Effects of environmental enrichment on the amyotrophic lateral sclerosis mouse model.

The manner in which an animal's environment is furnished may have significant implications for animal welfare as well as research outcomes. We evaluated four different housing conditions to determine the effects of what has been considered standard rodent enrichment and the exercise opportunities those environments allow on disease progression in the amyotrophic lateral sclerosis mouse model. Forty-eight copper/zinc superoxide dismutase mice (strain: B6SJL-TgN [SOD1-G931]1Gur) (SOD1) and 48 control (C) (strain: B6SJL-TgN[SOD1]2Gur) male mice were randomly assigned to four different conditions where 12 SOD1 and 12 C animals were allotted to each condition (n = 96). Conditions tested the effects of standard housing, a forced exercise regime, access to a mouse house and opportunity for ad libitum exercise on a running wheel. In addition to the daily all-occurrence behavioural sampling, mice were weighed and tested twice per week on gait and Rotor-Rod performance until the mice reached the age of 150 days (C) or met the criteria for our humane endpoint (SOD1). The SOD1 mice exposed to the forced exercise regime and wheel access did better in average lifespan and Rotor-Rod performance, than SOD1 mice exposed to the standard cage and mouse house conditions. In SOD1 mice, stride length remained longest throughout the progression of the disease in mice exposed to the forced exercise regime compared with other SOD1 conditions. Within the control group, mice in the standard cage and forced exercise regime conditions performed significantly less than the mice with the mouse house and wheels on the Rotor-Rod. Alpha motor neuron counts were highest in mice with wheels and in mice exposed to forced exercise regime in both mouse strains. All SOD1 mice had significantly lower alpha neuron counts than controls (P < 0.05). These data show that different enrichment strategies affect behaviour and disease progression in a transgenic mouse model, and may have implications for the effects of these strategies on experimental outcomes.

The 3' --> 5' exonuclease of T4 DNA polymerase removes premutagenic alkyl mispairs and contributes to futile cycling at O6-methylguanine lesions.

We have studied the processing of O(6)-methylguanine (m6G)-containing oligonucleotides and N-methyl-N-nitrosourea (MNU)-treated DNA templates by the 3' --> 5' exonuclease of T4 DNA polymerase. In vitro biochemical analyses demonstrate that the exonuclease can remove bases opposite a defined m6G lesion. The efficiency of excision of a terminal m6G.T was similar to that of m6G.C, and both were excised as efficiently as a G.T substrate. Partitioning assays between the polymerase and exonuclease activities, performed in the presence of dNTPs, resulted in repeated incorporation and excision events opposite the m6G lesion. This idling produces dramatically less full-length product, relative to natural substrates, indicating that the 3' --> 5' exonuclease may contribute to DNA synthesis inhibition by alkylating agents. Genetic data obtained using an in vitro herpes simplex virus-thymidine kinase assay support the inefficiency of the exonuclease as a "proofreading" activity for m6G, since virtually all mutations produced by the native enzyme using MNU-treated templates were G --> A transitions. Comparison of MNU dose-response curves for exonuclease-proficient and -deficient forms of T4 polymerase reveals that the exonuclease efficiently removes 50-86% of total premutagenic alkyl mispairs. We propose that idling of exonuclease-proficient polymerases at m6G lesions during repair DNA synthesis provides the biochemical explanation for cellular cytotoxicity of methylating agents.

Hydrophobic interactions in the hinge domain of DNA polymerase beta are important but not sufficient for maintaining fidelity of DNA synthesis.

