Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4): an archaeal DinB-like DNA polymerase with lesion-bypass properties akin to eukaryotic polη

Phylogenetic analysis of Y-family DNA polymerases suggests that it can be subdivided into several discrete branches consisting of UmuC/DinB/Rev1/Rad30/Rad30A and Rad30B. The most diverse is the DinB family that is found in all three kingdoms of life. Searches of the complete genome of the crenarchaeon Sulfolobus solfataricus P2 reveal that it possesses a DinB homolog that has been termed DNA polymerase IV (Dpo4). We have overproduced and purified native Dpo4 protein and report here its enzymatic characterization. Dpo4 is thermostable, but can also synthesize DNA at 37°C. Under these conditions, the enzyme exhibits misinsertion fidelities in the range of 8 × 10^–3 to 3 × 10^–4. Dpo4 is distributive but at high enzyme to template ratios can synthesize long stretches of DNA and can substitute for Taq polymerase in PCR. On damaged DNA templates, Dpo4 can facilitate translesion replication of an abasic site, a cis-syn thymine–thymine dimer, as well as acetyl aminofluorene adducted- and cisplatinated-guanine residues. Thus, although phylogenetically related to DinB polymerases, our studies suggest that the archaeal Dpo4 enzyme exhibits lesion-bypass properties that are, in fact, more akin to those of eukaryotic polη.     

Capacity of Nine Thermostable DNA Polymerases To Mediate DNA Amplification in the Presence of PCR-Inhibiting Samples

The PCR is an extremely powerful method for detecting microorganisms. However, its full potential as a rapid detection method is limited by the inhibition of the thermostable DNA polymerase from Thermus aquaticus by many components found in complex biological samples. In this study, we have compared the effects of known PCR-inhibiting samples on nine thermostable DNA polymerases. Samples of blood, cheese, feces, and meat, as well as various ions, were added to PCR mixtures containing various thermostable DNA polymerases. The nucleic acid amplification capacity of the nine polymerases, under buffer conditions recommended by the manufacturers, was evaluated by using a PCR-based detection method for Listeria monocytogenes in the presence of purified template DNA and different concentrations of PCR inhibitors. The AmpliTaq Gold and the Taq DNA polymerases from Thermus aquaticus were totally inhibited in the presence of 0.004% (vol/vol) blood in the PCR mixture, while the HotTub, Pwo, rTth, and Tfl DNA polymerases were able to amplify DNA in the presence of 20% (vol/vol) blood without reduced amplification sensitivity. The DNA polymerase from Thermotoga maritima (Ultma) was found to be the most susceptible to PCR inhibitors present in cheese, feces, and meat samples. When the inhibitory effect of K and Na ions was tested on the nine polymerases, HotTub from Thermus flavus and rTth from Thermus thermophilus were the most resistant. Thus, the PCR-inhibiting effect of various components in biological samples can, to some extent, be eliminated by the use of the appropriate thermostable DNA polymerase.     

A new thermostable DNA polymerase mixture for efficient amplification of long DNA fragments

The thermostable DNA polymerases have been used for amplification of DNA fragments since the invention of PCR. The constraint on the maximum size of the amplified fragments can be solved to certain level by the use of unbalanced mixtures of non-proofreading and proofreading thermostable DNA polymerases. In this study, we tested the use of a mixtures of N-terminal deletional variant of Taq polymerase—Klentaq278 and Tne polymerase from Thermotoga neapolitana. Klentaq278 and Tne polymerase genes were cloned and expressed in different expression vectors under tac promoter. The most efficient ratio of Klentaq278/Tne polymerase for amplification was 10: 1. The polymerase mixture of Klentaq278 and Tne polymerase is very effective in amplification of DNA fragments for up to 8 kb and is useful addition to a DNA polymerases used in long-range PCR.     

