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.     

New performance from an old member: SNP assay and de novo sequencing mediated by exo+ DNA polymerases

DNA polymerases without the 3' exonuclease function (exo(-) pol) have been widely used in sequencing and SNP genotyping. As a major player that expedited the coming of the postgenomic era, exo(-) polymerases worked remarkably well in the Human Genome Sequencing Project. However, it has become a challenge for this class of polymerases to efficiently screen the large number of SNPs that are found in the human genome. For more than three decades it has been recognized that polymerase fidelity varied according to the presence of proofreading activity that is mediated by its internal 3' exonuclease. Polymerases with proofreading function are famous for their high fidelity in DNA replication both in vivo and in vitro, but this well-known class of polymerases has been almost completely neglected in genetic analysis in the postgenomic era. We speculate that exo(+) polymerases may exhibit higher nucleotide identification ability when compared to exo- polymerases for an in vitro genetic analysis. With the application of exo(+) polymerases in SNP assays, a novel mechanism for the maintenance of DNA replication, the on/off switch, was discovered. Two new SNP assays have been developed to carry out genome-wide genotyping, taking advantage of the enzymatic properties of exo(+) polymerases. Furthermore, the on/off switch mechanism embodies a powerful nucleotide identification ability, which can be used to discriminate the bases that are upstream of the 3' terminus, and thus defines a new concept in de novo sequencing technology. Application of exo(+) polymerases to genetic analysis, and especially SNP assays, will greatly accelerate the pace to personalized medicine.     

Single-base discrimination mediated by proofreading inert allele specific Primers

The role of 3' exonuclease excision in DNA polymerization was evaluated for primer extension using inert allele specific primers with exonuclease-digestible ddNMP at their 3' termini. Efficient primer extension was observed in amplicons where the inert allele specific primers and their corresponding templates were mismatched. However, no primer-extended products were yielded by matched amplicons with inert primers. As a control, polymerase without proofreading activity failed to yield primer-extended products from inert primers regardless of whether the primers and templates were matched or mismatched. These data indicated that activation was undertaken for the inert allele specific primers through mismatch proofreading. Complementary to our previously developed SNP-operated on/off switch, in which DNA polymerization only occurs in matched amplicon, this new mutation detection assay mediated by exo(+) DNA polymerases has immediate applications in SNP analysis independently or in combination of the two assays.     

Proofreading genotyping assays mediated by high fidelity exo+ DNA polymerases

DNA polymerases with 3'-5' proofreading function mediate high fidelity DNA replication but their application for mutation detection was almost completely neglected before 1998. The obstacle facing the use of exo(+) polymerases for mutation detection could be overcome by primer-3'-termini modification, which has been tested using allele-specific primers with 3' labeling, 3' exonuclease-resistance and 3' dehydroxylation modifications. Accordingly, three new types of single nucleotide polymorphism (SNP) assays have been developed to carry out genome-wide genotyping making use of the fidelity advantage of exo(+) polymerases. Such SNP assays might also provide a novel approach for re-sequencing and de novo sequencing. These new mutation detection assays are widely adaptable to a variety of platforms, including real-time PCR, multi-well plate and microarray technologies. Application of exo(+) polymerases to genetic analysis could accelerate the pace of personalized medicine.     

Strong positional preference in the interaction of LNA oligonucleotides with DNA polymerase and proofreading exonuclease activities: implications for genotyping assays

The effect of locked nucleic acid (LNA) modification position upon representative DNA polymerase and exonuclease activities has been examined for potential use in primer extension genotyping applications. For the 3′→5′ exonuclease activities of four proofreading DNA polymerases (Vent, Pfu, Klenow fragment and T7 DNA polymerase) as well as exonuclease III, an LNA at the terminal (L-1) position of a primer is found to provide partial protection against the exonucleases of the two family B polymerases only. In contrast, an LNA residue at the penultimate (L-2) position generates essentially complete nuclease resistance. The polymerase active sites of these enzymes also display a distinct preference. An L-1 LNA modification has modest effects upon poly merization, but an L-2 LNA group slows dTTP incorporation somewhat while virtually abolishing extension with ddTTP or acyTTP terminators, even with A488L Vent DNA polymerase engineered for terminator incorporation. These observations on active site preference have been utilized to demonstrate two novel assays: exonuclease-mediated single base extension (E-SBE) and proofreading allele-specific extension (PRASE). We show that a model PRASE genotyping reaction with L-2 LNA primers offers greater specificity than existing non-proofreading assays, whether or not the non-proofreading reaction employs LNA-modified primers.     