We previously described a general mutator form of mammalian DNA polymerase beta containing a cysteine substitution for tyrosine 265. Residue 265 localizes to a hydrophobic hinge region predicted to mediate a polymerase conformational change that may aid in nucleotide selectivity. In this study we tested the hypothesis that van der Waals and hydrophobic contacts between Y265 and neighboring residues are important for DNA synthesis fidelity and catalysis, by altering interactions in the hinge domain via substitution at position 265. Consistent with the importance of hydrophobic interactions, we found that phenylalanine, leucine, and tryptophan substitutions did not alter significantly the steady-state catalytic efficiency of DNA synthesis, relative to wild type, while the polar serine substitution decreased catalytic efficiency 6-fold. However, we found that all substitutions other than phenylalanine increased the error frequency, relative to wild type, in the order serine > tryptophan = leucine. Therefore, maintenance of the hydrophobicity of residue 265 was not sufficient for maintaining fidelity of DNA synthesis. We conclude that while hydrophobic interactions in the hinge domain are important for fidelity, additional factors such as electrostatic and van der Waals interactions contributed by the tyrosine 265 aromatic ring are required to retain wild-type fidelity.

Mutational analyses of dinucleotide and tetranucleotide microsatellites in Escherichia coli: influence of sequence on expansion mutagenesis.

Mutagenesis at [GT/CA](10), [TC/AG](11) and [TTCC/AAGG](9) microsatellite sequences inserted in the herpes simplex virus thymidine kinase (HSV-tk) gene was analyzed in isogenic mutL(+) and mutL(-) Escherichia coli. In both strains, significantly more expansion than deletion mutations were observed at the [TTCC/AAGG](9) motif relative to either dinucleotide motif. As the HSV-tk coding sequence contains an endogenous [G/C](7) mononucleotide repeat and approximately 1000 bp of unique sequence, we were able to compare mutagenesis among various sequence motifs. We observed that the relative risk of mutation in E.COLI: is: [TTCC/AAGG](9) > [GT/CA](10) approximately [TC/AG](11) > unique approximately [G/C](7). The mutation frequency varied 1400-fold in mutL(+) cells between the tetranucleotide motif and the mononucleotide motif, but only 50-fold in mutL(-) cells. The [G/C](7) sequence was destabilized the greatest and the tetranucleotide motif the least by loss of mismatch repair. These results demonstrate that the quantitative risk of mutation at various microsatellites greatly depends on the DNA sequence composition. We suggest alternative models for the production of expansion mutations during lagging strand replication of the [TTCC/AAGG](9) microsatellite.

Somatic mutation rates and specificities at TC/AG and GT/CA microsatellite sequences in nontumorigenic human lymphoblastoid cells.

We have examined mutational events at TC/AG microsatellites, the second most abundant dinucleotide repetitive motif in the human genome. Mutational targets were constructed containing TC/AG alleles up to 20 units in-frame within the coding region of the herpes simplex virus thymidine kinase (HSV-tk) gene. These targets were incorporated into oriP shuttle vectors, which replicate episomally in human lymphoblastoid cells. The overall HSV-tk mutant frequencies measured after 10 population doublings in cells derived from a clinically normal donor were slightly increased over the background of mutations recovered in Escherichia coli. DNA sequence analyses revealed that replication of TC/AG vectors in human cells increased the mutation frequencies at the microsatellite motif up to 3-fold, relative to background. Additionally, the median HSV-tk mutation rate of single-cell clones carrying the [TC/AG]17 vector was significantly different from that of clones harboring the control vector. The median rate of allele length alterations within the [TC/AG]11 tract was 2 x 10(-6) mutations/cell generation, with an equivalent rate of deletion and expansion mutations. In contrast, a [GT/CA]10 vector showed no increase in microsatellite mutation frequency after replication in human cells, and mutation rates of clones carrying a [GT/CA]16 vector were not significantly different from controls. Intriguingly, replication in human cells of all microsatellite-containing vectors resulted in elevated mutation frequencies at the downstream HSV-tk coding sequence of up to 20-fold, an effect not observed for the control vector. These results demonstrate that the frequency of mutational events at TC/AG motifs is slightly greater than at GT/CA motifs of similar allele length. This is the first report to our knowledge of the mutation rates at TC/AG microsatellite alleles in eukaryotic or prokaryotic cells.

DNA polymerase mutagenic bypass and proofreading of endogenous DNA lesions.