Escherichia coli exonuclease III enhances long PCR amplification of damaged DNA templates

Recent development of the long PCR technology has provided an invaluable tool in many areas of molecular biology. However, long PCR amplification fails whenever the DNA template is imperfectly preserved. We report that Escherichia coli exonuclease III, a major repair enzyme in bacteria, strikingly improves the long PCR amplification of damaged DNA templates. Escherichia coli exonuclease III permitted or improved long PCR amplification with DNA samples submitted to different in vitro treatments known to induce DNA strand breaks and/or apurinic/apyrimidinic (AP) sites, including high temperature (99°C), depurination at low pH and near-UV radiation. Exonuclease III also permitted or improved amplification with DNA samples that had been isolated several years ago by the phenol/chloroform method. Amelioration of long PCR amplification was achieved for PCR products ranging in size from 5 to 15.4 kb and with DNA target sequences located either within mitochondrial DNA or the nuclear genome. Exonuclease III increased the amplification of damaged templates using either rTth DNA polymerase alone or rTth plus Vent DNA polymerases or Taq plus Pwo DNA polymerases. However, exonuclease III could not improve PCR amplification from extensively damaged DNA samples. In conclusion, supplementation of long PCR mixes with E.coli exonuclease III may represent a major technical advance whenever DNA samples have been partly damaged during isolation or subsequent storage.     

Global Conformational Dynamics of a Y-Family DNA Polymerase during Catalysis

Replicative DNA polymerases are stalled by damaged DNA while the newly discovered Y-family DNA polymerases are recruited to rescue these stalled replication forks, thereby enhancing cell survival. The Y-family DNA polymerases, characterized by low fidelity and processivity, are able to bypass different classes of DNA lesions. A variety of kinetic and structural studies have established a minimal reaction pathway common to all DNA polymerases, although the conformational intermediates are not well defined. Furthermore, the identification of the rate-limiting step of nucleotide incorporation catalyzed by any DNA polymerase has been a matter of long debate. By monitoring time-dependent fluorescence resonance energy transfer (FRET) signal changes at multiple sites in each domain and DNA during catalysis, we present here a real-time picture of the global conformational transitions of a model Y-family enzyme: DNA polymerase IV (Dpo4) from Sulfolobus solfataricus. Our results provide evidence for a hypothetical DNA translocation event followed by a rapid protein conformational change prior to catalysis and a subsequent slow, post-chemistry protein conformational change. Surprisingly, the DNA translocation step was induced by the binding of a correct nucleotide. Moreover, we have determined the directions, rates, and activation energy barriers of the protein conformational transitions, which indicated that the four domains of Dpo4 moved in a synchronized manner. These results showed conclusively that a pre-chemistry conformational change associated with domain movements was too fast to be the rate-limiting step. Rather, the rearrangement of active site residues limited the rate of correct nucleotide incorporation. Collectively, the conformational dynamics of Dpo4 offer insights into how the inter-domain movements are related to enzymatic function and their concerted interactions with other proteins at the replication fork.     

Unique Substrate Spectrum and PCR Application of Nanoarchaeum equitans Family B DNA Polymerase

The known archaeal family B DNA polymerases are unable to participate in the PCR in the presence of uracil. Here, we report on a novel archaeal family B DNA polymerase from Nanoarchaeum equitans that can successfully utilize deaminated bases such as uracil and hypoxanthine and on its application to PCR. N. equitans family B DNA polymerase (Neq DNA polymerase) produced λ DNA fragments up to 10 kb with an approximately 2.2-fold-lower error rate (5.53 × 10⁻⁶) than Taq DNA polymerase (11.98 × 10⁻⁶). Uniquely, Neq DNA polymerase also amplified λ DNA fragments using dUTP (in place of dTTP) or dITP (partially replaced with dGTP). To increase PCR efficiency, Taq and Neq DNA polymerases were mixed in different ratios; a ratio of 10:1 efficiently facilitated long PCR (20 kb). In the presence of dUTP, the PCR efficiency of the enzyme mixture was two- to threefold higher than that of either Taq and Neq DNA polymerase alone. These results suggest that Neq DNA polymerase and Neq plus DNA polymerase (a mixture of Taq and Neq DNA polymerases) are useful in DNA amplification and PCR-based applications, particularly in clinical diagnoses using uracil-DNA glycosylase.     

