Dissecting and tuning primer editing by proofreading polymerases

Proofreading polymerases have 3′ to 5′ exonuclease activity that allows the excision and correction of mis-incorporated bases during DNA replication. In a previous study, we demonstrated that in addition to correcting substitution errors and lowering the error rate of DNA amplification, proofreading polymerases can also edit PCR primers to match template sequences. Primer editing is a feature that can be advantageous in certain experimental contexts, such as amplicon-based microbiome profiling. Here we develop a set of synthetic DNA standards to report on primer editing activity and use these standards to dissect this phenomenon. The primer editing standards allow next-generation sequencing-based enzymological measurements, reveal the extent of editing, and allow the comparison of different polymerases and cycling conditions. We demonstrate that proofreading polymerases edit PCR primers in a concentration-dependent manner, and we examine whether primer editing exhibits any sequence specificity. In addition, we use these standards to show that primer editing is tunable through the incorporation of phosphorothioate linkages. Finally, we demonstrate the ability of primer editing to robustly rescue the drop-out of taxa with 16S rRNA gene-targeting primer mismatches using mock communities and human skin microbiome samples.     

Antimutator variants of DNA polymerases

Evolution balances DNA replication speed and accuracy to optimize replicative fitness and genetic stability. There is no selective pressure to improve DNA replication fidelity beyond the background mutation rate from other sources, such as DNA damage. However, DNA polymerases remain amenable to amino acid substitutions that lower intrinsic error rates. Here, we review these 'antimutagenic' changes in DNA polymerases and discuss what they reveal about mechanisms of replication fidelity. Pioneering studies with bacteriophage T4 DNA polymerase (T4 Pol) established the paradigm that antimutator amino acid substitutions reduce replication errors by increasing proofreading efficiency at the expense of polymerase processivity. The discoveries of antimutator substitutions in proofreading-deficient 'mutator' derivatives of bacterial Pols I and III and yeast Pol δ suggest there must be additional antimutagenic mechanisms. Remarkably, many of the affected amino acid positions from Pol I, Pol III, and Pol δ are similar to the original T4 Pol substitutions. The locations of antimutator substitutions within DNA polymerase structures suggest that they may increase nucleotide selectivity and/or promote dissociation of primer termini from polymerases poised for misincorporation, leading to expulsion of incorrect nucleotides. If misincorporation occurs, enhanced primer dissociation from polymerase domains may improve proofreading in cis by an intrinsic exonuclease or in trans by alternate cellular proofreading activities. Together, these studies reveal that natural selection can readily restore replication error rates to sustainable levels following an adaptive mutator phenotype.     

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.     

Antimutator variants of DNA polymerases

Evolution balances DNA replication speed and accuracy to optimize replicative fitness and genetic stability. There is no selective pressure to improve DNA replication fidelity beyond the background mutation rate from other sources, such as DNA damage. However, DNA polymerases remain amenable to amino acid substitutions that lower intrinsic error rates. Here, we review these ‘antimutagenic’ changes in DNA polymerases and discuss what they reveal about mechanisms of replication fidelity. Pioneering studies with bacteriophage T4 DNA polymerase (T4 Pol) established the paradigm that antimutator amino acid substitutions reduce replication errors by increasing proofreading efficiency at the expense of polymerase processivity. The discoveries of antimutator substitutions in proofreading-deficient ‘mutator’ derivatives of bacterial Pols I and III and yeast Pol δ suggest there must be additional antimutagenic mechanisms. Remarkably, many of the affected amino acid positions from Pol I, Pol III, and Pol δ are similar to the original T4 Pol substitutions. The locations of antimutator substitutions within DNA polymerase structures suggest that they may increase nucleotide selectivity and/or promote dissociation of primer termini from polymerases poised for misincorporation, leading to expulsion of incorrect nucleotides. If misincorporation occurs, enhanced primer dissociation from polymerase domains may improve proofreading in cis by an intrinsic exonuclease or in trans by alternate cellular proofreading activities. Together, these studies reveal that natural selection can readily restore replication error rates to sustainable levels following an adaptive mutator phenotype.     

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)     

Autonomous 3'-->5' exonucleases can proofread for DNA polymerase beta from rat liver

Autonomous 3'-->5'exonucleases are not bound covalently to DNA polymerases but are often involved in replicative complexes. Such exonucleases from rat liver, calf thymus and Escherichia coli (molecular masses of 28+/-2 kDa) are shown to increase more than 10-fold the accuracy of DNA polymerase beta (the most inaccurate mammalian polymerase) from rat liver in the course of reduplication of the primed DNA of bacteriophage phiX174 amber 3 in vitro. The extent of correction increases together with the rise in 3'-->5' exonuclease concentration. Extrapolation of the in vitro DNA replication fidelity to the cellular levels of rat exonuclease and beta-polymerase suggests that exonucleolytic proofreading could augment the accuracy of DNA synthesis by two orders of magnitude. These results are not explained by exonucleolytic degradation of the primers ("no synthesis-no errors"), since similar data are obtained with the use of the primers 15 or 150 nucleotides long in the course of a fidelity assay of DNA polymerases, both alpha and beta, in the presence of various concentrations of 3'-->5' exonuclease.     