DNA polymerases differentiate between correct and incorrect substrates during synthesis on undamaged DNA templates through the biochemical steps of base incorporation, primer-template extension and proofreading excision. Recent research examining DNA polymerase processing of abasic, alkylation and oxidative lesions is reviewed in light of these discrimination mechanisms. Inhibition of DNA synthesis results from correct polymerase discrimination against utilization of geometrically incorrect template bases or 3' terminal basepairs. The efficiency of translesion synthesis is thus related to the physical structure of the lesion containing DNA. However, variations in enzyme structure and kinetics result in translesion synthesis efficiencies that are also dependent upon the DNA polymerase. With a low probability, polymerase misinsertion events create a 3' lesion terminus which is geometrically favored over the correct lesion basepair, resulting in mutagenic translesion synthesis. For example, both polymerase alpha and polymerase beta appear to require the formation of a stable 3' primer-template structure for efficient abasic site translesion synthesis. However, the enzymes differ as to the precise molecular make-up of the stable DNA structure, resulting in different mutational specificities. Similar mechanisms may be applicable to oxidative damage, where mutational specificities dependent upon the DNA polymerase also have been observed. In vitro reaction conditions also influence DNA polymerase processing of lesions. Using an in vitro herpes simplex virus thymidine kinase (HSV-tk) gene forward mutation assay, we demonstrate that high dNTP substrate concentrations affect the mutagenic specificity of translesion synthesis using alkylated templates. The exonuclease-deficient Klenow polymerase error frequency for G-->A transition mutations using templates modified by N-ethyl-N-nitrosourea (ENU) was four-fold higher at 1000 microM [dNTP], relative to 50 microM [dNTP], consistent with an increased efficiency of extension of the etO6G.T mispair. Moreover, the frequency of other ENU-induced polymerase errors was suppressed when polymerase reactions contained 50 microM dNTP, relative to 1000 microM dNTP. The efficiency of proofreading as a polymerase error discrimination mechanism reflects a balance between the competing processes of 3'-->5' exonuclease removal of mispairs and polymerization of the next correct nucleotide. Polymerases that are devoid of a proofreading exonuclease generally display enhanced abasic site translesion synthesis relative to proofreading-proficient enzymes. In addition, the proofreading exonucleases of Escherichia coli Pol I and T4 DNA polymerases have been found to remove mispairs caused by abasic sites and oxidative lesions, respectively, resulting in lowered polymerase error rates. However, the magnitude of the exonuclease effect is small (less than 10-fold), and highly dependent upon the DNA polymerase-exonuclease. We have studied proofreading exonuclease removal of alkylation damage in the HSV-tk forward assay. We observed no significant reduction in the magnitude of the mutant frequency vs. dose-response curves when N-methyl-N-nitrosourea or ENU-treated templates were used in exonuclease-proficient Klenow polymerase reactions, as compared to the exonuclease-deficient polymerase reactions. Thus, available data suggest that proofreading excision of endogenous lesion mispairs does occur, but the efficiency is dependent upon the lesion and the DNA polymerase-exonuclease studied.

Alkylation-induced frameshift mutagenesis during in vitro DNA synthesis by DNA polymerases alpha and beta.

