T4 DNA Polymerase Research Articles

Preparation of long sticky ends for universal ligation-independent cloning: Sequential T4 DNA polymerase treatments

Ligation-independent cloning (LIC) is a useful method for efficient directional cloning of a PCR product. LIC requires a specially designed vector containing a long stretch of sequence that is missing any one of the four nucleotides. When the linearized vector is treated with T4 DNA polymerase, in the presence of the absent base, long single-stranded overhangs are generated that are suitable for cloning. In this study, long and efficient sticky ends for LIC were produced by sequential T4 DNA polymerase treatments at non-specific sequences on a commercially available vector. All restriction enzyme sites become available in the current LIC.

Different Behaviors In Vivo of Mutations in the β Hairpin Loop of the DNA Polymerases of the Closely Related Phages T4 and RB69

The T4 and RB69 DNA replicative polymerases are members of the B family and are highly similar. Both replicate DNA with high fidelity and employ the same mechanism that allows efficient switching of the primer terminus between the polymerase and exonuclease sites. Both polymerases have a β hairpin loop (hereafter called the β loop) in their exonuclease domains that plays an important role in active-site switching. The β loop is involved in strand separation and is needed to stabilize partially strand-separated exonuclease complexes. In T4 DNA polymerase, modification of the β-loop residue G255 to Ser confers a strong mutator phenotype in vivo due to a reduced ability to form editing complexes. Here, we describe the RB69 DNA polymerase mutant with the equivalent residue (G258) changed to Ser but showing only mild mutator activity in vivo. On the other hand, deletion of the tip of the RB69 β loop confers a strong mutator phenotype in vivo. Based on detailed mutational spectral analyses, DNA binding activities, and coupled polymerase/exonuclease assays, we define the differences between the T4 and RB69 polymerases. We propose that their β loops facilitate strand separation in both polymerases, while the residues that form the loop have low structural constraints.

Differential Effect of Extracellular Acidosis on the Release and Dispersal of Soluble and Membrane Proteins Secreted from the Weibel-Palade Body

Proteins secreted from Weibel-Palade bodies (WPBs) play important roles in regulating inflammatory and hemostatic responses. Inflammation is associated with the extracellular acidification of tissues and blood, conditions that can alter the behavior of secreted proteins. The effect of extracellular pH (pH(o)) on the release of von Willebrand factor (VWF), the VWF-propolypeptide (Proregion), interleukin-8, eotaxin-3, P-selectin, and CD63 from WPBs was investigated using biochemical approaches and by direct optical analysis of individual WPB fusion events in human endothelial cells expressing green or red fluorescent fusions of these different cargo proteins. Between pH(o) 7.4 and 7.0, ionomycin-evoked WPB exocytosis was characterized by the adhesion of VWF to the cell surface and the formation of long filamentous strands. The rapid dispersal of Proregion, interleukin-8, and eotaxin-3 into solution, and of P-selectin and CD63 into the plasma membrane, was unaltered over this pH(o) range. At pH(o) 6.8 or lower, Proregion remained associated with VWF, in many cases WPB failed to collapse fully and VWF failed to form filamentous strands. At pH(o) 6.5 dispersal of interleukin-8, eotaxin-3, and the membrane protein CD63 remained unaltered compared with that at pH(o) 7.4; however, P-selectin dispersal into the plasma membrane was significantly slowed. Thus, extracellular acidification to levels of pH(o) 6.8 or lower significantly alters the behavior of secreted VWF, Proregion, and P-selectin while rapid release of the small pro-inflammatory mediators IL-8 and eotaxin-3 is essentially unaltered. Together, these data suggest that WPB exocytosis during extracellular acidosis may favor the control of inflammatory processes.

