Proofreading, NTPases and translation: constraints on accurate biochemistry.

Two accurate individual reactions can work together as a more accurate overall mechanism. This is straightforward for a transient reaction, but the same accuracy in the steady state (termed proofreading) requires many preconditions. The preconditions for proofreading can be summarized as four statements, which allow experimental identification and subsequent confirmation of proofreading mechanisms.     

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.     

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.     

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.     

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.     

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.     

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.     

Contribution of 3' leads to 5' exonuclease activity of DNA polymerase III holoenzyme from Escherichia coli to specificity

The effects of deoxynucleoside monophosphates on the 3' leads to 5' exonuclease activity of DNA polymerase III holoenzyme have been correlated with their effects on the fidelity of DNA replication. In particular, dGMP inhibits the proofreading activity of the enzyme and decreases the fidelity in those cases where a "following nucleotide effect" is also noted. This is strong evidence for proofreading. However, the absence of the effects of proofreading inhibitors or following nucleotides need not be evidence against the occurrence of proofreading: a theoretical analysis shows that these effects may not be observed even though there is active proofreading. This is suggested to be the case with the phage T4 enzyme system. The proofreading activity of Pol III appears to be directed primarily towards removing purine x pyrimidine-mediated rather than purine x purine-mediated misincorporations. recA protein inhibits the proofreading activity of Pol III on synthetic templates containing mismatched 3' termini. This is paralleled by a decrease in the fidelity of DNA replication in vitro. The inhibition is increased in the presence of dGMP or dAMP but there is no further increase in the infidelity of replication. The presence of both dNMPs and recA protein does not enable Pol III to copy past pyrimidine photodimers.     

DNA polymerase proofreading: Multiple roles maintain genome stability

DNA polymerase proofreading is a spell-checking activity that enables DNA polymerases to remove newly made nucleotide incorporation errors from the primer terminus before further primer extension and also prevents translesion synthesis. DNA polymerase proofreading improves replication fidelity ∼ 100-fold, which is required by many organisms to prevent unacceptably high, life threatening mutation loads. DNA polymerase proofreading has been studied by geneticists and biochemists for > 35 years. A historical perspective and the basic features of DNA polymerase proofreading are described here, but the goal of this review is to present recent advances in the elucidation of the proofreading pathway and to describe roles of DNA polymerase proofreading beyond mismatch correction that are also important for maintaining genome stability.     

On the fidelity of DNA synthesis. Pyrophosphate-induced misincorporation allows detection of two proofreading mechanisms

The effect of pyrophosphate on the fidelity of in vitro DNA synthesis has been examined. Pyrophosphate enhances misincorporation by Escherichia coli DNA polymerase I in copying phi X174 DNA. The increased misincorporation is directly proportional to the extent of inhibition of the rate of polymerization. In contrast, pyrophosphate is not detectably mutagenic with avian myeloblastosis virus DNA polymerase or DNA polymerases alpha and beta from animal cells, which lack associated proofreading activities. This suggests that increased misincorporation by pyrophosphate is not due to an increase in misinsertions by DNA polymerase, but rather due to inhibition of proofreading by pyrophosphate. However, the pyrophosphate-induced infidelity has a different specificity from, and is not competitive with, two experimental markers of 3'----5' exonuclease proofreading; i.e. the effects of the next nucleotide or the addition of deoxynucleoside monophosphates. These distinctive features suggest a second mode of proofreading susceptible to inhibition by pyrophosphate. This concept is discussed in relation to models for proofreading described in the literature.     

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.     

Single-base mutations at position 2661 of Escherichia coli 23S rRNA increase efficiency of translational proofreading.

Two single-base substitutions were constructed in the 2660 loop of Escherichia coli 23S rRNA (G2661-->C or U) and were introduced into the rrnB operon cloned in plasmid pKK3535. Ribosomes were isolated from bacteria transformed with the mutated plasmids and assayed in vitro in a poly(U)-directed system for their response to the misreading effect of streptomycin, neomycin, and gentamicin, three aminoglycoside antibiotics known to impair the proofreading control of translational accuracy. Both mutations decreased the stimulation of misreading by these drugs, but neither interfered with their binding to the ribosome. The response of the mutant ribosomes to these drugs suggests that the 2660 loop, which belongs to the elongation factor Tu binding site, is involved in the proofreading step of the accuracy control. In vivo, both mutations reduced read-through of nonsense codons and frameshifting, which can also be related to the increased efficiency in proofreading control which they confer to ribosomes.     

The proofreading of hydroxy analogues of leucine and isoleucine by leucyl-tRNA synthetases from E. coli and yeast.

Three analogues each of leucine and isoleucine carrying hydroxy groups in gamma- or delta- or gamma- and delta-position have been synthesized, and tested in the aminoacylation by leucyl-tRNA synthetases from E. coli and yeast. Hydrolytic proofreading, as proposed in the chemical proofreading model, of these analogues and of homocysteine should result in a lactonisation of these compounds and therefore provide information regarding the proofreading mechanism of the two leucyl-tRNA synthetases. Leucyl-tRNA synthetase from E. coli shows a high initial substrate discrimination. Only two analogues, gamma-hydroxyleucine and homocysteine are activated and transferred to tRNALeu where a post-transfer proofreading occurs. Lactonisation of gamma-hydroxyleucine and homocysteine could be detected. Leucyl-tRNA synthetase from yeast has a relatively poor initial discrimination of these substrates, which is compensated by a very effective pre-transfer proofreading on the aminoacyl-adenylate level. No lactonisation nor mischarged tRNALeu is detectable.     

