Structural mechanism for coordination of proofreading and polymerase activities in archaeal DNA polymerases

A novel mechanism for controlling the proofreading and polymerase activities of archaeal DNA polymerases was studied. The 3'-5'exonuclease (proofreading) activity and PCR performance of the family B DNA polymerase from Thermococcus kodakaraensis KOD1 (previously Pyrococcus kodakaraensis KOD1) were altered efficiently by mutation of a "unique loop" in the exonuclease domain. Interestingly, eight different H147 mutants showed considerable variations in respect to their 3'-5'exonuclease activity, from 9% to 276%, as against that of the wild-type (WT) enzyme. We determined the 2.75A crystal structure of the H147E mutant of KOD DNA polymerase that shows 30% of the 3'-5'exonuclease activity, excellent PCR performance and WT-like fidelity. The structural data indicate that the properties of the H147E mutant were altered by a conformational change of the Editing-cleft caused by an interaction between the unique loop and the Thumb domain. Our data suggest that electrostatic and hydrophobic interactions between the unique loop of the exonuclease domain and the tip of the Thumb domain are essential for determining the properties of these DNA polymerases.     

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.     

Current understanding of UV-induced base pair substitution mutation in E. coli with particular reference to the DNA polymerase III complex

UV mutagenesis in E. coli is believed to occur in two discrete steps. The second step involves continued DNA synthesis beyond a blocking lesion in the template strand. This bypass step requires induced levels of umuD and umuC gene products and activated recA protein. DNA polymerase III may be involved since a dnaE mutator strain (believed to have defective base selection) is associated with enhanced UV mutagenesis in conjunction with a genetic background permitting the bypass step. In non-UV-mutable umu and lexA strains, UV mutagenesis can be demonstrated if delayed photorevesal is given. This is interpreted as indicating that an earlier misincorporation step can occur in such strains but the resulting mutations do not survive because the bypass step is blocked. The misincorporation step does not require any induced SOS gene products and can occur either at the replication fork or during repair replication following excision of a DNA lesion. Neither a dnaE mutator gene (leading to a defective subunit of DNA polymerase III holoenzyme) nor a mutD5 mutator gene (leading to a defective ε proofreading subunit) had any effect on he misincorporation step. Although this is consistent with DNA polymerase III holoenzyme not being involved in the misincorporation step, other interpretations involving the inhibition of ε proofreading activity by recA protein are possible.

In vitro studies are reported in which sites of termination of synthesis by DNA polymerase III holoenzyme on UV-irradiated M13 mp8 DNA were examined in the presence of inhibitors of the 3′–5′ proofreading exonuclease (including recA protein). No evidence was found for incorporation of bases opposite photoproducts suggesting that either inhibition is more complete in the cell and/or that other factors are involved in the misincorporation step.     

Sensing domain and extension rate of a family B-type DNA polymerase determine the stalling at a deaminated base

The uracil-sensing domain in archaeal family B-type DNA polymerases recognizes pro-mutagenic uracils in the DNA template, leading to stalling of DNA polymerases. Here, we describe our new findings regarding the molecular mechanism underpinning the stalling of polymerases. We observed that two successive deaminated bases were required to stall TNA1 and KOD1 DNA polymerases, whereas a single deaminated base was enough for stalling Pfu DNA polymerase, in spite of the virtually identical uracil-sensing domains. TNA1 and KOD1 DNA polymerases have a much higher extension rate than Pfu DNA polymerase; decreasing the extension rate resulted in stalling by TNA1 and KOD1 DNA polymerases at a single deaminated base. These results strongly suggest that these polymerases require two factors to stop DNA polymerization at a single deaminated base: the presence of the uracil-sensing domain and a relatively slow extension rate.     

Cloning,sequence analysis and expression in E. coli of the DNA polymerase I gene from Chloroflexus aurantiacus,a green: nonsulfur eubacterium