Mutations in the gene 5 DNA polymerase of bacteriophage T7 suppress the dominant lethal phenotype of gene 2.5 ssDNA binding protein lacking the C‐terminal phenylalanine

Gene 2.5 of bacteriophage T7 encodes a ssDNA binding protein (gp2.5) essential for DNA replication. The C-terminal phenylalanine of gp2.5 is critical for function and mutations in that position are dominant lethal. In order to identify gp2.5 interactions we designed a screen for suppressors of gp2.5 lacking the C-terminal phenylalanine. Screening for suppressors of dominant lethal mutations of essential genes is challenging as the phenotype prevents propagation. We select for phage encoding a dominant lethal version of gene 2.5, whose viability is recovered via second-site suppressor mutation(s). Functional gp2.5 is expressed in trans for propagation of the unviable phage and allows suppression to occur via natural selection. The isolated intragenic suppressors support the critical role of the C-terminal phenylalanine. Extragenic suppressor mutations occur in several genes encoding enzymes of DNA metabolism. We have focused on the suppressor mutations in gene 5 encoding the T7 DNA polymerase (gp5) as the gp5/gp2.5 interaction is well documented. The suppressor mutations in gene 5 are necessary and sufficient to suppress the lethal phenotype of gp2.5 lacking the C-terminal phenylalanine. The affected residues map in proximity to aromatic residues and to residues in contact with DNA in the crystal structure of T7 DNA polymerase-thioredoxin.

Polymerase-Tailored Variations in the Water-Mediated and Substrate-Assisted Mechanism for Nucleotidyl Transfer: Insights from a Study of T7 DNA Polymerase

The nucleotidyl transfer reaction catalyzed by DNA polymerases is the critical step governing the accurate transfer of genetic information during DNA replication, and its malfunctioning can cause mutations leading to human diseases, including cancer. Here, utilizing ab initio quantum mechanical/molecular mechanical calculations with free-energy perturbation, we carried out an extensive investigation of the nucleotidyl transfer reaction mechanism in the well-characterized high-fidelity replicative DNA polymerase from phage T7. Our defined mechanism entails an initial concerted deprotonation of a conserved crystal water molecule with protonation of the γ-phosphate of the deoxynucleotide triphosphate(dNTP) via a solvent water molecule, and then the proton on the primer 3′-terminus is transferred to the resulting hydroxide ion. Subsequently, the nucleophilic attack takes place, with the formation of a metastable pentacovalent phosphorane intermediate. Finally, the pyrophosphate leaves, facilitated by the relay of the proton on the γ-phosphate to the α–β bridging oxygen via solvent water. The computed activation free-energy barrier is consistent with kinetic data for the chemistry step with correct nucleotide incorporation in T7 DNA polymerase. This variant of the water-mediated and substrate-assisted mechanism has features tailored to the structure of the T7 DNA polymerase. However, a unifying theme in the water-mediated and substrate-assisted mechanism is the cycling through crystal and solvent water molecules of the proton originating from the primer 3′-terminus to the α–β bridging oxygen of the deoxynucleotide triphosphate; this neutralizes the evolving negative charge as pyrophosphate leaves and restores the polymerase to its pre-chemistry state. These unifying features are likely requisite elements for nucleotidyl transfer reactions.

Construction of a circular single-stranded DNA template containing a defined lesion

We report a concise and efficient method to make a circular single-stranded DNA containing a defined DNA lesion. In this protocol, phagemid DNA containing Uracil is used as a template to synthesize a complementary DNA strand using T7 DNA polymerase and an oligonucleotide primer including a site-specific DNA lesion. The ligated lesion-containing strand can be recovered after the phage-derived template DNA is degraded by treatment with E. coli Uracil DNA glycosylase and Exonucleases I and III. The resulting product is a circular single-stranded DNA containing a defined DNA lesion suitable for in vitro translesion replication assays.

