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.     

On the fidelity of DNA replication: herpes DNA polymerase and its associated exonuclease.

Procaryotic DNA polymerases contain an associated 3'----5' exonuclease activity which provides a proofreading function and contributes substantially to replication fidelity. DNA polymerases of the eucaryotic herpes-type viruses contain similar associated exonuclease activities. We have investigated the fidelity of polymerases purified from wild type herpes simplex virus, as well as from mutator and antimutator strains. On synthetic templates, the herpes enzymes show greater relative exonuclease activities, and greater ability to excise a terminal mismatched base, than procaryotic DNA polymerases which proofread. On a phi X174 natural DNA template, the herpes enzymes are more accurate than purified eucaryotic DNA polymerases; the error rate is similar to E. coli polymerase I. However, conditions which abnegate proofreading by E. coli polymerase I have little effect on the herpes enzymes. We conclude that either these viral polymerases are accurate in the absence of proofreading, or the conditions examined have little effect on proofreading by the herpes DNA polymerases.     

Effects of PCR cycle number and DNA polymerase type on the 16S rRNA gene pyrosequencing analysis of bacterial communities

The effects of PCR cycle number and DNA polymerase type on 16S rRNA gene pyrosequencing analysis were investigated using an artificially prepared bacterial community (mock community). The bacterial richness was overestimated at increased PCR cycle number mostly due to the occurence of chimeric sequences, and this was more serious with a DNA polymerase having proofreading activity than with Taq DNA polymerase. These results suggest that PCR cycle number must be kept as low as possible for accurate estimation of bacterial richness and that particular care must be taken when a DNA polymerase having proofreading activity is used.     

Proofreading, NTPases and translation: successful increase in specificity.

The discussion of proofreading started in the April issue of TIBS is completed by treating the two branched Michaelis-Menten enzymes that can proofread. The conditions required for proofreading can be seen to determine the expression of proofreading in its biological settings. There are surely instances of proofreading as yet unrecognized.     

Accuracy of proofreading with zero energy cost

Accuracy of biological discrimination at the molecular level is known in some systems to involve kinetic proofreading mechanisms. Hopfield and Ninio were the first to propose simple specific kinetic mechanisms for such proofreading and to demonstrate that an energy cost accompanies their improvement in accuracy. Savageau and Freter subsequently derived the explicit cost-accuracy relationship for a broad class of proofreading mechanisms, including the conventional Hopfield-Ninio mechanism just referred to. In other systems, the presence of proofreading mechanisms is in question because the diagnostic features of conventional kinetic proofreading are absent. However, Hopfield has recently proposed an alternative “energy-relay” mechanism, which lacks the characteristic features of conventional proofreading and yet is capable of improving accuracy. In this paper, I use the general cost-accuracy relationship that we have previously derived to examine the energy cost and accuracy of proofreading mechanisms involving an energy relay. The principal findings are the following. First, such mechanisms improve accuracy with a zero cost of proofreading, when “proofreading cost,” defined as the cost due specifically to proofreading, is separated from the costs of putting material through the system. Second, the basic energy-relay mechanism discussed by Hopfield has only a modest improvement in accuracy, but a comparable improvement by a conventional proofreading mechanism would have a cost of about 0·0352 (moles ATP per mole of total product output). Third, accuracy can be increased somewhat if multiple stages of conventional kinetic proofreading precede the energy-relay mechanism. The cost for this improvement is zero while a comparable increase in accuracy achieved by conventional proofreading alone has a cost of about 0·0385. Finally, I propose an alternative arrangement of energy-relay mechanisms that is capable of increasing accuracy still further. The maximum accuracy achieved by this scheme at zero energy cost is comparable to that achieved by an infinite expenditure of energy in a single stage of conventional proofreading.     