We have analyzed the mutational spectra produced during in vitro DNA synthesis by DNA polymerase alpha-primase and DNA polymerase beta. The polymerase mutation frequency as measured in the in vitro herpes simplex virus thymidine kinase (HSV-tk) forward assay was increased when reactions utilized single-stranded DNA templates randomly modified by 20 mM N-ethyl-N-nitrosourea (ENU), relative to solvent-treated templates. A 20- to 50-fold increase in the frequency of G-->A transition mutations was observed for both polymerases, as expected due to mispairing by O6-ethylguanine lesions. Strikingly, ENU treatment of the template also resulted in a five- to 12-fold increased frequency of frameshift errors at heteropolymeric (non-repetitive) template sequences produced by polymerase beta and polymerase alpha-primase, respectively. The increased proportion of frameshift mutations at heteropolymeric sequences relative to homopolymeric (repetitive) sequences produced by each polymerase in response to ENU damage was statistically significant. For polymerase alpha-primase, one-base deletion errors at template guanine residues was the second most frequent mutational event, observed at a frequency only four-fold lower than the G-->A transition frequency. In the polymerase beta reactions, the frequency of insertion errors at homopolymeric (repetitive) sequences was increased six-fold using alkylated templates, relative to solvent controls. The frequency of such insertion errors was only three-fold lower than the frequency of G-->A transition errors by polymerase beta. Although ENU is generally regarded as a potent base substitution mutagen, these data show that monofunctional alkylating agents are capable of inducing frameshift mutations in vitro. Alkylation-induced frameshift mutations occur in both repetitive and non-repetitive DNA sequences; however, the mutational specificity is dependent upon the DNA polymerase.

The mutator form of polymerase beta with amino acid substitution at tyrosine 265 in the hinge region displays an increase in both base substitution and frame shift errors.

This study describes the first complete in vitro error specificity analysis of a mutator DNA polymerase that is altered in a residue not predicted to contact either the DNA or dNTP substrate. We examined this mutator form of polymerase beta (Y265C) in order to elucidate the critical role tyrosine 265 plays in the accuracy of DNA synthesis. Our results demonstrate that an increase in both frame shift errors in homonucleotide repeat sequences and base substitution errors contribute nearly equally to the Y265C mutator phenotype. The models described for production of these errors, primer/template misalignment and base misincorporation, respectively, are distinctly different, suggesting the Y265C alteration affects discrimination against both types of error production pathways. In addition, Y265C displays a 530-fold increase in multiple errors within the 203-base pair target region examined, relative to that of wild type. Processivity studies revealed that Y265C retains the near distributive nature of DNA synthesis characteristic of the wild type polymerase beta. Therefore, multiple errors exhibited by Y265C most likely result from independent polymerase binding events. Localization of tyrosine 265 in the X-ray crystallographic structure suggests this residue may play a role in mediating a conformational change of the polymerase [Pelletier, H., et al. (1996) Biochemistry 35, 12742-12761]. A conformational change is predicted to enhance the accuracy of DNA synthesis by imposing an induced fit selection against premutational intermediates. The observed loss of discrimination against both misalignment-mediated and misincorporation-mediated errors produced by polymerase Y265C is consistent with such a model.

Development and use of an in vitro HSV-tk forward mutation assay to study eukaryotic DNA polymerase processing of DNA alkyl lesions.

We have developed an in vitro DNA polymerase forward mutation assay using damaged DNA templates that contain the herpes simplex virus type 1 thymidine kinase (HSV-tk) gene. The quantitative method uses complementary strand hybridization to gapped duplex DNA molecules and chloramphenicol selection. This design ensures exclusive analysis of mutations derived from the DNA strand produced during in vitro synthesis. We have examined the accuracy of DNA synthesis catalyzed by calf thymus polymerase alpha-primase, polymerase beta and exonuclease-deficient Klenow polymerase. Using unmodified DNA templates, polymerase beta displays a unique specificity for the loss of two bases in a dinucleotide repeat sequence within the HSV-tk locus. Treatment of the DNA template with N-ethyl-N-nitrosourea resulted in a dose-dependent inhibition of DNA synthesis concomitant with an increased mutation frequency. Similar dose-response curves were measured for the three polymerases examined; thus the identity of the DNA polymerase does not appear to affect the mutagenic potency of ethyl lesions. The HSV-tk system is unique in that damage-induced mutagenesis can be analyzed both quantitatively and qualitatively in human cells, in bacterial cells and in in vitro DNA synthesis reactions at a single target sequence.

Base miscoding and strand misalignment errors by mutator Klenow polymerases with amino acid substitutions at tyrosine 766 in the O helix of the fingers subdomain.