High-Density Gene Expression Analysis of Tumor-Associated Macrophages from Mouse Mammary Tumors

Clinical and experimental evidence indicates that tumor-associated macrophages (TAMs) promote malignant progression. In breast cancer, TAMs enhance tumor angiogenesis, tumor cell invasion, matrix remodeling, and immune suppression against the tumor. In this study, we examined late-stage mammary tumors from a transgenic mouse model of breast cancer. We used flow cytometry under conditions that minimized gene expression changes to isolate a rigorously defined TAM population previously shown to be associated with invasive carcinoma cells. The gene expression signature of this population was compared with a similar population derived from spleens of non-tumor-bearing mice using high-density oligonucleotide arrays. Using stringent selection criteria, transcript abundance of 460 genes was shown to be differentially regulated between the two populations. Bioinformatic analyses of known functions of these genes indicated that formerly ascribed TAM functions, including suppression of immune activation and matrix remodeling, as well as multiple mediators of tumor angiogenesis, were elevated in TAMs. Further bioinformatic analyses confirmed that a pure and valid TAM gene expression signature in mouse tumors could be used to assess expression of TAMs in human breast cancer. The data derived from these more physiologically relevant autochthonous tumors compared with previous studies in tumor xenografts suggest tactics by which TAMs may regulate tumor angiogenesis and thus provide a basis for exploring other transcriptional mediators of TAM trophic functions within the tumor microenvironment.

Coupling of two motors: T7 helicase-primase and DNA polymerase

T7 bacteriophage encodes all the proteins it needs to replicate its duplex DNA genome. The T7 replisome consists of the homo-hexameric T7 gp4 helicase-primase protein, T7 DNA polymerase (T7 gp5 complexed with E. coli Thioredoxin) and the T7 gene 2.5 protein (the single stranded DNA binding protein). The simplicity of the T7 replisome makes it a model enzyme complex to understand the mechanisms of DNA replication and the roles of the motor proteins in DNA replication. T7 helicase is a ring-shaped motor protein that binds ssDNA within its central channel and uses the energy of dTTP hydrolysis to move processively along ssDNA as well as to unwind the strands of duplex DNA.

Linear Diffusion of T7 DNA Polymerase: Thioredoxin is Required to Maintain Close Contact with DNA

The bacteriophage T7 DNA polymerase consists of a tight, 1:1 complex of T7 gp5, encoded by the phage, and thioredoxin, produced by the E. coli host. In the absence of thioredoxin, gp5 is capable of adding only a few nucleotides to the 3′ end of a primer before dissociating from the primer-template. But when complexed with thioredoxin, gp5 becomes highly processive, capable of polymerizing thousands of nucleotides complementary to the template strand. The mechanism by which thioredoxin acts as a processivity factor to gp5 is not fully understood. To understand the role of the thioredoxin in stabilizing polymerase-DNA interactions, we use a single-molecule imaging approach to observe individual, fluorescently labeled T7 DNA polymerase complexes diffusing along double-stranded DNA. Our results show that the average diffusion coefficient of T7 DNA polymerase complexes is insensitive to ionic strength and does not exceed the theoretical diffusion limit for a protein that tracks the helical pitch and rotates as it diffuses along the DNA helix. These results suggest that the T7 DNA polymerase slides along the DNA, remaining tightly bound to the DNA and tracking the helical pitch. However, the mean diffusion coefficients for fluorescently labeled T7 gp5 in the absence of thioredoxin increase with salt concentration, and exceed the theoretical limit for a protein tracking the DNA helix. Upon addition of unlabeled thioredoxin, the mean diffusion coefficient is restored to the value observed for the labeled T7 DNA polymerase, and becomes salt independent. These observations indicate that, in the absence of thioredoxin, T7 gp5 intermittently dissociates from the DNA as it diffuses, and that thioredoxin binding suppresses microscopic hopping on and off the DNA.