Backbone assignment of the catalytic core of a Y-family DNA polymerase

Sulfolobus solfataricus DNA polymerase IV (Dpo4), a model Y-family DNA polymerase, bypasses DNA lesions. Here, we report the assignments for the backbone nitrogen, carbon, and amide proton NMR signals of Dpo4’s catalytic core consisting of the finger, palm, and thumb domains. Our work provides the basis for further NMR spectroscopic studies of the interactions among Dpo4, DNA, and an incoming nucleotide.     

Crystal structure of a Y-family DNA polymerase in action: a mechanism for error-prone and lesion-bypass replication

Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) is a DinB homolog that belongs to the recently described Y-family of DNA polymerases, which are best characterized by their low-fidelity synthesis on undamaged DNA templates and propensity to traverse normally replication-blocking lesions. Crystal structures of Dpo4 in ternary complexes with DNA and an incoming nucleotide, either correct or incorrect, have been solved at 1.7 A and 2.1 A resolution, respectively. Despite a conserved active site and a hand-like configuration similar to all known polymerases, Dpo4 makes limited and nonspecific contacts with the replicating base pair, thus relaxing base selection. Dpo4 is also captured in the crystal translocating two template bases to the active site at once, suggesting a possible mechanism for bypassing thymine dimers.     

A High-Throughput Screening Method to Reengineer DNA Polymerases for Random Mutagenesis

A screening system for directed evolution of DNA polymerases employing a fluorescent Scorpion probe as a reporter has been developed. The screening system has been validated in a directed evolution experiment of a distributive polymerase from the Y-polymerase family (Dpo4 from Sulfolobus solfataricus) which was improved in elongation efficiency of consecutive mismatches. The engineering campaign yielded improved Dpo4 polymerase variants one of which was successfully benchmarked in a sequence saturation mutagenesis experiment especially with regard to the desirable consecutive transversion mutations (>2.5-fold increase in frequency relative to a reference library prepared with Dpo4 WT). The Scorpion probe screening system enables to reengineer polymerases with low processivity and fidelity, and no secondary activities (i.e. exonuclease activity or strand displacement activity) to match demands in diversity generation for directed protein evolution.     

Toward an International Standard for PCR-Based Detection of Food-Borne Thermotolerant Campylobacters: Assay Development and Analytical Validation

As part of a European research project (FOOD-PCR), we developed a standardized and robust PCR detection assay specific for the three most frequently reported food-borne pathogenic Campylobacter species, C. jejuni, C. coli, and C. lari. Fifteen published and unpublished PCR primers targeting the 16S rRNA gene were tested in all possible pairwise combinations, as well as two published primers targeting the 23S rRNA gene. A panel of 150 strains including target and nontarget strains was used in an in-house validation. Only one primer pair, OT1559 plus 18-1, was found to be selective. The inclusivity and exclusivity were 100 and 97%, respectively. In an attempt to find a thermostable DNA polymerase more resistant than Taq to PCR inhibitors present in chicken samples, three DNA polymerases were evaluated. The DNA polymerase Tth was not inhibited at a concentration of 2% (vol/vol) chicken carcass rinse, unlike both Taq DNA polymerase and DyNAzyme. Based on these results, Tth was selected as the most suitable enzyme for the assay. The standardized PCR test described shows potential for use in large-scale screening programs for food-borne Campylobacter species under the assay conditions specified.     

Backbone assignment of the binary complex of the full length <Emphasis Type="Italic">Sulfolobus solfataricus</Emphasis> DNA polymerase IV and DNA

Sulfolobus solfataricus DNA polymerase IV (Dpo4), a model Y-family DNA polymerase, bypasses a wide range of DNA lesions in vitro and in vivo. In this paper, we report the backbone chemical shift assignments of the full length Dpo4 in its binary complex with a 14/14-mer DNA substrate. Upon DNA binding, several β-stranded regions in the isolated catalytic core and little finger/linker fragments of Dpo4 become more structured. This work serves as a foundation for our ongoing investigation of conformational dynamics of Dpo4 and future determination of the first solution structures of a DNA polymerase and its binary and ternary complexes.     

