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T A Kunkel

Publications and source records attributed to T A Kunkel.

At least 19 recordsLinked to original sources
PubMed2000

DNA replication fidelity.

DNA replication fidelity is a key determinant of genome stability and is central to the evolution of species and to the origins of human diseases. Here we review our current understanding of replication fidelity, with emphasis on structural and biochemical studies of DNA polymerases that provide new insights into the importance of hydrogen bonding, base pair geometry, and substrate-induced conformational changes to fidelity. These studies also reveal polymerase interactions with the DNA minor groove at and upstream of the active site that influence nucleotide selectivity, the efficiency of exonucleolytic proofreading, and the rate of forming errors via strand misalignments. We highlight common features that are relevant to the fidelity of any DNA synthesis reaction, and consider why fidelity varies depending on the enzymes, the error, and the local sequence environment.

Base Pair Mismatch↗
PubMed1992

Biochemical studies on the reverse transcriptase and RNase H activities from human immunodeficiency virus strains resistant to 3'-azido-3'-deoxythymidine.

A series of biochemical investigations to compare the DNA polymerase and RNase H functions of the reverse transcriptases (RTs) corresponding to azidothymidine (AZT)-sensitive and -resistant human immunodeficiency virus (HIV) strains are described. Steady-state kinetic studies with purified recombinant enzymes utilizing several templates and three inhibitors, 3' azido-3' deoxythymidine triphosphate (AZTTP), 3-amino-thymidine 5'-triphosphate, and 2',3'-didehydro-2',3'-dideoxythymidine 5'-triphosphate, found consistent 2-4-fold differences between the enzymes from the two strains over a wide pH range. A strong pH dependence for all three inhibitors was found at pH values below 7.4 and suggested an ionizable group on the enzyme with a pK of about 7. The sensitivities of the RNase H activities of the two enzymes to AZTTP and AZTMP were also compared and found to be similar. The nucleotide incorporation fidelities of recombinant RTs corresponding to AZT-sensitive and -resistant clinical isolates were compared and the error specificities determined. No significant differences were found. Both enzymes were equally able to incorporate AZTTP into an elongating M13 DNA strand with concomitant chain termination. Purified wild-type and mutant virions from cell-culture supernatants were compared in "endogenous" DNA synthesis reactions, and the sensitivities of this activity to AZTTP were found to be similar. The contrast between the small differences found in this study and the high level of viral resistance in tissue culture presumably reflects an incomplete understanding of AZT inhibition of HIV in the cell.

Antiviral Agents↗
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↗
PubMed1992

The effects of dNTP pool imbalances on frameshift fidelity during DNA replication.

The use of unequal concentrations of the four deoxynucleoside triphosphates (dNTPs) in DNA polymerization reactions alters base substitution error rates in a predictable way. Less is known about the effects of substrate imbalances on base addition and deletion error rates. Thus, we examined pool bias effects on frameshift fidelity during DNA synthesis catalyzed by replicative DNA polymerases. Imbalanced pools altered the frameshift fidelity of the human immunodeficiency virus type-1 reverse transcriptase. Both mutagenic and antimutagenic effects were observed for minus-one, plus-one, and minus-two nucleotide errors, in a highly sequence-specific manner. Most of this specificity can be rationalized by either of two models. One involves frameshifts initiated by pool bias-induced nucleotide misinsertion, and the other involves pool bias-initiated template-primer slippage. Several examples of complex mutations were also recovered more than once in small mutant collections. These contained closely spaced single-base substitution and minus-one base frameshift changes. The two changes occurred at a frequency much higher than predicted if they were generated independently. This suggests that when the polymerase makes one mistake, the probability that it will make a second mistake within the next few incorporations increases significantly. Perturbation of dNTP pools also affected the frameshift fidelity of the replicative yeast DNA polymerase alpha. In reactions containing a low concentration of one dNTP, the error rate increased for one-nucleotide deletions at homopolymeric template nucleotides complementary to the dNTP whose concentration was low. We extended this approach to determine the frameshift fidelity of simian virus 40 origin-dependent semiconservative replication of double-stranded DNA in extracts of human cells. In reactions performed with an equal concentration of all four dNTPs, replication was highly accurate for minus-one-nucleotide errors. However, when the concentration of one dNTP was decreased, the replication error rate increased at complementary, homopolymeric template positions. This response provides an approach for describing frameshift accuracy during replication of the leading and lagging strands.