The high fidelity and unique error signature of human DNA polymerase epsilon

Bulk replicative DNA synthesis in eukaryotes is highly accurate and efficient, primarily because of two DNA polymerases (Pols): Pols δ and ε. The high fidelity of these enzymes is due to their intrinsic base selectivity and proofreading exonuclease activity which, when coupled with post-replication mismatch repair, helps to maintain human mutation rates at less than one mutation per genome duplication. Conditions that reduce polymerase fidelity result in increased mutagenesis and can lead to cancer in mice. Whereas yeast Pol ε has been well characterized, human Pol ε remains poorly understood. Here, we present the first report on the fidelity of human Pol ε. We find that human Pol ε carries out DNA synthesis with high fidelity, even in the absence of its 3'→5' exonucleolytic proofreading and is significantly more accurate than yeast Pol ε. Though its spectrum of errors is similar to that of yeast Pol ε, there are several notable exceptions. These include a preference of the human enzyme for T→A over A→T transversions. As compared with other replicative DNA polymerases, human Pol ε is particularly accurate when copying homonucleotide runs of 4-5 bases. The base pair substitution specificity and high fidelity for frameshift errors observed for human Pol ε are distinct from the errors made by human Pol δ.     

Trans-oligomerization of duplicated aminoacyl-tRNA synthetases maintains genetic code fidelity under stress

Aminoacyl-tRNA synthetases (aaRSs) play a key role in deciphering the genetic message by producing charged tRNAs and are equipped with proofreading mechanisms to ensure correct pairing of tRNAs with their cognate amino acid. Duplicated aaRSs are very frequent in Nature, with 25,913 cases observed in 26,837 genomes. The oligomeric nature of many aaRSs raises the question of how the functioning and oligomerization of duplicated enzymes is organized. We characterized this issue in a model prokaryotic organism that expresses two different threonyl-tRNA synthetases, responsible for Thr-tRNA^Thr synthesis: one accurate and constitutively expressed (T1) and another (T2) with impaired proofreading activity that also generates mischarged Ser-tRNA^Thr. Low zinc promotes dissociation of dimeric T1 into monomers deprived of aminoacylation activity and simultaneous induction of T2, which is active for aminoacylation under low zinc. T2 either forms homodimers or heterodimerizes with T1 subunits that provide essential proofreading activity in trans. These findings evidence that in organisms with duplicated genes, cells can orchestrate the assemblage of aaRSs oligomers that meet the necessities of the cell in each situation. We propose that controlled oligomerization of duplicated aaRSs is an adaptive mechanism that can potentially be expanded to the plethora of organisms with duplicated oligomeric aaRSs.     

Dissipation-error tradeoff in proofreading.

Chemical proofreading systems, of the kind believed responsible for the extremely high fidelity of DNA replication, achieve minimum error probability (equal to the product of the error probabilities of the writing and proofreading stages) only in the limit of infinite energy dissipation. However, a considerable degree of proofreading can be obtained in less strongly driven systems, dissipation only 0.1-1 kT/step.     

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)     

DNA合成的忠实性机制

DNA的忠实性合成对于基因组稳定和物种延续至关重要,否则可能会产生严重的后果。DNA合成具有极高的忠实性,这主要基于3个步骤:(1)基于氢键、碱基对构象或其他因素的核苷酸选择;(2)基于3′→5′外切酶活性的校对,方式有顺式校对和反式校对,可以去除错误掺入的核苷酸;(3)基于错配修复、切除修复、同源重组修复和跨损伤DNA合成的修复过程,可以纠正逃过校对的错误核苷酸。由于DNA聚合酶不仅可以作为抗病毒或抗癌药物的靶标,而且其忠实性还与抗药性或药物副作用有关,所以深入研究DNA合成的忠实性具有非常重要的意义。文章主要论述了DNA合成的忠实性机制,并对DNA聚合酶的应用前景做了展望。     

Proofreading systems of multiple stages for improved accuracy of biological discrimination