Single-molecule Observations of Replisome Structure and Function

DNA replication requires the coordinated activity of a large number of enzymes at the replication fork. Understanding the mechanisms controlling this organization requires a direct probing of the dynamics of fully functional replisomes during replication. Observations at the single-molecule level provide the most direct way to visualize the complex biochemistry of the replisome and to quantify the many transient intermediates essential to replication. We present a novel assay that combines the observation of individual fluorescently labeled proteins with the mechanical manipulation of DNA. Surface-tethered DNAs labeled with quantum dots are hydrodynamically stretched and imaged with a TIRF microscope. Activity of the replisome is observed as a change in the DNA length due to the differing force-dependent extension of single- and double-stranded DNA at low pico-Newton forces. We employ a two-color imaging scheme to monitor DNA length in real-time and to stroboscopically image fluorescently labeled single proteins interacting with DNA. Observation of labeled proteins in an ongoing replication reaction allows us to pose structural questions about the stoichiometry and exchange of proteins at the prokaryotic replication fork. We will discuss preliminary results on primer extension by the T7 DNA polymerase and strand-displacement synthesis by the coupled activity of the T7 helicase and polymerase.

Properties of pseudo-complementary DNA substituted with weakly pairing analogs of guanine or cytosine

A straightforward enzymatic protocol for converting regular DNA into pseudo-complementary DNA could improve the performance of oligonucleotide microarrays by generating readily hybridizable structure-free targets. Here we screened several highly destabilizing analogs of G and C for one that could be used with 2-aminoadenine (nA) and 2-thiothymine (sT) to generate structure-free DNA that is fully accessible to complementary probes. The analogs, which included bioactive bases such as 6-thioguanine (sG), 5-nitrocytosine (NitroC), 2-pyrimidinone (P; the free base of zebularine) and 6-methylfuranopyrimidinone (MefP), were prepared as dNTPs and evaluated as substrates for T7 and Phi29 DNA polymerases that lacked editor function. Pairing properties of the analogs were characterized by solution hybridization assays using modified oligonucleotides or primer extension products. P and MeP did not support robust primer extension whereas sG and NitroC did. In hybridization assays, however, sG lacked discrimination and NitroC paired too strongly to C. The dNTPs of two other base analogs, 7-nitro-7-deazahypoxanthine (NitrocH) and 2-thiocytosine (sC), exhibited the greatest promise. Either analog could be used with nA and sT to generate DNA that was nearly structure-free. Hybridization of probes to these modified DNAs will require the development of base analogs that pair strongly to NitrocH or sC.

Interactions of Escherichia coli Thioredoxin, the Processivity Factor, with Bacteriophage T7 DNA Polymerase and Helicase

Escherichia coli thioredoxin binds to a unique flexible loop of 71 amino acid residues, designated the thioredoxin binding domain (TBD), located in the thumb subdomain of bacteriophage T7 gene 5 DNA polymerase. The initial designation of thioredoxin as a processivity factor was premature. Rather it remodels the TBD for interaction with DNA and the other replication proteins. The binding of thioredoxin exposes a number of basic residues on the TBD that lie over the duplex region of the primer-template and increases the processivity of nucleotide polymerization. Two small solvent-exposed loops (loops A and B) located within TBD electrostatically interact with the acidic C-terminal tail of T7 gene 4 helicase-primase, an interaction that is enhanced by the binding of thioredoxin. Several basic residues on the surface of thioredoxin in the polymerase-thioredoxin complex lie in close proximity to the TBD. One of these residues, lysine 36, is located proximal to loop A. The substitution of glutamate for lysine has a dramatic effect on the binding of gene 4 helicase to a DNA polymerase-thioredoxin complex lacking charges on loop B; binding is decreased 15-fold relative to that observed with wild-type thioredoxin. This defective interaction impairs the ability of T7 DNA polymerase-thioredoxin together with T7 helicase to mediate strand displacement synthesis. This is the first demonstration that thioredoxin interacts with replication proteins other than T7 DNA polymerase.