We have cloned and sequenced the polA gene from Chloroflexus aurantiacus, a green nonsulfur eubacterium, and expressed the recombinant protein in Escherichia coli. One open reading frame encodes a protein with 942 amino acids showing 38% identity with DNA polymerase I from E. coli. Sequence alignments with other members of DNA polymerase family A and analysis of the separate domains show that the central 3′-5′ exonuclease domain is 30% identical to the corresponding E. coli domain and that three sequence motifs associated with 3′-5′ exonuclease activity are conserved. Also, a protein fraction from E. coli expressing the Chloroflexus polymerase contains a thermostable 3′-5′ exonucleolytic activity, indicating that this activity is present in the enzyme, in agreement with the sequence analysis. The N-terminal 5′-3′ exonuclease domain and the C-terminal polymerase domain show 31 and 46% identity, respectively, with the corresponding E. coli domains and all sequence motifs associated with these two enzymatic activities also are conserved. Since several DNA polymerase I enzymes lack the proofreading activity associated with the central domain it has been suggested that the ancestral polA gene contained only the two more conserved N- and C-terminal domains and that the proofreading 3′-5′ exonuclease domain was introduced later in those eubacterial branches that have this activity. Our data indicate a different scenario where the ancestral polA gene contained both the exonucleolytic activities in addition to the polymerase activity and where several eubacterial branches lost the polymerase-associated proofreading activity during evolution.     

Crystal structure of an archaebacterial DNA polymerase.

BACKGROUND: Members of the Pol II family of DNA polymerases are responsible for chromosomal replication in eukaryotes, and carry out highly processive DNA replication when attached to ring-shaped processivity clamps. The sequences of Pol II polymerases are distinct from those of members of the well-studied Pol I family of DNA polymerases. The DNA polymerase from the archaebacterium Desulfurococcus strain Tok (D. Tok Pol) is a member of the Pol II family that retains catalytic activity at elevated temperatures. RESULTS: The crystal structure of D. Tok Pol has been determined at 2.4 A resolution. The architecture of this Pol II type DNA polymerase resembles that of the DNA polymerase from the bacteriophage RB69, with which it shares less than approximately 20% sequence identity. As in RB69, the central catalytic region of the DNA polymerase is located within the 'palm' subdomain and is strikingly similar in structure to the corresponding regions of Pol I type DNA polymerases. The structural scaffold that surrounds the catalytic core in D. Tok Pol is unrelated in structure to that of Pol I type polymerases. The 3'-5' proofreading exonuclease domain of D. Tok Pol resembles the corresponding domains of RB69 Pol and Pol I type DNA polymerases. The exonuclease domain in D. Tok Pol is located in the same position relative to the polymerase domain as seen in RB69, and on the opposite side of the palm subdomain compared to its location in Pol I type polymerases. The N-terminal domain of D. Tok Pol has structural similarity to RNA-binding domains. Sequence alignments suggest that this domain is conserved in the eukaryotic DNA polymerases delta and epsilon. CONCLUSIONS: The structure of D. Tok Pol confirms that the modes of binding of the template and extrusion of newly synthesized duplex DNA are likely to be similar in both Pol II and Pol I type DNA polymerases. However, the mechanism by which the newly synthesized product transits in and out of the proofreading exonuclease domain has to be quite different. The discovery of a domain that seems to be an RNA-binding module raises the possibility that Pol II family members interact with RNA.     

Model for the catalytic domain of the proofreading epsilon subunit of Escherichia coli DNA polymerase III based on NMR structural data.

The DNA polymerase III holoenzyme (HE) is the primary replicative polymerase of Escherichia coli. The epsilon subunit of the HE complex provides the 3'-exonucleolytic proofreading activity for this enzyme complex. epsilon consists of two domains: an N-terminal domain containing the proofreading exonuclease activity (residues 1-186) and a C-terminal domain required for binding to the polymerase (alpha) subunit (residues 187-243). Multidimensional NMR studies of (2)H-, (13)C-, and (15)N-labeled N-terminal domains (epsilon186) were performed to assign the backbone resonances and measure H(N)-H(N) nuclear Overhauser effects (NOEs). NMR studies were also performed on triple-lableled [U-(2)H,(13)C,(15)N]epsilon186 containing Val, Leu, and Ile residues with protonated methyl groups, which allowed for the assignment of H(N)-CH(3) and CH(3)-CH(3) NOEs. Analysis of the (13)C(alpha), (13)C(beta), and (13)CO shifts, using chemical shift indexing and the TALOS program, allowed for the identification of regions of the secondary structure. H(N)-H(N) NOEs provided information on the assembly of the extended strands into a beta-sheet structure and confirmed the assignment of the alpha helices. Measurement of H(N)-CH(3) and CH(3)-CH(3) NOEs confirmed the beta-sheet structure and assisted in the positioning of the alpha helices. The resulting preliminary characterization of the three-dimensional structure of the protein indicated that significant structural homology exists with the active site of the Klenow proofreading exonuclease domain, despite the extremely limited sequence homology. On the basis of this analogy, molecular modeling studies of epsilon186 were performed using as templates the crystal structures of the exonuclease domains of the Klenow fragment and the T4 DNA polymerase and the recently determined structure of the E. coli Exonuclease I. A multiple sequence alignment was constructed, with the initial alignment taken from the previously published hidden Markov model and NMR constraints. Because several of the published structures included complexed ssDNA, we were also able to incorporate an A-C-G trinucleotide into the epsilon186 structure. Nearly all of the residues which have been identified as mutators are located in the portion of the molecule which binds the DNA, with most of these playing either a catalytic or structural role.     