The fidelity of DNA synthesis by the catalytic subunit of yeast DNA polymerase alpha alone and with accessory proteins

The fidelity of DNA synthesis catalyzed by the 180-kDa catalytic subunit (p180) of DNA polymerase alpha from Saccharomyces cerevisiae has been determined. Despite the presence of a 3'----5' exonuclease activity (Brooke et al., 1991, J. Biol. Chem., 266, 3005-3015), its accuracy is similar to several exonuclease-deficient DNA polymerases and much lower than other DNA polymerases that have associated exonucleolytic proofreading activity. Average error rates are 1/9900 and 1/12,000, respectively, for single base-substitution and minus-one nucleotide frameshift errors; the polymerase generates deletions as well. Similar error rates are observed with reactions containing the 180-kDa subunit plus an 86-kDa subunit (p86), or with these two polypeptides plus two additional subunits (p58 and p49) comprising the DNA primase activity required for DNA replication. Finally, addition of yeast replication factor-A (RF-A), a protein preparation that stimulates DNA synthesis and has single-stranded DNA-binding activity, yields a polymerization reaction with 7 polypeptides required for replication, yet fidelity remains low relative to error rates for semiconservative replication. The data suggest that neither exonucleolytic proofreading activity, the beta subunit, the DNA primase subunits nor RF-A contributes substantially to base substitution or frameshift error discrimination by the DNA polymerase alpha catalytic subunit.     

DNA replication fidelity

DNA replication fidelity plays fundamental role in faithful transmission of genetic material during cell division and during transfer of genetic material from parents to progeny. Replicative polymerases are the main guardian responsible for high replication fidelity of genomic DNA. DNA main replicative polymerases are also involved in many DNA repair processes. High fidelity of DNA replication is determined by correct nucleotide selectivity in polymerase active center, and exonucleolytic proofreading that removes mismatches from primer terminus. In this article we will focus on the mechanisms that are responsible for high fidelity of replications with the special emphasis on structural studies showing important conformational changes after substrate binding. We will also stress the importance of hydrogen bonding, base pair geometry, polymerase DNA interactions and the role of accessory proteins in replication fidelity.     

Replicative DNA polymerase defects in human cancers: Consequences,mechanisms, and implications for therapy

The fidelity of DNA replication relies on three error avoidance mechanisms acting in series: nucleotide selectivity of replicative DNA polymerases, exonucleolytic proofreading, and post-replicative DNA mismatch repair (MMR). MMR defects are well known to be associated with increased cancer incidence. Due to advances in DNA sequencing technologies, the past several years have witnessed a long-predicted discovery of replicative DNA polymerase defects in sporadic and hereditary human cancers. The polymerase mutations preferentially affect conserved amino acid residues in the exonuclease domain and occur in tumors with an extremely high mutation load. Thus, a concept has formed that defective proofreading of replication errors triggers the development of these tumors. Recent studies of the most common DNA polymerase variants, however, suggested that their pathogenicity may be determined by functional alterations other than loss of proofreading. In this review, we summarize our current understanding of the consequences of DNA polymerase mutations in cancers and the mechanisms of their mutator effects. We also discuss likely explanations for a high recurrence of some but not other polymerase variants and new ideas for therapeutic interventions emerging from the mechanistic studies.     

DNA polymerase alpha and models for proofreading.

Using a modified system to measure fidelity at an amber site in phi X174, we have employed DNA polymerase alpha to test different mechanisms for proofreading. DNA polymerase alpha does not exhibit the characteristics of "kinetic proofreading" seen with procaryotic polymerases. Polymerase alpha shows no evidence for a "next nucleotide" effect, and added deoxynucleoside monophosphates do not alter fidelity. Pyrophosphate, which increases error rates with a procaryotic polymerase, appears to weakly improve polymerase alpha fidelity. DNA polymerase alpha does exhibit a dramatic increase in error rate in the presence of a deoxycytidine thiotriphosphate (dCTP alpha S), but this enhanced mutagenesis also occurs under conditions where kinetic proofreading should be otherwise defeated. This particular effect with dCTP alpha S appears specific for DNA polymerase alpha and is not seen with the other polymerases tested.     

Molecular events during translocation and proofreading extracted from 200 static structures of DNA polymerase

DNA polymerases in family B are workhorses of DNA replication that carry out the bulk of the job at a high speed with high accuracy. A polymerase in this family relies on a built-in exonuclease for proofreading. It has not been observed at the atomic resolution how the polymerase advances one nucleotide space on the DNA template strand after a correct nucleotide is incorporated, that is, a process known as translocation. It is even more puzzling how translocation is avoided after the primer strand is excised by the exonuclease and returned back to the polymerase active site once an error occurs. The structural events along the bifurcate pathways of translocation and proofreading have been unwittingly captured by hundreds of structures in Protein Data Bank. This study analyzes all available structures of a representative member in family B and reveals the orchestrated event sequence during translocation and proofreading.     