Enzymatic conversion of long DNA to small DNA fragments for the construction of short hairpin RNA expression libraries

Several techniques to enzymatically construct a short hairpin RNA (shRNA) expression library have been reported as tools for comprehensive genetic analyses by RNA interference. Our technique constructs an shRNA expression library from 25- to 35-bp DNA fragments by fragmenting given double-stranded DNA (dsDNA). We compared the following two procedures to efficiently prepare such small DNA fragments: one is the cleavage of dsDNA with deoxyribonuclease I (DNase I) in the presence of Mn 2+ followed by blunting with T4 DNA polymerase, and the other is the introduction of nicks with DNase I in the presence of Mg 2+ followed by blunting with the Klenow fragment. Consequently, the latter yielded the DNA fragments more efficiently. However, these DNA fragments were contaminated with fused DNA fragments that had originated from two regions of original dsDNA. Therefore, we used single-strand-specific exonucleases and succeeded in suppressing the production of such fused DNA fragments. Our technique allows the efficient conversion of given dsDNA to small DNA fragments.

DNA‐Dependent DNA Polymerases

This unit presents characteristics and reaction conditions of the DNA-dependent DNA polymerases, including E. coli DNA polymerase I and its Klenow fragment, T4 DNA polymerase, native and modified T7 DNA polymerase, phi29 DNA polymerase, Bst DNA polymerase, and Taq DNA polymerase. The unit also provides overviews of other classes of thermophilic DNA polymerases used in PCR applications (described fully in UNIT 15.1), and the rapidly expanding class of lesion-bypass DNA polymerases that play a role in DNA damage repair.

Analysis of HSF4 Binding Regions Reveals Its Necessity for Gene Regulation during Development and Heat Shock Response in Mouse Lenses

Heat shock transcription factors (HSFs) regulate gene expression in response to heat shock and in physiological conditions. In mammals, HSF1 is required for heat-mediated induction of classic heat shock genes; however, we do not know the molecular mechanisms by which HSF4 regulates gene expression or the biological consequences of its binding to chromatin. Here, we identified that HSF4 binds to various genomic regions, including the introns and distal parts of protein-coding genes in vivo in mouse lenses, and a substantial numbers of the regions were also occupied by HSF1 and HSF2. HSF4 regulated expression of some genes at a developmental stage when HSF1 and HSF2 expression decreased. Although HSF4 binding did not affect expression of many genes, it induces demethylated status of histone H3K9 on the binding regions. Unexpectedly, a lot of HSF4 targets were induced by heat shock treatment, and HSF4 is required for induction of a set of non-classic heat shock genes in response to heat shock, in part by facilitating HSF1 binding through chromatin modification. These results suggest novel mechanisms of gene regulation controlled by HSF4 in non-classic heat shock response and in lens development.

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.

Role of Induced Fit in Enzyme Specificity: A Molecular Forward/Reverse Switch

Enzyme structures solved with and without bound substrate often show that substrate-induced conformational changes bring catalytic residues into alignment, alter the local environment, and position the substrate for catalysis. Although the structural data are compelling, the role of conformational changes in enzyme specificity has been controversial in that specificity is a kinetic property that is not easy to predict based upon structure alone. Recent studies on DNA polymerization have illuminated the role of substrate-induced conformational changes in enzyme specificity by showing that the rate at which the enzyme opens to release the bound substrate is a key kinetic parameter. The slow release of a correct substrate commits it to the forward reaction so that specificity is determined solely by the rate of substrate binding, including the isomerization step, and not by the slower rate of the chemical reaction. In contrast, fast dissociation of an incorrect substrate favors release rather than reaction. Thus, the conformational change acts as a molecular switch to select the right substrate and to recognize and disfavor the reaction of an incorrect substrate. A conformational switch may also favor release rather than reverse reaction of the product.

