Site-specific labeling of T7 DNA polymerase with a conformationally sensitive fluorophore and its use in detecting single-nucleotide polymorphisms

Like most enzymes, DNA polymerases undergo a large conformational change on the binding of a correct nucleotide. To determine how the conformational change contributes to substrate specificity, we labeled the T7 DNA polymerase with a conformationally sensitive fluorophore at a position that provides a signal coincident with structural changes following nucleotide binding and distinguishes correct base pairs from incorrect ones by the sign of the fluorescence change. Here we describe methods to document that only one site on the polymerase was labeled with the fluorophore based on mass spectral analysis of tryptic peptides. In addition, we show by equilibrium titrations of opposing signals that mismatches and correct bases compete for the same site. This analysis forms an essential basis for characterization of a fluorescently labeled enzyme intended for mechanistic studies. Finally, we show that the labeled enzyme can be used to identify single-nucleotide mutations in a procedure that could be automated.     

Construction of a highly error-prone DNA polymerase for developing organelle mutation systems

A novel family of DNA polymerases replicates organelle genomes in a wide distribution of taxa encompassing plants and protozoans. Making error-prone mutator versions of gamma DNA polymerases revolutionised our understanding of animal mitochondrial genomes but similar advances have not been made for the organelle DNA polymerases present in plant mitochondria and chloroplasts. We tested the fidelities of error prone tobacco organelle DNA polymerases using a novel positive selection method involving replication of the phage lambda cI repressor gene. Unlike gamma DNA polymerases, ablation of 3′–5′ exonuclease function resulted in a modest 5–8-fold error rate increase. Combining exonuclease deficiency with a polymerisation domain substitution raised the organelle DNA polymerase error rate by 140-fold relative to the wild type enzyme. This high error rate compares favourably with error-rates of mutator versions of animal gamma DNA polymerases. The error prone organelle DNA polymerase introduced mutations at multiple locations ranging from two to seven sites in half of the mutant cI genes studied. Single base substitutions predominated including frequent A:A (template: dNMP) mispairings. High error rate and semi-dominance to the wild type enzyme in vitro make the error prone organelle DNA polymerase suitable for elevating mutation rates in chloroplasts and mitochondria.     

Conformational Dynamics of Thermus aquaticus DNA Polymerase I during Catalysis

Despite the fact that DNA polymerases have been investigated for many years and are commonly used as tools in a number of molecular biology assays, many details of the kinetic mechanism they use to catalyze DNA synthesis remain unclear. Structural and kinetic studies have characterized a rapid, pre-catalytic open-to-close conformational change of the Finger domain during nucleotide binding for many DNA polymerases including Thermus aquaticus DNA polymerase I (Taq Pol), a thermostable enzyme commonly used for DNA amplification in PCR. However, little has been performed to characterize the motions of other structural domains of Taq Pol or any other DNA polymerase during catalysis. Here, we used stopped-flow Förster resonance energy transfer to investigate the conformational dynamics of all five structural domains of the full-length Taq Pol relative to the DNA substrate during nucleotide binding and incorporation. Our study provides evidence for a rapid conformational change step induced by dNTP binding and a subsequent global conformational transition involving all domains of Taq Pol during catalysis. Additionally, our study shows that the rate of the global transition was greatly increased with the truncated form of Taq Pol lacking the N-terminal domain. Finally, we utilized a mutant of Taq Pol containing a de novo disulfide bond to demonstrate that limiting protein conformational flexibility greatly reduced the polymerization activity of Taq Pol.     

Identification of Critical Residues for the Tight Binding of Both Correct and Incorrect Nucleotides to Human DNA Polymerase λ

DNA polymerase λ (Pol λ) is a novel X-family DNA polymerase that shares 34% sequence identity with DNA polymerase β. Pre-steady-state kinetic studies have shown that the Pol λ-DNA complex binds both correct and incorrect nucleotides 130-fold tighter, on average, than the DNA polymerase β-DNA complex, although the base substitution fidelity of both polymerases is 10^− 4 to 10^− 5. To better understand Pol λ's tight nucleotide binding affinity, we created single-substitution and double-substitution mutants of Pol λ to disrupt the interactions between active-site residues and an incoming nucleotide or a template base. Single-turnover kinetic assays showed that Pol λ binds to an incoming nucleotide via cooperative interactions with active-site residues (R386, R420, K422, Y505, F506, A510, and R514). Disrupting protein interactions with an incoming correct or incorrect nucleotide impacted binding to each of the common structural moieties in the following order: triphosphate ? base > ribose. In addition, the loss of Watson-Crick hydrogen bonding between the nucleotide and the template base led to a moderate increase in K_d. The fidelity of Pol λ was maintained predominantly by a single residue, R517, which has minor groove interactions with the DNA template.     