Kinetic proofreading of ligand-FcepsilonRI interactions may persist beyond LAT phosphorylation

Cells may discriminate among ligands with different dwell times for receptor binding through a mechanism called kinetic proofreading in which the formation of an activated receptor complex requires a progression of events that is aborted if the ligand dissociates before completion. This mechanism explains how, at equivalent levels of receptor occupancy, a rapidly dissociating ligand can be less effective than a more slowly dissociating analog at generating distal cellular responses. Simple mathematical models predict that kinetic proofreading is limited to the initial complex; once the signal passes to second messengers, the dwell time no longer regulates the signal. This suggests that an assay for kinetic proofreading might be used to determine which activation events occur within the initial signaling complex. In signaling through the high affinity IgE receptor FcepsilonRI, the transmembrane adaptor called linker for activation of T cells (LAT) is thought to nucleate a distinct secondary complex. Experiments in which the concentrations of two ligands with different dwell times are adjusted to equalize the level of LAT phosphorylation in rat basophilic leukemia 2H3 cells show that Erk2 phosphorylation, intracellular Ca(2+), and degranulation exhibit kinetic proofreading downstream of LAT phosphorylation. These results suggest that ligand-bound FcepsilonRI and LAT form a complex that is required for effective signal transmission.     

Streptomycin preferentially perturbs ribosomal proofreading

Summary We have studied the influence of streptomycin (Sm) on the kinetics and accuracy of translation by wild-type as well as Ram-mutant ribosomes in an in vitro system that mimics the performance characteristics of ribosomes in bacteria. It can be shown in this system that the accuracy of translation is made up of an initial selection step and one or more proofreading steps. The data show that the antibiotic has only a small influence on the initial selectivity step of wild-type or mutant ribosomes. Streptomycin stimulates the missense rate primarily by suppressing the proofreading of the ribosomes. The kinetic effects of Sm and of Ram alteration are not additive, but seem to be overlapping if not identical.     

Developing controllable hypermutable Clostridium cells through manipulating its methyl-directed mismatch repair system

Development of controllable hypermutable cells can greatly benefit understanding and harnessing microbial evolution. However, there have not been any similar systems developed for Clostridium, an important bacterial genus. Here we report a novel two-step strategy for developing controllable hypermutable cells of Clostridium acetobutylicum, an important and representative industrial strain. Firstly, the mutS/L operon essential for methyldirected mismatch repair (MMR) activity was inactivated from the genome of C. acetobutylicum to generate hypermutable cells with over 250-fold increased mutation rates. Secondly, a proofreading control system carrying an inducibly expressed mutS/L operon was constructed. The hypermutable cells and the proofreading control system were integrated to form a controllable hypermutable system SMBMutC, of which the mutation rates can be regulated by the concentration of anhydrotetracycline (aTc) . Duplication of the miniPthl-tetR module of the proofreading control system further significantly expanded the regulatory space of the mutation rates, demonstrating hypermutable Clostridium cells with controllable mutation rates are generated. The developed C. acetobutylicum strain SMBMutC2 showed higher survival capacities than the control strain facing butanol-stress, indicating greatly increased evolvability and adaptability of the controllable hypermutable cells under environmental challenges.     

On the fidelity of DNA replication. Effect of the next nucleotide on proofreading

The contribution of proofreading to the fidelity by which Escherichia coli DNA polymerase I copies natural DNA has been analyzed by two independent criteria. With phi X174 am 3 DNA as a template, there is approximately a 25-fold increase in noncomplementary base substitutions at position 587 when the concentration of the next correct nucleotide, dATP, is increased. Sequence analysis indicates that the mistakes represent misincorporation of C in place of T at position 587. This mutagenic response is presumed to result from a decrease in the probability of excision by the 3' leads to 5' exonuclease of Pol I and is considered within the context of current theories on proofreading. No enhanced mutagenicity is observed with avian myeloblastosis virus DNA polymerase, which lacks a 3' leads to 5' exonuclease. Using a second approach, an enhancement in mutagenesis as large as 30-fold is observed to result from the addition of deoxynucleoside monophosphates to the Pol I reaction. This mutagenicity occurs with any of the four deoxynucleoside monophosphates and is independent of a significant inhibition of DNA synthesis, thus supporting proofreading models in which sites of excision and incorporation are independent. The results of both approaches suggest that the exonucleolytic activity of Pol I can increase fidelity by approximately 30-fold on natural DNA, a value much higher than previous estimates with polynucleotide templates. The effect of the next correct nucleotide in decreasing accuracy provides an in vitro probe for screening eukaryotic cells for putative proofreading functions.     

The ‘A rule’ of mutagen specificity: A consequence of DNA polymerase bypass of non-instructional lesions?

The replicative bypass of lesions in DNA and the induction of mutations by agents which react with DNA to produce damaged bases can be understood on the basis of a simple kinetic model. Bypass can be analyzed by separately considering three processes: a) addition of a base opposite a lesion, b) a proofreading excision process, and c) a rate limiting elongation step. Adenine nucleotides are preferentially added opposite many lesions making it possible to predict mutational specificity. Replicative bypass (translesion synthesis) is dependent on modulation of proofreading exonuclease activity but loss of exonuclease activity alone is not sufficient to ensure bypass. Frameshift mutation is the result of the failure of translesion synthesis accompanied by rearrangement of the template, particularly at repetitive sites.