Base Sequence↗
PubMed1992

Biological asymmetries and the fidelity of eukaryotic DNA replication.

A diploid human genome contains approximately six billion nucleotides. This enormous amount of genetic information can be replicated with great accuracy in only a few hours. However, because DNA strands are oriented antiparallel while DNA polymerization only occurs in the 5'----3' direction, semi-conservative replication of double-stranded DNA is an asymmetric process, i.e., there is a leading and a lagging strand. This provides a considerable opportunity for non-random error rates, because the architecture of the two strands as well as the DNA polymerases that replicate them may be different. In addition, the proteins that start or finish chains may well be different from those that perform the bulk of chain elongation. Furthermore, while replication fidelity depends on the absolute and relative concentrations of the four deoxyribonucleotide precursors, these are not equal in vivo, not constant throughout the cell cycle, and not necessarily equivalent in all cell types. Finally, the fidelity of DNA synthesis is sequence-dependent and the eukaryotic nuclear genome is a heterogeneous substrate. It contains repetitive and non-repetitive sequences and can actually be considered as two subgenomes that differ in nucleotide composition and gene content and that replicate at different times. The effects that each of these asymmetries may have on error rates during replication of the eukaryotic genome are discussed.

DNA Replication↗
PubMed1991

Fidelity of mammalian DNA replication and replicative DNA polymerases.

Current models suggest that two or more DNA polymerases may be required for high-fidelity semiconservative DNA replication in eukaryotic cells. In the present study, we directly compare the fidelity of SV40 origin-dependent DNA replication in human cell extracts to the fidelity of mammalian DNA polymerases alpha, delta, and epsilon using lacZ alpha of M13mp2 as a reporter gene. Their fidelity, in decreasing order, is replication greater than or equal to pol epsilon greater than pol delta greater than pol alpha. DNA sequence analysis of mutants derived from extract reactions suggests that replication is accurate when considering single-base substitutions, single-base frameshifts, and larger deletions. The exonuclease-containing calf thymus DNA polymerase epsilon is also highly accurate. When high concentrations of deoxynucleoside triphosphates and deoxyguanosine monophosphate are included in the pol epsilon reaction, both base substitution and frameshift error rates increase. This response suggests that exonucleolytic proofreading contributes to the high base substitution and frameshift fidelity. Exonuclease-containing calf thymus DNA polymerase delta, which requires proliferating cell nuclear antigen for efficient synthesis, is significantly less accurate than pol epsilon. In contrast to pol epsilon, pol delta generates errors during synthesis at a relatively modest concentration of deoxynucleoside triphosphates (100 microM), and the error rate did not increase upon addition of adenosine monophosphate. Thus, we are as yet unable to demonstrate that exonucleolytic proofreading contributes to accuracy during synthesis by DNA polymerase delta. The four-subunit DNA polymerase alpha-primase complex from both HeLa cells and calf thymus is the least accurate replicative polymerase. Fidelity is similar whether the enzyme is assayed immediately after purification or after being stored frozen.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1991

Eukaryotic DNA polymerase amino acid sequence required for 3'----5' exonuclease activity.

