is introduced to measure DNA polymerase insertion kinetics at single sites along a DNA template strand. The assay is used to analyze the kinetics of inserting deoxynucleotides opposite a synthetic abasic (apurinic/apyrimidinic) lesions using Drosophila DNA polymerase alpha. The location of the abasic lesion next to different nearest-neighbor bases allows the effects of base stacking on the specificity of insertion to be evaluated. The specificity of nucleotide insertion, Vmax/Km, is 6-11 times greater for A over G and about 20-50 times greater for A over C and T. The insertion specificity at the abasic lesion appears to depend more on differences in Vmax than Km. Apparent Michaelis constants for inserting A and G deoxynucleotides are similar to within about a factor of 2. The insertion of A or G occurs most efficiently at the abasic lesion when T is the 5'-nearest neighbor on the primer strand and least efficiently when G is the 5'-nearest neighbor. The presence of different base stacking partners adjacent to the site of insertion has up to a 4-fold effect on specificity.

Animals↗

PubMed1987

Structural and dynamic properties of a bromouracil-adenine base pair in DNA studied by proton NMR.

We have synthesized and studied by proton NMR a duplex heptaoligonucleotide containing a 5-bromouracil (brU)-adenine base pair. This represents the first structural characterization of a B-form DNA containing brU. The brU.A base pair is Watson-Crick rather than Hoogsteen as seen for the monomers in the crystalline state. From analysis of the NOESY sepctra at very short mixing times evidence is presented that substitution of brU for T induces significant conformational changes from that of a normal B DNA. The helix twist between brU4.A11 and G3.C12 is ca. 15 degrees and for both brU4 and G3 the glycosyl torsion angles are significantly changed. The imino proton of the bru.A base pair shows a pH insensitive line with which shows that the pK of brU in this base pair is very much higher than that of the monomer.

Adenine↗

PubMed1986

Synthesis and properties of oligonucleotides containing 2'-deoxynebularine and 2'-deoxyxanthosine.

The preparation of synthetic oligonucleotides containing 2'-deoxynebularine (dN) and 2'-deoxyxanthosine (dX) is described. The thermal stabilities of duplexes containing dX, dN, and 2'-deoxyinosine (dI) base-paired with the four natural bases have been measured. Xanthine base pairs have stabilities at pH 5.5 that are similar to those of dI-containing duplexes at neutral pH. When xanthine is paired with adenine or cytosine an unusual stabilization of the duplex structure is observed at acid pH. Incorporation of base mispairs opposite template xanthine sites were measured using Drosophila DNA polymerase alpha. The relative nucleoside incorporation rates are in the order: T greater than C much greater than A approximately equal to G. These rates do not correlate with relative thermodynamic stabilities of base mispairs with xanthine obtained from Tm measurements: T greater than G greater than A approximately equal to C. We suggest that DNA polymerase misinsertion rates are greatest when the base mispair can be formed in accordance with Watson-Crick as opposed to other base pairing geometries even though other geometries, e.g. wobble, may result in a more stable final DNA product.

Chemical Phenomena↗

PubMed1986

Synthesis and properties of defined DNA oligomers containing base mispairs involving 2-aminopurine.

DNA heptamers containing the mutagenic base analogue 2-aminopurine (AP) have been chemically synthesized and physically characterized. We report on the relative stabilities of base pairs between AP and each of the common DNA bases, as determined from heptamer duplex melts at 275 and 330 nm. Base pairs are ranked in order of decreasing stability: AP.T greater than AP.A greater than AP.C greater than AP.G. It is of interest that AP.A is more stable than AP.C even though DNA polymerase strongly favors the formation of AP.C over AP.A base pairs. Comparisons of melting profiles at 330 nm and 275 nm indicate that AP.T, AP.A, and AP.C base pairs are annealed in heptamer duplexes and melt 2-3 degrees prior to surrounding base pairs, whereas AP.G appears not to be annealed.

2-Aminopurine↗

PubMed1986

Variation of nonexchangeable proton resonance chemical shifts as a probe of aberrant base pair formation in DNA.