Crystal structure of the protein from Arabidopsis thaliana gene At5g06450, a putative DnaQ‐like exonuclease domain‐containing protein with homohexameric assembly

Arabidopsis thaliana gene At5g06450 encodes a putative DnaQ‐like 3′‐5′ exonuclease domain‐containing protein (AtDECP). The DnaQ‐like 3′‐5′ exonuclease domain is often found as a proofreading domain of DNA polymerases. The overall structure of AtDECP adopts an RNase H fold that consists of a mixed β‐sheet flanked by α‐helices. Interestingly, AtDECP forms a homohexameric assembly with a central six fold symmetry, generating a central cavity. The ring‐shaped structure and comparison with WRN‐exo, the best structural homologue of AtDECP, suggest a possible mechanism for implementing its exonuclease activity using positively charged patch on the N‐terminal side of the homohexameric assembly. The homohexameric structure of AtDECP provides unique information about the interaction between the DnaQ‐like 3′‐5′ exonuclease and its substrate nucleic acids.Proteins 2013. © 2013 Wiley Periodicals, Inc.     

Exonucleolytic proofreading enhances the fidelity of DNA synthesis by chick embryo DNA polymerase-gamma

The high fidelity of chick embryo DNA polymerase-gamma (pol-gamma) observed during in vitro DNA synthesis (Kunkel, T. A. (1985) J. Biol. Chem. 260, 12866-12874) has led us to examine this DNA polymerase for the presence of an exonuclease activity capable of proofreading errors. Highly purified chick embryo pol-gamma preparations do contain exonuclease activity capable of digesting radiolabeled DNA in a 3'----5' direction, releasing deoxynucleoside 5'-monophosphates. The polymerase and exonuclease activities cosediment during centrifugation in a glycerol gradient containing 0.5 M KCl. In the absence of dNTP substrates, this exonuclease excises both matched and mismatched primer termini, with a preference for mismatched bases. Excision is inhibited by the addition of nucleoside 5'-monophosphates to the digestion reaction. In the presence of dNTP substrates to permit competition between excision and polymerization from the mismatched primer, the exonuclease excises mismatched bases from preformed terminal mispairs with greater than 98% efficiency. The preference for excision over polymerization can be diminished by addition of either high concentrations of dNTP substrates or nucleoside 5'-monophosphates to the exonuclease/polymerase reaction. To determine if this exonuclease is capable of proofreading misinsertions produced during a normal polymerization reaction, a sensitive base substitution fidelity assay was developed based on reversion of an M13mp2 lacZ alpha nonsense codon. In this assay using reaction conditions that permit highly active exonucleolytic proofreading, pol-gamma exhibits a fidelity of less than one error for every 260,000 bases polymerized. As for terminal mismatch excision, fidelity is reduced by the addition to the synthesis reaction of high concentrations of dNTP substrates or nucleoside 5'-monophosphates, both hallmarks of exonucleolytic proofreading by prokaryotic enzymes. Taken together, these observations suggest that the 3'----5' exonuclease present in highly purified chick embryo pol-gamma preparations proofreads base substitution errors during DNA synthesis. It remains to be determined if the polymerase and exonuclease activities reside in the same or different polypeptides.     