A mutant derivative of Klenow fragment DNA polymerase containing serine substituted for tyrosine at residue 766 has been shown by kinetic analysis to have an increased misinsertion rate relative to wild-type Klenow fragment, but a decreased rate of extension from the resulting mispairs (Carroll, S. S., Cowart, M., and Benkovic, S. J. (1991) Biochemistry 30, 804-813). In the present study we use an M13mp2-based fidelity assay to study the error specificity of this mutator polymerase. Despite its compromised ability to extend mispairs, the Y766S polymerase and a Y766A mutant both have elevated base substitution error rates. The magnitude of the mutator effect is mispair-specific, from no effect for some mispairs to rates elevated by 60-fold for misincorporation of TMP opposite template G. The results with the Y766S mutant are remarkably consistent with the earlier kinetic analysis of misinsertion, demonstrating that either approach can be used to identify and characterize mutator polymerases. Both the Y766S and Y766A mutant polymerases are also frameshift mutators, having elevated rates for two-base deletions and a 276-base deletion between a direct repeat sequence. However, neither mutant polymerase has an increased error rate for single-base frameshifts in repetitive sequences. This error specificity suggests that the deletions generated by the mutator polymerases are initiated by misinsertion rather than by strand slippage. When considered with recent structure-function studies of other polymerases, the data indicate that the nucleotide misinsertion and strand-slippage mechanisms for polymerization infidelity are differentially affected by changes in distinct structural elements of DNA polymerases that share similar subdomain structures.

A genetic system to identify DNA polymerase beta mutator mutants.

DNA polymerase beta (pol beta) is a 39-kDa protein that functions in DNA repair processes in mammalian cells. As a first step toward understanding mechanisms of polymerase fidelity, we developed a genetic method to identify mammalian pol beta mutator mutants. This screen takes advantage of a microbial genetics assay and the ability of rat pol beta to substitute for Escherichia coli DNA polymerase I in DNA replication in vivo. Using this screen, we identified 13 candidate pol beta mutator mutants. Three of the candidate mutator mutants were further characterized in vivo and shown to confer an increased spontaneous mutation frequency over that of wild-type pol beta to our bacterial strain. Purification and subsequent analysis of one of our putative mutator proteins, the pol beta-14 protein, showed that it possesses intrinsic mutator activity in four different assays that measure the fidelity of DNA synthesis. Therefore, residue 265, which is altered in pol beta-14 and another of our mutant proteins, pol beta-166, is probably critical for accurate DNA synthesis by pol beta. Thus, our genetic method of screening for pol beta mutator mutants is useful in identifying active mammalian DNA polymerase mutants that encode enzymes that catalyze DNA synthesis with altered fidelity compared with the wild-type pol beta enzyme.

Factors affecting fidelity of DNA synthesis during PCR amplification of d(C-A)n.d(G-T)n microsatellite repeats.

The susceptibility of microsatellite DNA sequences to insertions and deletions in vivo makes them useful for genetic mapping and for detecting genomic instability in tumors. An in vitro manifestation of this instability is the production of undesirable frameshift products during amplification of (dC-dA)n x (dG-dT)n microsatellites in the polymerase chain reaction (PCR). These products differ from the primary product by multiples of 2 nucleotides. We have tested the hypothesis that factors known to affect the fidelity of DNA synthesis may affect (dC-dA)n x (dG-dT)n frameshifting during the PCR. Neither modifications of pH, dNTP concentration, and Mg++ concentration using Amplitaq, nor the use of thermophilic DNA polymerases including UITma, Pfu, Vent and Deep Vent significantly decreased the production of frameshift products during amplification. However, 3'-->5' exonuclease activity in thermophilic DNA polymerases inhibited the accumulation of PCR products containing non-templated 3' terminal nucleotides. Most interestingly, extension temperatures of 37 degrees C during amplification using the thermolabile DNA polymerases Sequenase 1.0, Sequenase 2.0, and 3'-->5' exonuclease-deficient Klenow fragment greatly decreased the production of frameshift products. This method can improve the resolution of heterozygous or mutant (dC-dA)n x (dG-dT)n alleles differing in size by one or two repeat units.