Structures of mismatch replication errors observed in a DNA polymerase

Accurate DNA replication is essential for genomic stability. One mechanism by which high-fidelity DNA polymerases maintain replication accuracy involves stalling of the polymerase in response to covalent incorporation of mismatched base pairs, thereby favoring subsequent mismatch excision. Some polymerases retain a "short-term memory" of replication errors, responding to mismatches up to four base pairs in from the primer terminus. Here we a present a structural characterization of all 12 possible mismatches captured at the growing primer terminus in the active site of a polymerase. Our observations suggest four mechanisms that lead to mismatch-induced stalling of the polymerase. Furthermore, we have observed the effects of extending a mismatch up to six base pairs from the primer terminus and find that long-range distortions in the DNA transmit the presence of the mismatch back to the enzyme active site, suggesting the structural basis for the short-term memory of replication errors.     

A single highly mutable catalytic site amino acid is critical for DNA polymerase fidelity

DNA polymerases contain active sites that are structurally superimposable and conserved in amino acid sequence. To probe the biochemical and structure-function relationship of DNA polymerases, a large library (200,000 members) of mutant Thermus aquaticus DNA polymerase I (Taq pol I) was created containing random substitutions within a portion of the dNTP binding site (Motif A; amino acids 605-617), and a fraction of all selected active Taq pol I (291 out of 8000) was tested for base pairing fidelity; seven unique mutants that efficiently misincorporate bases and/or extend mismatched bases were identified and sequenced. These mutants all contain substitutions of one specific amino acid, Ile-614, which forms part of the hydrophobic pocket that binds the base and ribose portions of the incoming nucleotide. Mutant Taq pol Is containing hydrophilic substitution I614K exhibit 10-fold lower base misincorporation fidelity, as well as a high propensity to extend mispairs. In addition, these low fidelity mutants containing hydrophilic substitution for Ile-614 can bypass damaged templates that include an abasic site and vinyl chloride adduct ethenoA. During polymerase chain reaction, Taq pol I mutant I614K exhibits an error rate that is >20-fold higher relative to the wild-type enzyme and efficiently catalyzes both transition and transversion errors. These studies have generated polymerase chain reaction-proficient mutant polymerases containing substitutions within the active site that confers low base pairing fidelity and a high error rate. Considering the structural and sequence conservation of Motif A, it is likely that a similar substitution will yield active low fidelity DNA polymerases that are mutagenic.     

Structural Mechanism of Replication Stalling on a Bulky Amino-Polycyclic Aromatic Hydrocarbon DNA Adduct by a Y Family DNA Polymerase

Polycyclic aromatic hydrocarbons and their nitro derivatives are culprits of the detrimental health effects of environmental pollution. These hydrophobic compounds metabolize to reactive species and attach to DNA producing bulky lesions, such as N-[deoxyguanosine-8-yl]-1-aminopyrene (APG), in genomic DNA. The bulky adducts block DNA replication by high-fidelity polymerases and compromise replication fidelities and efficiencies by specialized lesion bypass polymerases. Here we present three crystal structures of the DNA polymerase Dpo4, a model translesion DNA polymerase of the Y family, in complex with APG-lesion-containing DNA in pre-insertion and extension stages. APG is captured in two conformations in the pre-insertion complex; one is highly exposed to the solvent, whereas the other is harbored in a shallow cleft between the finger and unique Y family little finger domain. In contrast, APG is in a single conformation at the extension stage, in which the pyrene ring is sandwiched between the little finger domain and a base from the turning back single-stranded template strand. Strikingly, a nucleotide intercalates the DNA helix to form a quaternary complex with Dpo4, DNA, and an incoming nucleotide, which stabilizes the distorted DNA structure at the extension stage. The unique APG DNA conformations in Dpo4 inhibit DNA translocation through the polymerase active site for APG bypass. We also modeled an insertion complex that illustrates a solvent-exposed pyrene ring contributing to an unstable insertion state. The structural work combined with our lesion replication assays provides a novel structural mechanism on bypass of DNA adducts containing polycyclic aromatic hydrocarbon moieties.     