Structure of Escherichia coli exonuclease I suggests how processivity is achieved

Exonuclease I (ExoI) from Escherichia coli is a monomeric enzyme that processively degrades single stranded DNA in the 3' to 5' direction and has been implicated in DNA recombination and repair. Determination of the structure of ExoI to 2.4 A resolution using X-ray crystallography verifies the expected correspondence between a region of ExoI and the exonuclease (or proofreading) domains of the DNA polymerases. The overall fold of ExoI also includes two other regions, one of which extends the exonuclease domain and another that can be described as an elaborated SH3 domain. These three regions combine to form a molecule that is shaped like the letter C, although it also contains a segment that effectively converts the C into an O-like shape. The structure of ExoI thus provides additional support for the idea that DNA metabolizing enzymes achieve processivity by completely enclosing the DNA.     

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.     

The Family X DNA Polymerase from Deinococcus radiodurans Adopts a Non-standard Extended Conformation

Deinococcus radiodurans is an extraordinarily radioresistant bacterium that is able to repair hundreds of radiation-induced double-stranded DNA breaks. One of the players in this pathway is an X family DNA polymerase (PolX_Dr). Deletion of PolX_Dr has been shown to decrease the rate of repair of double-stranded DNA breaks and increase cell sensitivity to gamma-rays. A 3′→5′ exonuclease activity that stops cutting close to DNA loops has also been demonstrated. The present crystal structure of PolX_Dr solved at 2.46-Å resolution reveals that PolX_Dr has a novel extended conformation in stark contrast to the closed “right hand” conformation commonly observed for DNA polymerases. This extended conformation is stabilized by the C-terminal PHP domain, whose putative nuclease active site is obstructed by its interaction with the polymerase domain. The overall conformation and the presence of non standard residues in the active site of the polymerase X domain makes PolX_Dr the founding member of a novel class of polymerases involved in DNA repair but whose detailed mode of action still remains enigmatic.DNA replication and repair are functions that are of vital importance for the maintenance of cellular life. These functions are carried out by various DNA replicating engines, most of them acting as multiprotein complexes. Deinococcus radiodurans, a Gram-positive bacterium, is characterized by an extraordinary resistance to ionizing radiation and desiccation. After radiation induced cutting of its 3.28-megabase genome into hundreds of small fragments, it is capable of reassembling it completely (1). Different hypotheses have been suggested to explain this radioresistance. A recently proposed mechanism involves the creation of long linear DNA intermediates by an extended synthesis-dependent strand annealing process, where overlapping chromosomal fragments are used both as primers and as templates for synthesis of complementary single strands (2). Recircularization of chromosomes would be assured by homologous recombination. Although DNA polymerase I is one of the main enzymes involved in this process, it was shown that other proteins affect double strand break repair efficiency in D. radiodurans. One of these is an X family DNA polymerase (PolX_Dr)⁵ (3). Cells devoid of PolX_Dr protein show increased sensitivity to γ-irradiation and a longer delay in the restoration of an intact genome after irradiation. It was therefore proposed that PolX_Dr has an important role in double strand break repair in D. radiodurans. The contribution of PolX_Dr may become essential for instance when damage gets too important or, alternatively, it may act in different repair pathways from polymerase I. Indeed, some of the X DNA polymerases, such as Saccharomyces cerevisiae Pol4 and human polymerase λ (4) have been proposed to play important roles in different DNA repair processes, including non-homologous end-joining (5). It was shown that PolX_Dr also has strong 3′→5′ exonuclease activity that is stimulated by Mn²⁺ (6). This activity is associated with proofreading mechanisms in other polymerase families and encoded by protein domains or subunits distinct from the polymerase catalytic domain (7). Curiously the exonuclease activity of PolX_Dr is modulated upon encounter of a stem-loop structure. The combination of both activities leads to the hypothesis that PolX_Dr might be involved in DNA repair, potentially non-homologous end-joining, by processing damaged DNA or repair intermediates, thus generating substrates for other repair proteins (6). Very recently an orthologue of PolX from Bacillus subtilis was characterized. It was shown that PolX_Bs is a template-directed DNA polymerase acting on DNA gaps with a downstream 5′ phosphate group, suggesting it may play a role in base excision repair (8).DNA polymerases all combine a catalytic palm domain, a thumb domain, binding double-stranded DNA, and a finger domain that fixes the incoming nucleotide. The polymerase domain of the X family belongs to the Polβ-like nucleotidyltransferase superfamily, sharing ∼25% amino acid identity with the DNA polymerase domains of Polλ, Pol4, and Polβ. PolX_Dr has a second domain at the C terminus called PHP, with strong sequence identity with the histidinol phosphatase involved in histidine transport in bacteria. Due to its similarity to histidinol phosphatase and the presence of a trinuclear zinc site, the PolX_Dr PHP domain is thought to function as phosphoesterase (9). In the context of DNA polymerases, this activity might be responsible for the degradation of pyrophosphate, thus driving the polymerization reaction, or contributes to a nuclease reaction that would be involved in proofreading the newly synthesized strand. The deletion of the PHP domain also had a negative effect on survival of γ-irradiated cells suggesting that this domain possesses a function in DNA repair. Unexpectedly, deletion of the PHP domain destroys structure modulated but not the general 3′→5′ exonuclease activity (6). No activity could be demonstrated for the PHP domain alone.In this report we present the crystal structure of PolX_Dr at 2.46-Å resolution. Surprisingly, PolX_Dr adopts a stretched out conformation instead of the commonly observed closed right hand conformation. In the active site of the polymerase catalytic domain, the two universally conserved aspartates are replaced by two glutamates, whereas the active site of the PHP domain is obstructed by its interaction with the polymerase domain.     