Fidelity of DNA synthesis catalyzed by human DNA polymerase alpha and HIV-1 reverse transcriptase: effect of reaction pH.

The accuracy of DNA synthesis catalyzed by the Thermus aquaticus DNA polymerase and the 3'-->5' exonuclease-deficient Klenow fragment of Escherichia coli DNA polymerase I varies as a function of reaction pH (Eckert, K.A. and Kunkel, T.A. (1990) Nucleic Acids Res. 18, 3739-3744; Eckert, K.A. and Kunkel, T.A. (1993) J. Biol. Chem. 268, 13462-13471). In the current study, we demonstrate that the fidelity of human DNA polymerase alpha increases 10-fold when the pH of the in vitro synthesis reaction is lowered from pH 8.6 to pH 6.1 (37 degrees C), as determined using a base substitution reversion assay to score polymerase errors within the lacZ alpha gene of bacteriophage M13mp2. Similarly, the base substitution fidelity of DNA-dependent DNA synthesis by the human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) was improved nine-fold at pH 6.5 relative to pH 8.0 (37 degrees C). A detailed comparison of HIV-1 RT error specificity at neutral and low pH in a lacZ alpha forward mutation assay revealed that low pH suppresses both mispairing-mediated and misalignment-mediated mutations; however, the characteristic HIV-1 RT pattern of mutational hotspots at homopolymeric sequences is retained at the lower pH. Consistent with the presumption that these mutations result, in part, from increased termination of DNA synthesis within the hotspot sequences relative to other homopolymeric sequences, the HIV-1 RT termination pattern during processive DNA synthesis is not altered by low pH. The HIV-1 RT results are in agreement with our previous hypothesis that the observed increase in polymerase fidelity at low pH results from a decreased efficiency of continuing DNA synthesis from premutational DNA intermediates.

Effect of reaction pH on the fidelity and processivity of exonuclease-deficient Klenow polymerase.

We have examined as a function of pH the fidelity of DNA synthesis catalyzed by the 3'-->5' exonuclease-deficient form of the Klenow fragment of Escherichia coli DNA polymerase I. Increasing the pH of in vitro gap-filling reactions from pH 6.2 through 9.8 (37 degrees C increased the frequency of base substitution and minus-one-base frameshift mutations 50- and 40-fold, respectively, as measured by reversion of a nonsense or frameshift mutation within the lacZ alpha gene of bacteriophage M13mp2. To understand the mechanisms of high fidelity at low pH, we have examined the biochemical events associated with DNA synthesis at pH 6.2 that might be responsible for the observed accuracy in vitro. We show that while the steady-state frequency of T.dGTP misinsertion at the lacZ alpha opal codon is 20-fold lower at pH 6.2 than at pH 7.6, pH-dependent changes in the frequencies of G-dATP and A-dCTP base misinsertions at the lacZ alpha nonsense codon are insufficient to explain the fidelity changes observed in the gap-filling assay. However, the efficiency of steady-state extension synthesis from template-primers containing 3'-terminal T.G, G.A, and A.C (template-primer) mispairs was reduced up to 160-fold at pH 6.2 relative to pH 7.6. Analyses of the processivity of DNA polymerization versus pH demonstrated that at low pH the termination probability was decreased at specific template positions. Concomitantly, at sites where the termination probability was lower at pH 6.2, a decreased error rate was observed for base substitution mutations at three template positions and for minus-one-base frameshift mutations at two homopolymeric sequences relative to pH 7.6. We suggest that the observed increase in error discrimination by the exonuclease-deficient Klenow polymerase results from altered template binding properties of the enzyme at pH 6.2.

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.

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)

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.

N-ethyl-N-nitrosourea induces A:T to C:G transversion mutations as well as transition mutations in SOS-induced Escherichia coli.