On the fidelity of DNA replication. Lack of primer position effect on the fidelity of mammalian DNA polymerases

Mechanisms for the fidelity of DNA replication in eucaryotes are not adequately understood. Certain hypotheses can be tested by examining whether the first nucleotide inserted is incorporated with a significantly higher error rate than subsequent nucleotides. Using synthetic oligodeoxynucleotides, we have measured the effect of primer position on single-base misinsertion frequencies at an amber site in phi X174 DNA. Our results show a lack of position effect, indicating that processivity and the most direct "energy relay" proofreading mechanisms are not important determinants in eucaryotic replication fidelity.     

DNA replication fidelity: proofreading in trans

Proofreading is the primary guardian of DNA polymerase fidelity. New work has revealed that polymerases with intrinsic proofreading activity may cooperate with non-proofreading polymerases to ensure faithful DNA replication.     

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.     

DNA polymerase Family X: Function,structure, and cellular roles

The X family of DNA polymerases in eukaryotic cells consists of terminal transferase and DNA polymerases β, λ, and μ. These enzymes have similar structural portraits, yet different biochemical properties, especially in their interactions with DNA. None of these enzymes possesses a proofreading subdomain, and their intrinsic fidelity of DNA synthesis is much lower than that of a polymerase that functions in cellular DNA replication. In this review, we discuss the similarities and differences of three members of Family X: polymerases β, λ, and μ. We focus on biochemical mechanisms, structural variation, fidelity and lesion bypass mechanisms, and cellular roles. Remarkably, although these enzymes have similar three-dimensional structures, their biochemical properties and cellular functions differ in important ways that impact cellular function.     

DNA polymerase activity at the single-molecule level

DNA polymerases are essential enzymes responsible for replication and repair of DNA in all organisms. To replicate DNA with high fidelity, DNA polymerases must select the correct incoming nucleotide substrate during each cycle of nucleotide incorporation, in accordance with the templating base. When an incorrect nucleotide is sometimes inserted, the polymerase uses a separate 3'→5' exonuclease to remove the misincorporated base (proofreading). Large conformational rearrangements of the polymerase-DNA complex occur during both the nucleotide incorporation and proofreading steps. Single-molecule fluorescence spectroscopy provides a unique tool for observation of these dynamic conformational changes in real-time, without the need to synchronize a population of DNA-protein complexes.     

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.     

Evidence from mutational specificity studies that yeast DNA polymerases delta and epsilon replicate different DNA strands at an intracellular replication fork

Although polymerases delta and epsilon are required for DNA replication in eukaryotic cells, whether each polymerase functions on a separate template strand remains an open question. To begin examining the relative intracellular roles of the two polymerases, we used a plasmid-borne yeast tRNA gene and yeast strains that are mutators due to the elimination of proofreading by DNA polymerases delta or epsilon. Inversion of the tRNA gene to change the sequence of the leading and lagging strand templates altered the specificities of both mutator polymerases, but in opposite directions. That is, the specificity of the polymerase delta mutator with the tRNA gene in one orientation bore similarities to the specificity of the polymerase epsilon mutator with the tRNA gene in the other orientation, and vice versa. We also obtained results consistent with gene orientation having a minor influence on mismatch correction of replication errors occurring in a wild-type strain. However, the data suggest that neither this effect nor differential replication fidelity was responsible for the mutational specificity changes observed in the proofreading-deficient mutants upon gene inversion. Collectively, the data argue that polymerases delta and epsilon each encounter a different template sequence upon inversion of the tRNA gene, and so replicate opposite strands at the plasmid DNA replication fork.     

Proofreading of DNA polymerase eta-dependent replication errors

Human DNA polymerase eta, the product of the skin cancer susceptibility gene XPV, bypasses UV photoproducts in template DNA that block synthesis by other DNA polymerases. Pol eta lacks an intrinsic proofreading exonuclease and copies DNA with low fidelity, such that pol eta errors could contribute to mutagenesis unless they are corrected. Here we provide evidence that pol eta can compete with other human polymerases during replication of duplex DNA, and in so doing it lowers replication fidelity. However, we show that pol eta has low processivity and extends mismatched primer termini less efficiently than matched termini. These properties could provide an opportunity for extrinsic exonuclease(s) to proofread pol eta-induced replication errors. When we tested this hypothesis during replication in human cell extracts, pol eta-induced replication infidelity was found to be modulated by changing the dNTP concentration and to be enhanced by adding dGMP to a replication reaction. Both effects are classical hallmarks of exonucleolytic proofreading. Thus, pol eta is ideally suited for its role in reducing UV-induced mutagenesis and skin cancer risk, in that its relaxed base selectivity may facilitate efficient bypass of UV photoproducts, while subsequent proofreading by extrinsic exonuclease(s) may reduce its mutagenic potential.