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.     

Involvement of the TPR2 subdomain movement in the activities of ?29 DNA polymerase

The polymerization domain of ϕ29 DNA polymerase acquires a toroidal shape by means of an arch-like structure formed by the specific insertion TPR2 (Terminal Protein Region 2) and the thumb subdomain. TPR2 is connected to the fingers and palm subdomains through flexible regions, suggesting that it can undergo conformational changes. To examine whether such changes take place, we have constructed a ϕ29 DNA polymerase mutant able to form a disulfide bond between the apexes of TPR2 and thumb to limit the mobility of TPR2. Biochemical analysis of the mutant led us to conclude that TPR2 moves away from the thumb to allow the DNA polymerase to replicate circular ssDNA. Despite the fact that no TPR2 motion is needed to allow the polymerase to use the terminal protein (TP) as primer during the initiation of ϕ29 TP–DNA replication, the disulfide bond prevents the DNA polymerase from entering the elongation phase, suggesting that TPR2 movements are necessary to allow the TP priming domain to move out from the polymerase during transition from initiation to elongation. Furthermore, the TPR2-thumb bond does not affect the equilibrium between the polymerization and exonuclease activities, leading us to propose a primer-terminus transference model between both active sites.     

Probing the mechanisms of two exonuclease domain mutators of DNA polymerase ϵ

We report the properties of two mutations in the exonuclease domain of the Saccharomyces cerevisiae DNA polymerase ϵ. One, pol2-Y473F, increases the mutation rate by about 20-fold, similar to the catalytically dead pol2-D290A/E290A mutant. The other, pol2-N378K, is a stronger mutator. Both retain the ability to excise a nucleotide from double-stranded DNA, but with impaired activity. pol2-Y473F degrades DNA poorly, while pol2-N378K degrades single-stranded DNA at an elevated rate relative to double-stranded DNA. These data suggest that pol2-Y473F reduces the capacity of the enzyme to perform catalysis in the exonuclease active site, while pol2-N378K impairs partitioning to the exonuclease active site. Relative to wild-type Pol ϵ, both variants decrease the dNTP concentration required to elicit a switch between proofreading and polymerization by more than an order of magnitude. While neither mutation appears to alter the sequence specificity of polymerization, the N378K mutation stimulates polymerase activity, increasing the probability of incorporation and extension of a mismatch. Considered together, these data indicate that impairing the primer strand transfer pathway required for proofreading increases the probability of common mutations by Pol ϵ, elucidating the association of homologous mutations in human DNA polymerase ϵ with cancer.     

Kinetics and fidelity of polymerization by DNA polymerase III from Sulfolobus solfataricus

We have biochemically and kinetically characterized the polymerase and exonuclease activities of the third B-family polymerase (Dpo3) from the hyperthermophilic Crenarchaeon, Sulfolobus solfataricus (Sso). We have established through mutagenesis that despite incomplete sequence conservation, the polymerase and exonuclease active sites are functionally conserved in Dpo3. Using pre-steady-state kinetics, we can measure the fidelity of nucleotide incorporation by Dpo3 from the polymerase active site alone to be 10(3)-10(4) at 37 °C. The functional exonuclease proofreading active site will increase fidelity by at least 10(2), making Dpo3 comparable to other DNA polymerases in this family. Additionally, Dpo3's exonuclease activity is modulated by temperature, where a loss of promiscuous degradation activity can be attributed to a reorganization of the exonuclease domain when it is bound to primer-template DNA at high temperatures. Unexpectedly, the DNA binding affinity is weak compared with those of other DNA polymerases of this family. A comparison of the fidelity, polymerization kinetics, and associated functional exonuclease domain with those previously reported for other Sso polymerases (Dpo1 and Dpo4) illustrates that Dpo3 is a potential player in the proper maintenance of the archaeal genome.     