The fixation of DNA lesions induced in Escherichia coli by N-ethyl-N-nitrosourea (ENU) occurs by both SOS-dependent and SOS-independent pathways. To determine whether these pathways result in differential processing of ENU-induced lesions, we have analyzed the DNA sequence changes of mutations induced at a plasmid-encoded herpes simplex virus type 1 thymidine kinase gene by ENU treatment of plasmid-bearing RecA- and RecA+ bacteria, and by transformation of RecA-, RecA+ and SOS-induced RecA+ bacteria with ENU-modified plasmid DNA. Transition mutations were the predominant types of base substitution mutations observed for wild-type and RecA- E. coli, consistent with the SOS-independent mispairing of O6-ethylguanine and O4-ethylthymine adducts during DNA replication. Under conditions of SOS processing of ENU lesions, however, we observed the frequent induction of A:T----C:G transversion mutations. The proportion of A:T----C:G transversion mutations (42%) observed after transformation of SOS-induced bacteria with ENU modified DNA was approximately equal to that of the G:C----A:T transitions (46%). The frequencies of these mutations were increased 20- and 5-fold respectively over that observed for non-induced RecA+ cells. We suggest that ethylated DNA lesions which normally block DNA replication can be processed to yield A:T----C:G transversion mutations in SOS-induced E. coli.

Molecular analysis of mutations induced in human cells by N-ethyl-N-nitrosourea.

Mutational activation of cellular proto-oncogenes is an important event in the pathogenesis of chemically induced tumors. We have used the ori P-tk shuttle vector, pHET, to analyze the types of DNA sequence changes induced after treating mammalian cells with the carcinogen N-ethyl-N-nitrosourea (ENU). This shuttle vector contains the putative replication origin of the Epstein-Barr virus (EBV) and is stably maintained as a plasmid in EBV-transformed human lymphoblastoid cells. Populations of plasmid-bearing cells were treated with ENU, and plasmid DNA was isolated approximately 7-8 population doublings after treatment for analysis of mutations induced at the herpes simplex virus type 1 thymidine kinase (HSV-tk) target gene. After ENU treatment, frequencies of four of the six possible base substitution mutations significantly increased. Transition mutations were the most common sequence change: 48% of the 46 mutants sequenced were GC----AT transitions and 17% were AT----GC transitions. In addition, the number of AT----TA (20%) and AT----CG (9%) transversion mutations significantly increased after ENU treatment. Based on the comparison of mutations induced by ENU in human cells with the types of base pair changes previously reported for other alkylating agents, we propose that the O2-ethylthymine adduct may be a significant premutagenic lesion in mammalian cells, capable of resulting in AT base pair transversion mutations. Studies from other laboratories have demonstrated the importance of AT----TA transversion mutations in the activation of cellular proto-oncogenes by ENU.

recA-dependent and recA-independent N-ethyl-N-nitrosourea mutagenesis at a plasmid-encoded herpes simplex virus thymidine kinase gene in Escherichia coli.

We have compared isogenic recA13/recA+ Escherichia coli K-12 strains for the induction by N-ethyl-N-nitrosourea (ENU) of forward mutations at a plasmid-encoded herpes simplex virus type 1 thymidine kinase (HSV-tk) gene. Treatment of plasmid-bearing bacteria with ENU resulted in a dose-dependent increase in the mutant frequencies of the chromosomal udk locus and of the plasmid HSV-tk locus in both recA13 and recA+ strains. Although the recA13 strain was considerably more sensitive to the cytotoxic effects of ENU treatment than was the recA+ strain, the ENU-induced mutation frequency at both loci was greater for the recA+ strain than for the recA13 strain. When plasmid DNA modified by in vitro reaction with ENU was used to transform recA13, recA+, and UV pre-irradiated recA+ strains, an increase in the HSV-tk mutant frequency was observed in all 3 cases. The induction of mutations in recA13 and recA+ strains followed a similar dose-response, while the ENU-induced HSV-tk mutant frequency was significantly greater for UV pre-irradiated recA+ bacteria. These results indicate that fixation of ENU-induced premutagenic lesions can occur by both recA-dependent and recA-independent pathways.