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.     

Purification and identification of subunit structure of the human mitochondrial DNA polymerase.

The mitochondrial DNA polymerase of HeLa cells was purified 18,000-fold to near homogeneity. The purified polymerase cofractionated with two polypeptides that had molecular mass of 140 and 54 kDa. The 140-kDa subunit was specifically radiolabeled in a photoaffinity cross-linking assay and is most likely the catalytic subunit of the mitochondrial DNA polymerase. The purified enzyme exhibited properties that have been attributed to DNA polymerase gamma and shows a preference for replicating primed poly(pyrimidine) DNA templates in the presence of 0.5 mM MgCl2. As in the case of mitochondrial DNA polymerases from other animal cells, human DNA polymerase gamma cofractionated with a 3'----5' exonuclease activity. However, it has not been possible to determine if the two enzymatic activities reside in the same polypeptide. The exonuclease activity preferentially removes mismatched nucleotides from the 3' end of a duplex DNA and is not active toward DNA with matched 3' ends. These properties are consistent with the notion that the exonuclease activity plays a proofreading function in the replication of the organelle genome.     

On/off switch mediated by Exo+ polymerases: experimental analysis for its physiological and technological implications

The potential physiological role and technological application of the premature termination of DNA polymerization through the off-switch of exo+ polymerases were studied using 3' phosphorothioate-modified or unmodified primers with single base mismatch distal to the 3' terminus. With exonuclease-digestible unmodified primers, a gradient premature termination of DNA polymerization was observed when amplified with exo+ polymerases. With 3' allele specific phosphorothioate-modified primers, an efficient off-switch effect occurred in the discrimination of a single nucleotide polymorphism when directly using genomic DNA. Clearly, the off-switch of exo+ polymerases is useful in biomedical research.     

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.     

SNP discrimination through proofreading and OFF-switch of Exo+ polymerase

Single nucleotide polymorphisms (SNPs) are useful physical markers for genetic studies as well as the cause of some genetic diseases. To develop more reliable SNP assays, we examined the underlying molecular mechanisms by which deoxyribonucleic acid (DNA) polymerases with 3' exonuclease activity maintain the high fidelity of DNA replication. In addition to mismatch removal by proofreading, we have discovered a premature termination of polymerization mediated by a novel OFF-switch mechanism. Two SNP assays were developed, one based on proofreading using 3' end-labeled primer extension and the other based on the newly identified OFF-switch, respectively. These two new assays are well suited for conventional techniques, such as electrophoresis and microplates detection systems as well as the sophisticated microchips. Application of these reliable SNP assays will greatly facilitate genetic and biomedical studies in the postgenome era.     

Hydrolysis of 3'-terminal mispairs in vitro by the 3'----5' exonuclease of DNA polymerase delta permits subsequent extension by DNA polymerase alpha

Purified DNA polymerase alpha, the major replicating enzyme found in mammalian cells, lacks an associated 3'----5' proofreading exonuclease that, in bacteria, contributes significantly to the accuracy of DNA replication. Calf thymus DNA polymerase alpha cannot remove mispaired 3'-termini, nor can it extend them efficiently. We designed a biochemical assay to search in cell extracts for a putative proofreading exonuclease that might function in concert with DNA polymerase alpha in vivo but dissociates from it during purification. Using this assay, we purified a 3'----5' exonuclease from calf thymus that preferentially hydrolyzes mispaired 3'-termini, permitting subsequent extension of the correctly paired 3'-terminus by DNA polymerase alpha. This exonuclease copurifies with a DNA polymerase activity that is biochemically distinct from DNA polymerase alpha and exhibits characteristics described for a second replicative DNA polymerase, DNA polymerase delta. In related studies, we showed that the 3'----5' exonuclease of authentic DNA polymerase delta, like the purified exonuclease, removes terminal mispairs, allowing extension by DNA polymerase alpha. These data suggest that a single proofreading exonuclease could be shared by DNA polymerases alpha and delta, functioning at the site of DNA replication in mammalian cells.     