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.     

Substrate specificity and proposed structure of the proofreading complex of T7 DNA polymerase

Faithful replication of genomic DNA by high-fidelity DNA polymerases is crucial for the survival of most living organisms. While high-fidelity DNA polymerases favor canonical base pairs over mismatches by a factor of ∼1 × 10⁵, fidelity is further enhanced several orders of magnitude by a 3′–5′ proofreading exonuclease that selectively removes mispaired bases in the primer strand. Despite the importance of proofreading to maintaining genome stability, it remains much less studied than the fidelity mechanisms employed at the polymerase active site. Here we characterize the substrate specificity for the proofreading exonuclease of a high-fidelity DNA polymerase by investigating the proofreading kinetics on various DNA substrates. The contribution of the exonuclease to net fidelity is a function of the kinetic partitioning between extension and excision. We show that while proofreading of a terminal mismatch is efficient, proofreading a mismatch buried by one or two correct bases is even more efficient. Because the polymerase stalls after incorporation of a mismatch and after incorporation of one or two correct bases on top of a mismatch, the net contribution of the exonuclease is a function of multiple opportunities to correct mistakes. We also characterize the exonuclease stereospecificity using phosphorothioate-modified DNA, provide a homology model for the DNA primer strand in the exonuclease active site, and propose a dynamic structural model for the transfer of DNA from the polymerase to the exonuclease active site based on MD simulations.     

Crystal structure of DNA polymerase from hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1

The crystal structure of family B DNA polymerase from the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 (KOD DNA polymerase) was determined. KOD DNA polymerase exhibits the highest known extension rate, processivity and fidelity. We carried out the structural analysis of KOD DNA polymerase in order to clarify the mechanisms of those enzymatic features. Structural comparison of DNA polymerases from hyperthermophilic archaea highlighted the conformational difference in Thumb domains. The Thumb domain of KOD DNA polymerase shows an "opened" conformation. The fingers subdomain possessed many basic residues at the side of the polymerase active site. The residues are considered to be accessible to the incoming dNTP by electrostatic interaction. A beta-hairpin motif (residues 242-249) extends from the Exonuclease (Exo) domain as seen in the editing complex of the RB69 DNA polymerase from bacteriophage RB69. Many arginine residues are located at the forked-point (the junction of the template-binding and editing clefts) of KOD DNA polymerase, suggesting that the basic environment is suitable for partitioning of the primer and template DNA duplex and for stabilizing the partially melted DNA structure in the high-temperature environments. The stabilization of the melted DNA structure at the forked-point may be correlated with the high PCR performance of KOD DNA polymerase, which is due to low error rate, high elongation rate and processivity.     

Characterization of the 3' exonuclease subunit DP1 of Methanococcus jannaschii replicative DNA polymerase D

The B-subunits associated with the replicative DNA polymerases are conserved from Archaea to humans, whereas the corresponding catalytic subunits are not related. The latter belong to the B and D DNA polymerase families in eukaryotes and archaea, respectively. Sequence analysis places the B-subunits within the calcineurin-like phosphoesterase superfamily. Since residues implicated in metal binding and catalysis are well conserved in archaeal family D DNA polymerases, it has been hypothesized that the B-subunit could be responsible for the 3′-5′ proofreading exonuclease activity of these enzymes. To test this hypothesis we expressed Methanococcus jannaschii DP1 (MjaDP1), the B-subunit of DNA polymerase D, in Escherichia coli, and demonstrate that MjaDP1 functions alone as a moderately active, thermostable, Mn²⁺-dependent 3′-5′ exonuclease. The putative polymerase subunit DP2 is not required. The nuclease activity is strongly reduced by single amino acid mutations in the phosphoesterase domain indicating the requirement of this domain for the activity. MjaDP1 acts as a unidirectional, non-processive exonuclease preferring mispaired nucleotides and single-stranded DNA, suggesting that MjaDP1 functions as the proofreading exonuclease of archaeal family D DNA polymerase.