Resistance to Nucleotide Excision Repair of Bulky Guanine Adducts Opposite Abasic Sites in DNA Duplexes and Relationships between Structure and Function

The nucleotide excision repair of certain bulky DNA lesions is abrogated in some specific non-canonical DNA base sequence contexts, while the removal of the same lesions by the nucleotide excision repair mechanism is efficient in duplexes in which all base pairs are complementary. Here we show that the nucleotide excision repair activity in human cell extracts is moderate-to-high in the case of two stereoisomeric DNA lesions derived from the pro-carcinogen benzo[a]pyrene (cis- and trans-B[a]P-N ²-dG adducts) in a normal DNA duplex. By contrast, the nucleotide excision repair activity is completely abrogated when the canonical cytosine base opposite the B[a]P-dG adducts is replaced by an abasic site in duplex DNA. However, base excision repair of the abasic site persists. In order to understand the structural origins of these striking phenomena, we used NMR and molecular spectroscopy techniques to evaluate the conformational features of 11mer DNA duplexes containing these B[a]P-dG lesions opposite abasic sites. Our results show that in these duplexes containing the clustered lesions, both B[a]P-dG adducts adopt base-displaced intercalated conformations, with the B[a]P aromatic rings intercalated into the DNA helix. To explain the persistence of base excision repair in the face of the opposed bulky B[a]P ring system, molecular modeling results suggest how the APE1 base excision repair endonuclease, that excises abasic lesions, can bind productively even with the trans-B[a]P-dG positioned opposite the abasic site. We hypothesize that the nucleotide excision repair resistance is fostered by local B[a]P residue—DNA base stacking interactions at the abasic sites, that are facilitated by the absence of the cytosine partner base in the complementary strand. More broadly, this study sets the stage for elucidating the interplay between base excision and nucleotide excision repair in processing different types of clustered DNA lesions that are substrates of nucleotide excision repair or base excision repair mechanisms.     

Consecutive incorporation of fluorophore-labeled nucleotides by mammalian DNA polymerase β

In the present study, we investigated mammalian polymerases that consecutively incorporate various fluorophore-labeled nucleotides. We found that rat DNA polymerase β (pol β) consecutively incorporated fluorophore-labeled nucleotides to a greater extent than four bacterial polymerases, Sequenase Version 2.0, Vent_R (exo-), DNA polymerase IIIα and the Klenow fragment, and the mammalian polymerases DNA polymerase α and human DNA polymerase δ, under mesophilic conditions. Furthermore, we investigated the kinetics of correct or mismatched incorporation with labeled nucleotides during synthesis by rat pol β. The kinetic parameters K_m and k_cat were measured and used for evaluating: (i) the discrimination against correct pair incorporation of labeled nucleotides relative to unlabeled nucleotides; and (ii) the fidelity for all nucleotide combinations of mismatched pairs in the presence of labeled or unlabeled nucleotides. We also investigated the effect of fluorophore-labeled nucleotides on terminal deoxynucleotidyl transferase activity of rat pol β. We have demonstrated for the first time that mammalian pol β can consecutively incorporate various fluorophore-labeled dNTPs. These findings suggest that pol β is useful for high-density labeling of DNA probes and single-molecule sequencing for high-speed genome analysis.     

Specificity and Catalytic Mechanism in Family 5 Uracil DNA Glycosylase

UDGb belongs to family 5 of the uracil DNA glycosylase (UDG) superfamily. Here, we report that family 5 UDGb from Thermus thermophilus HB8 is not only a uracil DNA glycosyase acting on G/U, T/U, C/U, and A/U base pairs, but also a hypoxanthine DNA glycosylase acting on G/I, T/I, and A/I base pairs and a xanthine DNA glycosylase acting on all double-stranded and single-stranded xanthine-containing DNA. Analysis of potentials of mean force indicates that the tendency of hypoxanthine base flipping follows the order of G/I > T/I, A/I > C/I, matching the trend of hypoxanthine DNA glycosylase activity observed in vitro. Genetic analysis indicates that family 5 UDGb can also act as an enzyme to remove uracil incorporated into DNA through the existence of dUTP in the nucleotide pool. Mutational analysis coupled with molecular modeling and molecular dynamics analysis reveals that although hydrogen bonding to O₂ of uracil underlies the UDG activity in a dissociative fashion, Tth UDGb relies on multiple catalytic residues to facilitate its excision of hypoxanthine and xanthine. This study underscores the structural and functional diversity in the UDG superfamily.     