A trimeric DNA polymerase complex increases the native replication processivity

DNA polymerases are essential enzymes in all domains of life for both DNA replication and repair. The primary DNA replication polymerase from Sulfolobus solfataricus (SsoDpo1) has been shown previously to provide the necessary polymerization speed and exonuclease activity to replicate the genome accurately. We find that this polymerase is able to physically associate with itself to form a trimer and that this complex is stabilized in the presence of DNA. Analytical gel filtration and electrophoretic mobility shift assays establish that initially a single DNA polymerase binds to DNA followed by the cooperative binding of two additional molecules of the polymerase at higher concentrations of the enzyme. Protein chemical crosslinking experiments show that these are specific polymerase–polymerase interactions and not just separate binding events along DNA. Isothermal titration calorimetry and fluorescence anisotropy experiments corroborate these findings and show a stoichiometry where three polymerases are bound to a single DNA substrate. The trimeric polymerase complex significantly increases both the DNA synthesis rate and the processivity of SsoDpo1. Taken together, these results suggest the presence of a trimeric DNA polymerase complex that is able to synthesize long DNA strands more efficiently than the monomeric form.     

Optimal conditions to use Pfu exo– DNA polymerase for highly efficient ligation-mediated polymerase chain reaction protocols

Ligation-Mediated Polymerase Chain Reaction (LMPCR) is the most sensitive sequencing technique available to map single-stranded DNA breaks at the nucleotide level of resolution using genomic DNA. LMPCR has been adapted to map DNA damage and reveal DNA–protein interactions inside living cells. However, the sequence context (GC content), the global break frequency and the current combination of DNA polymerases used in LMPCR affect the quality of the results. In this study, we developed and optimized an LMPCR protocol adapted for Pyrococcus furiosus exo^– DNA polymerase (Pfu exo^–). The relative efficiency of Pfu exo^– was compared to T7-modified DNA polymerase (Sequenase 2.0) at the primer extension step and to Thermus aquaticus DNA polymerase (Taq) at the PCR amplification step of LMPCR. At all break frequencies tested, Pfu exo^– proved to be more efficient than Sequenase 2.0. During both primer extension and PCR amplification steps, the ratio of DNA molecules per unit of DNA polymerase was the main determinant of the efficiency of Pfu exo^–, while the efficiency of Taq was less affected by this ratio. Substitution of NaCl for KCl in the PCR reaction buffer of Taq strikingly improved the efficiency of the DNA polymerase. Pfu exo^– was clearly more efficient than Taq to specifically amplify extremely GC-rich genomic DNA sequences. Our results show that a combination of Pfu exo^– at the primer extension step and Taq at the PCR amplification step is ideal for in vivo DNA analysis and DNA damage mapping using LMPCR.     

DNA lesion alters global conformational dynamics of Y-family DNA polymerase during catalysis

A major product of oxidative damage to DNA, 8-oxo-7,8-dihydro-2'-deoxyguanine (8-oxoG), can lead to genomic mutations if it is bypassed unfaithfully by DNA polymerases in vivo. However, our pre-steady-state kinetic studies show that DNA polymerase IV (Dpo4), a prototype Y-family enzyme from Sulfolobus solfataricus, can bypass 8-oxoG both efficiently and faithfully. For the first time, our stopped-flow FRET studies revealed that a DNA polymerase altered its synchronized global conformational dynamics in response to a DNA lesion. Relative to nucleotide incorporation into undamaged DNA, three of the four domains of Dpo4 undertook different conformational transitions during 8-oxoG bypass and the subsequent extension step. Moreover, the rapid translocation of Dpo4 along DNA induced by nucleotide binding was significantly hindered by the interactions between the embedded 8-oxoG and Dpo4 during the extension step. These results unprecedentedly demonstrate that a Y-family DNA polymerase employs different global conformational dynamics when replicating undamaged and damaged DNA.     