We have identified an amino-proximal sequence motif, Phe-Asp-Ile-Glu-Thr, in Saccharomyces cerevisiae DNA polymerase II that is almost identical to a sequence comprising part of the 3'----5' exonuclease active site of Escherichia coli DNA polymerase I. Similar motifs were identified by amino acid sequence alignment in related, aphidicolin-sensitive DNA polymerases possessing 3'----5' proofreading exonuclease activity. Substitution of Ala for the Asp and Glu residues in the motif reduced the exonuclease activity of partially purified DNA polymerase II at least 100-fold while preserving the polymerase activity. Yeast strains expressing the exonuclease-deficient DNA polymerase II had on average about a 22-fold increase in spontaneous mutation rate, consistent with a presumed proofreading role in vivo. In multiple amino acid sequence alignments of this and two other conserved motifs described previously, five residues of the 3'----5' exonuclease active site of E. coli DNA polymerase I appeared to be invariant in aphidicolin-sensitive DNA polymerases known to possess 3'----5' proofreading exonuclease activity. None of these residues, however, appeared to be identifiable in the catalytic subunits of human, yeast, or Drosophila alpha DNA polymerases.

Amino Acid Sequence↗
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↗
PubMed1991

Heteroduplex repair in extracts of human HeLa cells.

A general repair process for DNA heteroduplexes has been detected in HeLa cell extracts. Using a variety of M13mp2 DNA substrates containing single-base mismatches and extra nucleotides, extensive repair is observed after incubation with HeLa cell cytoplasmic extracts and subsequent transfection of bacterial cells with the treated DNA. Most, but not all, mispairs as well as two frameshift heteroduplexes are repaired efficiently. Parallel measurements of repair in HeLa extracts and in Escherichia coli suggest that repair specificities are similar for the two systems. The presence of a nick in the molecule is required for efficient repair in HeLa cell extracts, and the strand containing the nick is the predominantly repaired strand. Mismatch-dependent DNA synthesis is observed when radiolabeled restriction fragments, produced by reaction of the extract with heteroduplex and homoduplex molecules, are compared. Specific labeling of fragments, representing a region of approximately 1,000 base pairs and containing the nick and the mismatch, is detected for the heteroduplex substrate but not the homoduplex. The repair reaction is complete after 20 min and requires added Mg2+, ATP, and an ATP-regenerating system, but not dNTPs, which are present at sufficient levels in the extract. An inhibitor of DNA polymerase beta, dideoxythimidine 5'-triphosphate, does not inhibit mismatch-specific DNA synthesis. Aphidicolin, an inhibitor of DNA polymerases alpha, delta, and epsilom, inhibits both semiconservative replication and repair synthesis in the extract. Butylphenyl-dGTP also inhibits both replicative and repair synthesis but at a concentration known to inhibit DNA polymerase alpha preferentially rather than delta or epsilon. This suggests that DNA polymerase alpha may function in mismatch repair.

Adenosine Triphosphate↗
PubMed1991

DNA polymerase fidelity and the polymerase chain reaction.

High-fidelity DNA synthesis conditions are those that exploit the inherent ability of polymerases to discriminate against errors. This review has described several experimental approaches for controlling the fidelity of enzymatic DNA amplification. One of the most important parameters to consider is the choice of which polymerase to use in PCR. As demonstrated by the data in Tables 2 and 3, high-fidelity DNA amplification will be best achieved by using a polymerase with an active 3'-->5' proofreading exonuclease activity (Fig. 1E). For those enzymes that are proofreading-deficient, the in vitro reaction conditions can significantly influence the polymerase error rates. To maximize fidelity at the dNTP insertion step (Fig. 1A,B), any type of deoxynucleoside triphosphate pool imbalance should be avoided. Similarly, stabilization of errors by polymerase extension from mispaired or misaligned primer-termini (Fig. 1D) can be minimized by reactions using short synthesis times, low dNTP concentrations, and low enzyme concentrations. Additional improvements in fidelity can be made by further manipulating the reaction conditions. To perform high-fidelity PCR with Taq polymerase, reactions should contain a low MgCl2 concentration, not in large excess over the total concentration of dNTP substrates, and be buffered to approximately pH 6 (70 degrees C) using Bis-Tris Propane or PIPES (Table 2). These buffers have a pKa between pH 6 and pH 7 and a small temperature coefficient (delta pKa/degree C), allowing the pH to be maintained stably throughout the PCR cycle. For amplifications in which fidelity is the critical issue, one should avoid the concept that conditions generating more DNA product are the better conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA↗
PubMed1990

The fidelity of DNA synthesis catalyzed by derivatives of Escherichia coli DNA polymerase I.