Steric gate residues of Y-family DNA polymerases DinB and pol kappa are crucial for dNTP-induced conformational change

Discrimination against ribonucleotides by DNA polymerases is critical to preserve DNA integrity. For many DNA polymerases, including those of the Y family, rNTP discrimination has been attributed to steric clashes between a residue near the active site, the steric gate, and the 2′-hydroxyl of the incoming rNTP. Here we used hydrogen/deuterium exchange (HDX) mass spectrometry (MS) to probe the effects of the steric gate in the Y-family DNA polymerases Escherichia coli DinB and human DNA pol κ. Formation of a ternary complex with a G:dCTP base pair in the active site resulted in slower hydrogen exchange relative to a ternary complex with G:rCTP in the active site. The protection from exchange was localized to regions both distal and proximal to the active site, suggesting that DinB and DNA pol κ adopt different conformations depending on the sugar of the incoming nucleotide. In contrast, when the respective steric gate residues were mutated to alanine, the differences in HDX between the dNTP- and rNTP-bound ternary complexes were attenuated such that for DinB(F13A) and pol κ(Y112A), ternary complexes with either G:dCTP or G:rCTP base pairs had similar HDX profiles. Furthermore, the HDX in these ternary complexes resembled that of the rCTP-bound state rather than the dCTP-bound state of the wild-type enzymes. Primer extension assays confirmed that DinB(F13A) and pol κ(Y112A) do not discriminate against rNTPs to the same extent as the wild-type enzymes. Our observations indicate that the steric gate is crucial for rNTP discrimination because of its role in specifically promoting a dNTP-induced conformational change and that rNTP discrimination occurs in a relatively closed state of the polymerases.     

Discrimination against the cytosine analog tC by Escherichia coli DNA polymerase IV DinB

The cytosine analog 1,3-diaza-2-oxophenothiazine (tC) is a fluorescent nucleotide that forms Watson-Crick base pairs with dG. The Klenow fragment of DNA polymerase I (an A-family polymerase) can efficiently bypass tC on the template strand and incorporate deoxyribose-triphosphate-tC into the growing primer terminus. Y-family DNA polymerases are known for their ability to accommodate bulky lesions and modified bases and to replicate beyond such nonstandard DNA structures in a process known as translesion synthesis. We probed the ability of the Escherichia coli Y-family DNA polymerase DinB (Pol IV) to copy DNA containing tC and to incorporate tC into a growing DNA strand. DinB selectively adds dGTP across from tC in template DNA but cannot extend beyond the newly formed G:tC base pair. However, we find that DinB incorporates the tC deoxyribonucleotide triphosphate opposite template G and extends from tC. Therefore, DinB displays asymmetry in terms of its ability to discriminate against the modification of the DNA template compared to the incoming nucleotide. In addition, our finding that DinB (a lesion-bypass DNA polymerase) specifically discriminates against tC in the template strand may suggest that DinB discriminates against template modifications in the major groove of DNA.     

Incorporation of dUTP does not mediate mutation of A:T base pairs in Ig genes in vivo

Activation-induced cytidine deaminase (AID) protein initiates Ig gene mutation by deaminating cytosines, converting them into uracils. Excision of AID-induced uracils by uracil-N-glycosylase is responsible for most transversion mutations at G:C base pairs. On the other hand, processing of AID-induced G:U mismatches by mismatch repair factors is responsible for most mutation at Ig A:T base pairs. Why mismatch processing should be error prone is unknown. One theory proposes that long patch excision in G1-phase leads to dUTP-incorporation opposite adenines as a result of the higher G1-phase ratio of nuclear dUTP to dTTP. Subsequent base excision at the A:U base pairs produced could then create non-instructional templates leading to permanent mutations at A:T base pairs (1). This compelling theory has remained untested. We have developed a method to rapidly modify DNA repair pathways in mutating mouse B cells in vivo by transducing Ig knock-in splenic mouse B cells with GFP-tagged retroviruses, then adoptively transferring GFP⁺ cells, along with appropriate antigen, into primed congenic hosts. We have used this method to show that dUTP-incorporation is unlikely to be the cause of AID-induced mutation of A:T base pairs, and instead propose that A:T mutations might arise as an indirect consequence of nucleotide paucity during AID-induced DNA repair.     

Characterisation of the spectrum and genetic dependence of collateral mutations induced by translesion DNA synthesis

Translesion DNA synthesis (TLS) is a fundamental damage bypass pathway that utilises specialised polymerases with relaxed template specificity to achieve replication through damaged DNA. Misinsertions by low fidelity TLS polymerases may introduce additional mutations on undamaged DNA near the original lesion site, which we termed collateral mutations. In this study, we used whole genome sequencing datasets of chicken DT40 and several human cell lines to obtain evidence for collateral mutagenesis in higher eukaryotes. We found that cisplatin and UVC radiation frequently induce close mutation pairs within 25 base pairs that consist of an adduct-associated primary and a downstream collateral mutation, and genetically linked their formation to TLS activity involving PCNA ubiquitylation and polymerase κ. PCNA ubiquitylation was also indispensable for close mutation pairs observed amongst spontaneously arising base substitutions in cell lines with disrupted homologous recombination. Collateral mutation pairs were also found in melanoma genomes with evidence of UV exposure. We showed that collateral mutations frequently copy the upstream base, and extracted a base substitution signature that describes collateral mutagenesis in the presented dataset regardless of the primary mutagenic process. Using this mutation signature, we showed that collateral mutagenesis creates approximately 10–20% of non-paired substitutions as well, underscoring the importance of the process.     