Primer-terminus stabilization at the 3'-5' exonuclease active site of phi29 DNA polymerase. Involvement of two amino acid residues highly conserved in proofreading DNA polymerases.

By site-directed mutagenesis in phi29 DNA polymerase, we have analyzed the functional importance of two evolutionarily conserved residues belonging to the 3'-5' exonuclease domain of DNA-dependent DNA polymerases. In Escherichia coli DNA polymerase I, these residues are Thr358 and Asn420, shown by crystallographic analysis to be directly acting as single-stranded DNA (ssDNA) ligands at the 3'-5' exonuclease active site. On the basis of these structural data, single substitution of the corresponding residues of phi29 DNA polymerase, Thr15 and Asn62, produced enzymes with a very reduced or altered capacity to bind ssDNA. Analysis of the residual 3'-5' exonuclease activity of these mutant derivatives on ssDNA substrates allowed us to conclude that these two residues do not play a direct role in the catalysis of the reaction. On the other hand, analysis of the 3'-5' exonuclease activity on either matched or mismatched primer/template structures showed a critical role of these two highly conserved residues in exonucleolysis under polymerization conditions, i.e. in the proofreading of DNA polymerization errors, an evolutionary advantage of most DNA-dependent DNA polymerases. Moreover, in contrast to the dual role in 3'-5' exonucleolysis and strand displacement previously observed for phi29 DNA polymerase residues acting as metal ligands, the contribution of residues Thr15 and Asn62 appears to be restricted to the proofreading function, by stabilization of the frayed primer-terminus at the 3'-5' exonuclease active site.     

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.     

Accessory proteins assist exonuclease-deficient bacteriophage T4 DNA polymerase in replicating past an abasic site

Replicative DNA polymerases, such as T4 polymerase, possess both elongation and 3'-5' exonuclease proofreading catalytic activities. They arrest at the base preceding DNA damage on the coding DNA strand and specialized DNA polymerases have evolved to replicate across the lesion by a process known as TLS (translesion DNA synthesis). TLS is considered to take place in two steps that often require different enzymes, insertion of a nucleotide opposite the damaged template base followed by extension from the inserted nucleotide. We and others have observed that inactivation of the 3'-5' exonuclease function of T4 polymerase enables TLS across a single site-specific abasic [AP (apurinic/apyrimidinic)] lesion. In the present study we report a role for auxiliary replicative factors in this reaction. When replication is performed with a large excess of DNA template over DNA polymerase in the absence of auxiliary factors, the exo- polymerase (T4 DNA polymerase deficient in the 3'-5' exonuclease activity) inserts one nucleotide opposite the AP site but does not extend past the lesion. Addition of the clamp processivity factor and the clamp loader complex restores primer extension across an AP lesion on a circular AP-containing DNA substrate by the exo- polymerase, but has no effect on the wild-type enzyme. Hence T4 DNA polymerase exhibits a variety of responses to DNA damage. It can behave as a replicative polymerase or (in the absence of proofreading activity) as a specialized DNA polymerase and carry out TLS. As a specialized polymerase it can function either as an inserter or (with the help of accessory proteins) as an extender. The capacity to separate these distinct functions in a single DNA polymerase provides insight into the biochemical requirements for translesion DNA synthesis.     