The phenomenal accuracy of biological discrimination is due in many cases to specific proofreading mechanisms. We have previously developed a macroscopic theory of such mechanisms and applied it to the case of single-stage proofreading. In this article we apply the theory to systems with multiple stages of proofreading. A specific relationship between improved accuracy due to proofreading and the associated energy cost is given. This is a macroscopic relationship that must be satisfied regardless of the details of the underlying mechanisms. Five factors in the design of such systems are shown to influence their overall accuracy: (1) initial discrimination, (2) number of proofreading stages, (3) proofreading discrimination of each stage, (4) distribution of proofreading effort among the stages, and (5) total energy expended for proofreading. We show that there is an optimal distribution of proofreading effort that, for a given degree of accuracy, minimizes the energy cost of proofreading. We also provide a simple physical interpretation of this minimum condition. These results are used to examine proofreading in two experimental systems for which there is appropriate data available in the literature: the valyl-tRNA synthetase catalyzed misacylation of tRNA^Val with threonine and the isoleucyl-tRNA synthetase catalyzed misacylation of tRNA^Ile with valine. The correlation between the magnitude of a discrimination factor and the size of the corresponding enzymatic cavity is discussed.     

Single-base discrimination mediated by proofreading 3' phosphorothioate-modified primers

It has been well known for decades that deoxyribonucleic acid (DNA) polymerases with proofreading function have a higher fidelity in primer extension as compared to those without 3' exonuclease activities. However, polymerases with proofreading function have not been used in single nucleotide polymorphism (SNP) assays. Here, we describe a new method for single-base discrimination by proofreading the 3' phosphorothioate-modified primers using a polymerase with proofreading function. Our data show that the combination of a polymerase with 3' exonuclease activity and the 3' phosphorothioate-modified primers work efficiently as a single-base mismatch-operated on/off switch. DNA polymerization only occurred from matched primers, whereas mismatched primers were not extended at the broad range of annealing temperature tested in our study. This novel single-base discrimination method has potential in SNP assays.     

Fidelity of DNA synthesis by the Thermococcus litoralis DNA polymerase--an extremely heat stable enzyme with proofreading activity

We demonstrate that the DNA polymerase isolated from Thermococcus litoralis (VentTM DNA polymerase) is the first thermostable DNA polymerase reported having a 3'----5' proofreading exonuclease activity. This facilitates a highly accurate DNA synthesis in vitro by the polymerase. Mutational frequencies observed in the base substitution fidelity assays were in the range of 30 x 10(-6). These values were 5-10 times lower compared to other thermostable DNA polymerases lacking the proofreading activity. All classes of DNA polymerase errors (transitions, transversions, frameshift mutations) were assayed using the forward mutational assay (1). The mutation frequencies of Thermococcus litoralis DNA polymerase varied between 15-35 x 10(-4) being 2-4 times lower than the respective values obtained using enzymes without proofreading activity. We also noticed that the fidelity of the DNA polymerase from Thermococcus litoralis responds to changes in dNTP concentration, units of enzyme used per one reaction and the concentration of MgSO4 relative to the total concentration of dNTPs present in the reaction. The high fidelity DNA synthesis in vitro by Thermococcus litoralis DNA polymerase provides good possibilities for maintaining the genetic information of original target DNA sequences intact in the DNA amplification applications.     

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.     

Single-base discrimination mediated by proofreading 3′ phosphorothioate-modified primers

It has been well known for decades that deoxyribonucleic acid (DNA) polymerases with proofreading function have a higher fidelity in primer extension as compared to those without 3′ exonuclease activities. However, polymerases with proofreading function have not been used in single nucleotide polymorphism (SNP) assays. Here, we describe a new method for single-base discrimination by proofreading the 3′ phosphorothioate-modified primers using a polymerase with proofreading function. Our data show that the combination of a polymerase with 3′ exonuclease activity and the 3′ phosphorothioate-modified primers work efficiently as a single-base mismatch-operated on/off switch. DNA polymerization only occurred from matched primers, whereas mismatched primers were not extended at the broad range of annealing temperature tested in our study. This novel single-base discrimination method has potential in SNP assays.     