Molecular breeding of polymerases for amplification of ancient DNA

In the absence of repair, lesions accumulate in DNA. Thus, DNA persisting in specimens of paleontological, archaeological or forensic interest is inevitably damaged. We describe a strategy for the recovery of genetic information from damaged DNA. By molecular breeding of polymerase genes from the genus Thermus (Taq (Thermus aquaticus), Tth (Thermus thermophilus) and Tfl (Thermus flavus)) and compartmentalized self-replication selection, we have evolved polymerases that can extend single, double and even quadruple mismatches, process non-canonical primer-template duplexes and bypass lesions found in ancient DNA, such as hydantoins and abasic sites. Applied to the PCR amplification of 47,000-60,000-year-old cave bear DNA, these outperformed Taq DNA polymerase by up to 150% and yielded amplification products at sample dilutions at which Taq did not. Our results demonstrate that engineered polymerases can expand the recovery of genetic information from Pleistocene specimens and may benefit genetic analysis in paleontology, archeology and forensic medicine.     

The Y-family DNA polymerase Dpo4 uses a template slippage mechanism to create single-base deletions

The Y-family polymerases help cells tolerate DNA damage by performing translesion synthesis, yet they also can be highly error prone. One distinctive feature of the DinB class of Y-family polymerases is that they make single-base deletion errors at high frequencies in repetitive sequences, especially those that contain two or more identical pyrimidines with a 5' flanking guanosine. Intriguingly, different deletion mechanisms have been proposed, even for two archaeal DinB polymerases that share 54% sequence identity and originate from two strains of Sulfolobus. To reconcile these apparent differences, we have characterized Dpo4 from Sulfolobus solfataricus using the same biochemical and crystallographic approaches that we have used previously to characterize Dbh from Sulfolobus acidocaldarius. In contrast to previous suggestions that Dpo4 uses a deoxynucleoside triphosphate (dNTP)-stabilized misalignment mechanism when creating single-base deletions, we find that Dpo4 predominantly uses a template slippage deletion mechanism when replicating repetitive DNA sequences, as was previously shown for Dbh. Dpo4 stabilizes the skipped template base in an extrahelical conformation between the polymerase and the little-finger domains of the enzyme. This contrasts with Dbh, in which the extrahelical base is stabilized against the surface of the little-finger domain alone. Thus, despite sharing a common deletion mechanism, these closely related polymerases use different contacts with the substrate to accomplish the same result.     

Translesion Synthesis across 1,N6-(2-Hydroxy-3-hydroxymethylpropan-1,3-diyl)-2′-deoxyadenosine (1,N6-γ-HMHP-dA) Adducts by Human and Archebacterial DNA Polymerases

The 1,N⁶-(2-Hydroxy-3-hydroxymethylpropan-1,3-diyl)-2′-deoxyadenosine (1,N⁶-γ-HMHP-dA) adducts are formed upon bifunctional alkylation of adenine nucleobases in DNA by 1,2,3,4-diepoxybutane, the putative ultimate carcinogenic metabolite of 1,3-butadiene. The presence of a substituted 1,N⁶-propano group on 1,N⁶-γ-HMHP-dA is expected to block the Watson-Crick base pairing of the adducted adenine with thymine, potentially contributing to mutagenesis. In this study, the enzymology of replication past site-specific 1,N⁶-γ-HMHP-dA lesions in the presence of human DNA polymerases (hpols) β, η, κ, and ι and archebacterial polymerase Dpo4 was investigated. Run-on gel analysis with all four dNTPs revealed that hpol η, κ, and Dpo4 were able to copy the modified template. In contrast, hpol ι inserted a single base opposite 1,N⁶-γ-HMHP-dA but was unable to extend beyond the damaged site, and a complete replication block was observed with hpol β. Single nucleotide incorporation experiments indicated that although hpol η, κ, and Dpo4 incorporated the correct nucleotide (dTMP) opposite the lesion, dGMP and dAMP were inserted with a comparable frequency. HPLC-ESI-MS/MS analysis of primer extension products confirmed the ability of bypass polymerases to insert dTMP, dAMP, or dGMP opposite 1,N⁶-γ-HMHP-dA and detected large amounts of −1 and −2 deletion products. Taken together, these results indicate that hpol η and κ enzymes bypass 1,N⁶-γ-HMHP-dA lesions in an error-prone fashion, potentially contributing to A→T and A→C transversions and frameshift mutations observed in cells following treatment with 1,2,3,4-diepoxybutane.     