The fidelity of DNA synthesis by an exonuclease-proficient DNA polymerase results from the selectivity of the polymerization reaction and from exonucleolytic proofreading. We have examined the contribution of these two steps to the fidelity of DNA synthesis catalyzed by the large Klenow fragment of Escherichia coli DNA polymerase I, using enzymes engineered by site-directed mutagenesis to inactivate the proofreading exonuclease. Measurements with two mutant Klenow polymerases lacking exonuclease activity but retaining normal polymerase activity and protein structure demonstrate that the base substitution fidelity of polymerization averages one error for each 10,000 to 40,000 bases polymerized, and can vary more than 30-fold depending on the mispair and its position. Steady-state enzyme kinetic measurements of selectivity at the initial insertion step by the exonuclease-deficient polymerase demonstrate differences in both the Km and the Vmax for incorrect versus correct nucleotides. Exonucleolytic proofreading by the wild-type enzyme improves the average base substitution fidelity by 4- to 7-fold, reflecting efficient proofreading of some mispairs and less efficient proofreading of others. The wild-type polymerase is highly accurate for -1 base frameshift errors, with an error rate of less than or equal to 10(-6). The exonuclease-deficient polymerase is less accurate, suggesting that proofreading also enhances frameshift fidelity. Even without a proofreading exonuclease, Klenow polymerase has high frameshift fidelity relative to several other DNA polymerases, including eucaryotic DNA polymerase-alpha, an exonuclease-deficient, 4-subunit complex whose catalytic subunit is almost three times larger. The Klenow polymerase has a large (46 kDa) domain containing the polymerase active site and a smaller (22 kDa) domain containing the active site for the 3'----5' exonuclease. Upon removal of the small domain, the large polymerase domain has altered base substitution error specificity when compared to the two-domain but exonuclease-deficient enzyme. It is also less accurate for -1 base errors at reiterated template nucleotides and for a 276-nucleotide deletion error. Thus, removal of a protein domain of a DNA polymerase can affect its fidelity.

Base Sequence↗
PubMed1990

High fidelity DNA synthesis by the Thermus aquaticus DNA polymerase.

We demonstrate that despite lacking a 3'----5' proofreading exonuclease, the Thermus aquaticus (Taq) DNA polymerase can catalyze highly accurate DNA synthesis in vitro. Under defined reaction conditions, the error rate per nucleotide polymerized at 70 degrees C can be as low as 10(-5) for base substitution errors and 10(-6) for frameshift errors. The frequency of mutations produced during a single round of DNA synthesis of the lac Z alpha gene by Taq polymerase responds to changes in dNTP concentration, pH, and the concentration of MgCl2 relative to the total concentration of deoxynucleotide triphosphates present in the reaction. Both base substitution and frameshift error rates of less than 1/100,000 were observed at pH 5-6 (70 degrees C) or when MgCl2 and deoxynucleotide triphosphates were present at equimolar concentrations. These high fidelity reaction conditions for DNA synthesis by the Taq polymerase may be useful for specialized uses of DNA amplified by the polymerase chain reaction.

Base Composition↗
PubMed1990

Lysine 188 of the catabolite gene activator protein (CAP) plays no role in specificity at base pair 7 of the DNA half site.

Two similar, but not identical, models have been proposed for the amino acid-base pair contacts in the CAP-DNA complex ('model I,' Weber, I. and Steitz, T., Proc. Natl. Acad. Sci. USA, 81, 3973-3977, 1984; 'model II,' Ebright, et al., Proc. Natl. Acad. Sci. USA, 81, 7274-7278, 1984). The most important difference between the two models involves Lys188 of CAP. Model I predicts that Lys188 of CAP makes a specificity determining contact with base pair 7 of the DNA half site. In contrast, model II predicts that Lys188 makes no contact with base pair 7 of the DNA half site. In the present work, we have used site-directed mutagenesis to replace Lys188 of CAP by Asn, an amino acid unable to make the putative contact. We have assessed the specificities of the following proteins, both in vitro and in vivo: wild-type CAP, [Asn188]CAP, [Val181]CAP, and [Val181;Asn188]CAP. The results indicate that Lys188 makes no contribution to specificity at base pair 7 of the DNA half site. We propose, contrary to model I, that Lys188 makes no contact with base pair 7 of the DNA half site.