Variation of nonexchangeable proton resonance chemical shifts for deoxycytidine and deoxy-adenosine as a function of protonation and imino tautomer formation has been determined. Protonation induces downfield shifts of proton resonances whereas formation of the rare imino tautomer induces upfield shifts. Titration curves are constructed on the basis of spectrophotometrically determined pK values. Excellent fit is obtained between theoretical titration curves and experimental data, which indicates that chemical shifts of base protons may be used to quantitatively determine the relative concentrations of either rare imino tautomeric conformations or protonated base forms. These data may be utilized as an aid in the elucidation of the nature of hydrogen bonding between mismatched base pairs in DNA oligomers containing cytosine or adenine residues. These data, in conjunction with the oligonucleotide study of Patel et al. [Patel, D. J., Kozlowski, S.A., Ikuta, S., & Itakura, K. (1984) Biochemistry 23, 3218-3226], have been used to rigorously argue the existence of a "protonated" adenine residue in the A-C mismatch. This structure allows reconciliation of the NMR solution data with crystallographic data [Hunter, W.N., Brown, T., Anand, N.N., & Kennard, O. (1986) Nature (London) 320, 552-555], which support the protonated base pair.

Base Composition↗

PubMed1986

Base pairing and mutagenesis: observation of a protonated base pair between 2-aminopurine and cytosine in an oligonucleotide by proton NMR.

2-Aminopurine (AP), a potent mutagenic base analogue, most frequently pairs with thymine. In the AP X T base pair, both bases adopt normal tautomeric forms. The mechanism for the mutagenic activity arises from its observed pairing with cytosine, which has been ascribed to an enhanced tendency to adopt the rare imino tautomeric form. NMR studies in H2O on all the exchangeable protons in an oligonucleotide duplex containing an AP X T base pair show Watson-Crick hydrogen bonding. When the thymine is replaced by cytosine in the duplex, we observe an AP X C base pair. Both amino protons of AP are seen excluding the rare tautomeric form. Although several alternative structures are possible, it is shown that the second hydrogen bond is formed by protonation of the AP X C base pair and that this is the dominant species under physiological conditions.

2-Aminopurine↗

PubMed1986

Comparison of nucleotide interactions in water, proteins, and vacuum: model for DNA polymerase fidelity.

We propose a model for DNA polymerase fidelity in which free energy differences, delta delta G, between matched and mismatched nucleotides are magnified at the enzyme's active site. Both hydrogen bonding and stacking components of the interaction energy are amplified, with the most profound effect being on the magnitude of hydrogen-bonding interactions. Magnification in delta delta G values follows from the exclusion of water around base pairs in the active site cleft of the enzyme. After showing that base-pair dissociation energies calculated from hydrogen-bonding and base-stacking interactions in vacuo are greatly reduced by water, it is proposed that water removal results in a proportional restoration of these contributions to base pairing. Assuming approximately equal to 40% exclusion of surrounding water, one predicts magnified values of delta delta G sufficient to account for polymerase insertion and proofreading fidelity, thereby avoiding the need to postulate additional active site constraints in order to select or reject nucleotides.

Base Composition↗

PubMed1985

Influence of neighboring bases on DNA polymerase insertion and proofreading fidelity.

We propose a model to describe the frequencies of site-specific base substitution errors by DNA polymerase. In the model, nucleotide misinsertion frequencies are determined by 5'-nearest-neighbor base stacking and 3'-exonucleolytic proofreading efficiencies are governed by the relative proportions of G . C base pairs in the region surrounding the misinserted nucleotide. The model is used to analyze the frequency of replacing dAMP by 2-aminopurine deoxyribonucleotide with purified bacteriophage T4 L141 antimutator DNA polymerase at 57 sites on phi X174 DNA (Pless, R. C., and Bessman, M.J. (1983) Biochemistry 22, 4905-4915). A linear least-squares fit yields a correlation coefficient of 0.83 and a standard deviation of 2.8% between predicted and observed results. Four to five base pairs on each side of the 2-aminopurine incorporation site, approximately one double-helical turn, are found to exert a maximal influence on proofreading. Thermal melting data on native and synthetic DNA are used to deduce base-stacking energies for nearest-neighbor doublets including those involving 2-aminopurine. The inclusion of base-stacking energies in the model calculations leads to predictions similar to those based on the use of empirical parameters for individual base pairs.

Bacteriophage phi X 174↗