A novel strategy to engineer DNA polymerases for enhanced processivity and improved performance in vitro

Mechanisms that allow replicative DNA polymerases to attain high processivity are often specific to a given polymerase and cannot be generalized to others. Here we report a protein engineering-based approach to significantly improve the processivity of DNA polymerases by covalently linking the polymerase domain to a sequence non-specific dsDNA binding protein. Using Sso7d from Sulfolobus solfataricus as the DNA binding protein, we demonstrate that the processivity of both family A and family B polymerases can be significantly enhanced. By introducing point mutations in Sso7d, we show that the dsDNA binding property of Sso7d is essential for the enhancement. We present evidence supporting two novel conclusions. First, the fusion of a heterologous dsDNA binding protein to a polymerase can increase processivity without compromising catalytic activity and enzyme stability. Second, polymerase processivity is limiting for the efficiency of PCR, such that the fusion enzymes exhibit profound advantages over unmodified enzymes in PCR applications. This technology has the potential to broadly improve the performance of nucleic acid modifying enzymes.     

Kinetic basis of sugar selection by a Y-family DNA polymerase from Sulfolobus solfataricus P2

DNA polymerases use either a bulky active site residue or a backbone segment to select against ribonucleotides in order to faithfully replicate cellular genomes. Here, we demonstrated that an active site mutation (Y12A) within Sulfolobus solfataricus DNA polymerase IV (Dpo4) caused an average increase of 220-fold in matched ribonucleotide incorporation efficiency and an average decrease of 9-fold in correct deoxyribonucleotide incorporation efficiency, leading to an average reduction of 2000-fold in sugar selectivity. Thus, the bulky side chain of Tyr12 is important for both ribonucleotide discrimination and efficient deoxyribonucleotide incorporation. Other than synthesizing DNA as the wild-type Dpo4, the Y12A Dpo4 mutant incorporated more than 20 consecutive ribonucleotides into primer/template (DNA/DNA) duplexes, suggesting that this mutant protein possesses both a DNA-dependent DNA polymerase activity and a DNA-dependent RNA polymerase activity. Moreover, the binary and ternary crystal structures of Dpo4 have revealed that this DNA lesion bypass polymerase can bind up to eight base pairs of double-stranded DNA which is entirely in B-type. Thus, the DNA binding cleft of Dpo4 is flexible and can accommodate both A- and B-type oligodeoxyribonucleotide duplexes as well as damaged DNA.     

Evaluation of PCR-Generated Chimeras, Mutations, and Heteroduplexes with 16S rRNA Gene-Based Cloning

To evaluate PCR-generated artifacts (i.e., chimeras, mutations, and heteroduplexes) with the 16S ribosomal DNA (rDNA)-based cloning approach, a model community of four species was constructed from alpha, beta, and gamma subdivisions of the division Proteobacteria as well as gram-positive bacterium, all of which could be distinguished by HhaI restriction digestion patterns. The overall PCR artifacts were significantly different among the three Taq DNA polymerases examined: 20% for Z-Taq, with the highest processitivity; 15% for LA-Taq, with the highest fidelity and intermediate processitivity; and 7% for the conventionally used DNA polymerase, AmpliTaq. In contrast to the theoretical prediction, the frequency of chimeras for both Z-Taq (8.7%) and LA-Taq (6.2%) was higher than that for AmpliTaq (2.5%). The frequencies of chimeras and of heteroduplexes for Z-Taq were almost three times higher than those of AmpliTaq. The total PCR artifacts increased as PCR cycles and template concentrations increased and decreased as elongation time increased. Generally the frequency of chimeras was lower than that of mutations but higher than that of heteroduplexes. The total PCR artifacts as well as the frequency of heteroduplexes increased as the species diversity increased. PCR artifacts were significantly reduced by using AmpliTaq and fewer PCR cycles (fewer than 20 cycles), and the heteroduplexes could be effectively removed from PCR products prior to cloning by polyacrylamide gel purification or T7 endonuclease I digestion. Based upon these results, an optimal approach is proposed to minimize PCR artifacts in 16S rDNA-based microbial community studies.