DNA聚合酶高保真机理的新发现及其在SNP分析中的应用

高保真DNA聚合酶在遗传与进化等生命活动中具有十分重要的生理与病理意义。高保真聚合酶除具有广为人知的校正功能外,最近的实验进一步表明, 由不能及时校正或难于纠正的错配碱基引发的“关”闭DNA聚合反应的效应, 同样保证了DNA聚合反应终产物的纯度。高保真聚合酶这一“关”闭DNA聚合反应的能力, 促成了其与耐外切酶消化的3´末端碱基特异性引物共同构成一个SNP敏感性纳米级复合分子“开/关”,高保真聚合酶分子中相距三纳米的聚合中心和3´→5´外切酶酶解中心则既合作又独立地起到了复合分子开关中“开”和“关”的效能:对于配对的引物,则直接在该酶的聚合中心进行聚合反应,即“开”的效应;而对于3´末端错配的引物,则从该酶的聚合中心转移至3´→5´外切酶的酶解中心,由于引物修饰了的3´末端耐外切酶的特点,继而出现了一种长时间无酶解产物的酶解过程,最后因酶的聚合中心空转而“关”闭DNA聚合反应,即“关”的效应。这一新的复合分子“开/关”在很大程度上满足了后基因时代对SNP分析的要求。该SNP分子开关的应用, 使基因诊断提高到单碱基水平。同时, 利用该方法通过SNP对基因组扫描, 在单基因遗传病病因研究及法医学鉴定上具有很强的理论和实用价值。     

Polymerization activity of an alpha-like DNA polymerase requires a conserved 3''-5'' exonuclease active site.

Most DNA polymerases are multifunctional proteins that possess both polymerizing and exonucleolytic activities. For Escherichia coli DNA polymerase I and its relatives, polymerase and exonuclease activities reside on distinct, separable domains of the same polypeptide. The catalytic subunits of the alpha-like DNA polymerase family share regions of sequence homology with the 3'-5' exonuclease active site of DNA polymerase I; in certain alpha-like DNA polymerases, these regions of homology have been shown to be important for exonuclease activity. This finding has led to the hypothesis that alpha-like DNA polymerases also contain a distinct 3'-5' exonuclease domain. We have introduced conservative substitutions into a 3'-5' exonuclease active site homology in the gene encoding herpes simplex virus DNA polymerase, an alpha-like polymerase. Two mutants were severely impaired for viral DNA replication and polymerase activity. The mutants were not detectably affected in the ability of the polymerase to interact with its accessory protein, UL42, or to colocalize in infected cell nuclei with the major viral DNA-binding protein, ICP8, suggesting that the mutation did not exert global effects on protein folding. The results raise the possibility that there is a fundamental difference between alpha-like DNA polymerases and E. coli DNA polymerase I, with less distinction between 3'-5' exonuclease and polymerase functions in alpha-like DNA polymerases.     

Phosphorothioate primers improve the amplification of DNA sequences by DNA polymerases with proofreading activity.

Two thermostable DNA polymerases with proofreading activity--Vent DNA polymerase and Pfu DNA polymerase--have attracted recent attention, mainly because of their enhanced fidelities during amplification of DNA sequences by the polymerase chain reaction. A severe disadvantage for their practical application, however, results from the observation that due to their 3' to 5' exonuclease activities these enzymes degrade the oligodeoxynucleotides serving as primers for the DNA synthesis. It is demonstrated that this exonucleolytic attack on the primer molecules can be efficiently prevented by the introduction of single phosphorothioate bonds at their 3' termini. This strategy, which can be easily accomplished using routine DNA synthesis methodology, may open the way to a widespread use of these novel enzymes in the polymerase chain reaction.     

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.     

DNA polymerase gamma from Xenopus laevis. II. A 3'----5' exonuclease is tightly associated with the DNA polymerase activity

Xenopus laevis DNA polymerase gamma co-purifies with a tightly associated 3'----5' exonuclease. The purified enzyme lacks 5'----3' exonuclease and endonuclease activity. The ratio of the 3'----5' exonuclease activity to DNA polymerase gamma activity remains constant over the final three chromatographic procedures. In addition, these activities co-sediment under partially denaturing conditions in the presence of ethylene glycol. The associated 3'----5' exonuclease activity removes a terminally mismatched nucleotide more rapidly than a correctly base-paired 3'-terminal residue, as expected if this exonuclease has a proofreading function. The 3'----5' exonuclease has the ability to release a terminal phosphorothioated nucleotide, a property shared with T4 DNA polymerase, but not with Escherichia coli DNA polymerase I.