Mechanisms of aminoacyl-tRNA synthetases: a critical consideration of recent results

During the last 10 years intensive and detailed studies on mechanisms and specificities of aminoacyl-tRNA synthetases have been carried out. Physical measurements, chemical modification of substrates, site-directed mutagenesis, and determination of kinetic parameters in misacylation reactions with noncognate amino acids have provided extensive knowledge which is now considered critically for its consistency. A common picture emerges: (1) The enzymes work with different catalytic cycles, kinetic constants, and specificities under different assay conditions. (2) Chemical modifications of substrates can have comparable influence on catalysis as can changes in assay conditions. (3) All enzymes show a specificity for the 2'- or 3'-position of the tRNA. (4) Hydrolytic proofreading is achieved in a pre- and a posttransfer process. In most cases pretransfer proofreading is the main step; posttransfer proofreading is often marginal. (5) Initial discrimination of substrates takes place in a two-step binding process. For some investigated enzymes, initial discrimination factors were found to depend on hydrophobic interaction and hydrogen bonds. (6) The overall recognition of amino acids is achieved in a process of at least four steps. At present, only a rough overall picture of aminoacyl-tRNA synthetase action can be given.     

Genetic factors affecting the impact of DNA polymerase delta proofreading activity on mutation avoidance in yeast

Base selectivity, proofreading, and postreplication mismatch repair are important for replication fidelity. Because proofreading plays an important role in error correction, we have investigated factors that influence its impact in the yeast Saccharomyces cerevisiae. We have utilized a sensitive mutation detection system based on homonucleotide runs of 4 to 14 bases to examine the impact of DNA polymerase delta proofreading on mutation avoidance. The contribution of DNA polymerase delta proofreading on error avoidance was found to be similar to that of DNA polymerase epsilon proofreading in short homonucleotide runs (A4 and A5) but much greater than the contribution of DNA polymerase epsilon proofreading in longer runs. We have identified an intraprotein interaction affecting mutation prevention that results from mutations in the replication and the proofreading regions, resulting in an antimutator phenotype relative to a proofreading defect. Finally, a diploid strain with a defect in DNA polymerase delta proofreading exhibits a higher mutation rate than a haploid strain. We suggest that in the diploid population of proofreading defective cells there exists a transiently hypermutable fraction that would be inviable if cells were haploids.     

A kinetic view of clathrin assembly and endocytic cargo sorting

Specificity and sensitivity in biochemical reactions can be achieved through regulation of equilibrium binding affinity or through proofreading mechanisms that allow for the dissociation of unwanted intermediates. In this essay, we aim to provide our perspectives on how the concept of kinetic proofreading might apply in the context of cargo sorting in clathrin-mediated endocytosis.     

Integrated function of a kinetic proofreading mechanism: double-stage proofreading by isoleucyl-tRNA synthetase

Experimental measurements for isoleucyl-tRNA synthetase proofreading valyl-tRNAIle in Escherichia coli previously have been incorporated into the conventional Michaelis-Menten model for this system. This model was augmented to include two stages of proofreading--the aminoacyl adenylate and aminoacyl-tRNA stages--and used to predict the values of four additional rate constants that have been determined experimentally. The results suggest that two stages of conventional kinetic proofreading with binding sites designed for isoleucine (the "correct" substrate) are inconsistent with the experimental data, that a double-stage mechanism in which one stage (the "double-sieve") involves a binding site designed for valine (the "incorrect" substrate) and the other involves a binding site designed for isoleucine is consistent with all the experimental data, and that the experimental data are not sufficiently accurate to distinguish the stage at which the double-sieve mechanism operates in vivo. Furthermore, analysis of the model suggests that four parameters have the most questionable values and that experimental refinement of their estimates will be needed to determine which of the two stages involves the double-sieve mechanism.