Physiological and Biochemical Defects in Carboxyl-terminal Mutants of Mitochondrial DNA Helicase

Mitochondrial DNA helicase, also called Twinkle, is essential for mtDNA maintenance. Its helicase domain shares high homology with helicases from superfamily 4. Structural analyses of helicases from this family indicate that carboxyl-terminal residues contribute to NTP hydrolysis required for translocation and DNA unwinding, yet genetic and biochemical information is very limited. Here, we evaluate the effects of overexpression in Drosophila cell culture of variants carrying a series of deletion and alanine substitution mutations in the carboxyl terminus and identify critical residues between amino acids 572 and 596 of the 613 amino acid polypeptide that are essential for mitochondrial DNA helicase function in vivo. Likewise, amino acid substitution mutants K574A, R576A, Y577A, F588A, and F595A show dose-dependent dominant-negative phenotypes. Arg-576 and Phe-588 are analogous to the arginine finger and base stack of other helicases, including the bacteriophage T7 gene 4 protein and bacterial DnaB helicase, respectively. We show here that representative human recombinant proteins that are analogous to the alanine substitution mutants exhibit defects in nucleotide hydrolysis. Our findings may be applicable to understand the role of the carboxyl-terminal region in superfamily 4 DNA helicases in general.

Mechanism and Dynamics of Translesion DNA Synthesis Catalyzed by the Escherichia coli Klenow Fragment

Translesion DNA synthesis represents the ability of a DNA polymerase to incorporate and extend beyond damaged DNA. In this report, the mechanism and dynamics by which the Escherichia coli Klenow fragment performs translesion DNA synthesis during the misreplication of an abasic site were investigated using a series of natural and non-natural nucleotides. Like most other high-fidelity DNA polymerases, the Klenow fragment follows the "A-rule" of translesion DNA synthesis by preferentially incorporating dATP opposite the noninstructional lesion. However, several 5-substituted indolyl nucleotides lacking classical hydrogen-bonding groups are incorporated approximately 100-fold more efficiently than the natural nucleotide. In general, analogues that contain large substituent groups in conjunction with significant pi-electron density display the highest catalytic efficiencies ( k cat/ K m) for incorporation. While the measured K m values depend upon the size and pi-electron density of the incoming nucleotide, k cat values are surprisingly independent of both biophysical features. As expected, the efficiency by which these non-natural nucleotides are incorporated opposite templating nucleobases is significantly reduced. This reduction reflects minimal increases in K m values coupled with large decreases in k cat values. The kinetic data obtained with the Klenow fragment are compared to that of the high-fidelity bacteriophage T4 DNA polymerase and reveal distinct differences in the dynamics by which these non-natural nucleotides are incorporated opposite an abasic site. These biophysical differences argue against a unified mechanism of translesion DNA synthesis and suggest that polymerases employ different catalytic strategies during the misreplication of damaged DNA.

Peptide ligands specific to the oxidized form of Escherichia coli thioredoxin

Thioredoxin (Trx) is a highly conserved redox protein involved in several essential cellular processes. In this study, our goal was to isolate peptide ligands to Escherichia coli Trx that mimic protein–protein interactions, specifically the T7 polymerase-Trx interaction. To do this, we subjected Trx to affinity selection against a panel of linear and cysteine-constrained peptides using M13 phage display. A novel cyclized conserved peptide sequence, with a motif of C(D/N/S/T/G)D(S/T)-hydrophobic-C-X-hydrophobic-P, was isolated to Trx. These peptides bound specifically to the E. coli Trx when compared to the human and spirulina homologs. An alanine substitution of the active site cysteines (CGPC) resulted in a significant loss of peptide binding affinity to the Cys-32 mutant. The peptides were also characterized in the context of Trx's role as a processivity factor of the T7 DNA polymerase (gp5). As the interaction between gp5 and Trx normally takes place under reducing conditions, which might interfere with the conformation of the disulfide-bridged peptides, we made use of a 22 residue deletion mutant of gp5 in the thioredoxin binding domain (gp5Δ22) that bypassed the requirements of reducing conditions to interact with Trx. A competition study revealed that the peptide selectively inhibits the interaction of gp5Δ22 with Trx, under oxidizing conditions, with an IC50 of ∼ 10 μM.