Base Sequence↗
PubMed1990

A sensitive genetic assay for the detection of cytosine deamination: determination of rate constants and the activation energy.

Previously it has not been possible to determine the rate of deamination of cytosine in DNA at 37 degrees C because this reaction occurs so slowly. We describe here a sensitive genetic assay to measure the rate of cytosine deamination in DNA at a single cytosine residue. The assay is based on reversion of a mutant in the lacZ alpha gene coding sequence of bacteriophage M13mp2 and employs ung- bacterial strains lacking the enzyme uracil glycosylase. The assay is sufficiently sensitive to allow us to detect, at a given site, a single deamination event occurring with a background frequency as low as 1 in 200,000. With this assay, we determined cytosine deamination rate constants in single-stranded DNA at temperatures ranging from 30 to 90 degrees C and then calculated that the activation energy for cytosine deamination in single-stranded DNA is 28 +/- 1 kcal/mol. At 80 degrees C, deamination rate constants at six sites varied by less than a factor of 3. At 37 degrees C, the cytosine deamination rate constants for single- and double-stranded DNA at pH 7.4 are 1 x 10(-10) and about 7 x 10(-13) per second, respectively. (In other words, the measured half-life for cytosine in single-stranded DNA at 37 degrees C is ca. 200 years, while in double-stranded DNA it is on the order of 30,000 years.) Thus, cytosine is deaminated approximately 140-fold more slowly when present in the double helix. These and other data indicate that the rate of deamination is strongly dependent upon DNA structure and the degree of protonation of the cytosine. The data suggest that agents which perturb DNA structure or facilitate direct protonation of cytosine may induce deamination at biologically significant rates. The assay provides a means to directly test the hypothesis.

Bacteriophages↗
PubMed1990

Purification and characterization of DNA polymerase II from the yeast Saccharomyces cerevisiae. Identification of the catalytic core and a possible holoenzyme form of the enzyme.

We have purified yeast DNA polymerase II to near homogeneity as a 145-kDa polypeptide. During the course of this purification we have detected and purified a novel form of DNA polymerase II that we designate as DNA polymerase II. The most highly purified preparations of DNA polymerase II are composed of polypeptides with molecular masses of 200, 80, 34, 30, and 29 kDa. Immunological analysis and peptide mapping of DNA polymerase II and the 200-kDa subunit of DNA polymerase II indicate that the 145-kDa DNA polymerase II polypeptide is derived from the 200-kDa polypeptide of DNA polymerase II. Activity gel analysis shows that the 145- and the 200-kDa polypeptides have catalytic function. The polypeptides present in the DNA polymerase II preparation copurify with the polymerase activity with a constant relative stoichiometry during chromatography over five columns and co-sediment with the activity during glycerol gradient centrifugation, suggesting that this complex may be a holoenzyme form of DNA polymerase II. Both forms of DNA polymerase II possess a 3'-5' exonuclease activity that remains tightly associated with the polymerase activity during purification. DNA polymerase II is similar to the proliferating cell nuclear antigen (PCNA)-independent form of mammalian DNA polymerase delta in its resistance to butylpheny-dGTP, template specificity, stimulation of polymerase and exonuclease activity by KCl, and high processivity. Although calf thymus PCNA does not stimulate the activity of DNA polymerase II on poly(dA):oligo(dT), possibly due to the limited length of the template, the high processivity of yeast DNA polymerase II on this template can be further increased by the addition of PCNA, suggesting that conditions may exist for interactions between PCNA and yeast DNA polymerase II.

Base Composition↗