Building better polymerases: Engineering the replication of expanded genetic alphabets

DNA polymerases are today used throughout scientific research, biotechnology, and medicine, in part for their ability to interact with unnatural forms of DNA created by synthetic biologists. Here especially, natural DNA polymerases often do not have the “performance specifications” needed for transformative technologies. This creates a need for science-guided rational (or semi-rational) engineering to identify variants that replicate unnatural base pairs (UBPs), unnatural backbones, tags, or other evolutionarily novel features of unnatural DNA. In this review, we provide a brief overview of the chemistry and properties of replicative DNA polymerases and their evolved variants, focusing on the Klenow fragment of Taq DNA polymerase (Klentaq). We describe comparative structural, enzymatic, and molecular dynamics studies of WT and Klentaq variants, complexed with natural or noncanonical substrates. Combining these methods provides insight into how specific amino acid substitutions distant from the active site in a Klentaq DNA polymerase variant (ZP Klentaq) contribute to its ability to replicate UBPs with improved efficiency compared with Klentaq. This approach can therefore serve to guide any future rational engineering of replicative DNA polymerases.     

Error induction and correction by mutant and wild type T4 DNA polymerases. Kinetic error discrimination mechanisms.

The fidelity of DNA synthesis as determined by the misincorporation of the base analogue 2-aminopurine in competition with adenine has been measured as a function of deoxynucleoside triphosphate substrate concentrations using purified mutator (L56), antimutator (L141), and wild type (T4D) T4 DNA polymerases. Although the rates of both incorporation and turnover of aminopurine and adenine decrease as substrate concentrations are decreased, the ratio of turnover/polymerase activity is increased. Thus, the nuclease/polymerase ratio of each of these three DNA polymerases can be controlled. The misincorporation of aminopurine decreases with decreasing substrate concentrations such that all three enzymes approach nearly identical misincorporation frequencies at the lowest substrate concentration. The increased accuracy of DNA synthesis corresponds to conditions producing a high nuclease/polymerase ratio. The misinsertion frequency for aminopurine is independent of substrate concentrations and enzyme phenotype; therefore, the increased accuracy of DNA synthesis with decreasing substrate concentrations is shown to be a result of increased nuclease activity and not increased polymerase or nuclease specificity. The data are analyzed in terms of a kinetic model of DNA polymerase accuracy which proposes that discrimination in nucleotide insertion and removal is based on the free energy difference between matched and mismatched base pairs. A value of 1.1 kcal/mol free energy difference, delta G, between adenine: thymine and aminopurine:thymine base pairs is predicted by model analysis of the cocentration dependence of aminopurine misincorporation and removal frequencies. An independent estimate of this free energy difference based on the 6-fold higher apparent Km of T4 DNA polymerase for aminopurine compared to adenine also gives a value of 1.1 kcal/mol. It is shown that the aminopurine misinsertion frequency for an enzyme having either extremely low 3'-exonuclease activity, Escherichia coli DNA polymerase I, or no measurable exonuclease activity, calf thymus DNA polymerase alpha, is 12 to 15%, which is similar to that for the T4 polymerases and consistent with delta G approximately 1.1 kcal/mol.     

Structure of human DNA polymerase iota and the mechanism of DNA synthesis

Cellular DNA polymerases belong to several families and carry out different functions. Highly accurate replicative DNA polymerases play the major role in cell genome replication. A number of new specialized DNA polymerases were discovered at the turn of XX–XXI centuries and have been intensively studied during the last decade. Due to the special structure of the active site, these enzymes efficiently perform synthesis on damaged DNA but are characterized by low fidelity. Human DNA polymerase iota (Pol ι) belongs to the Y-family of specialized DNA polymerases and is one of the most error-prone enzymes involved in DNA synthesis. In contrast to other DNA polymerases, Pol ι is able to use noncanonical Hoogsteen interactions for nucleotide base pairing. This allows it to incorporate nucleotides opposite various lesions in the DNA template that impair Watson-Crick interactions. Based on the data of X-ray structural analysis of Pol ι in complexes with various DNA templates and dNTP substrates, we consider the structural peculiarities of the Pol ι active site and discuss possible mechanisms that ensure the unique behavior of the enzyme on damaged and undamaged DNA.