Realizing directional cloning using sticky ends produced by 3′-5′ exonuclease of Klenow fragment

The Klenow fragment (KF) has been used to make the blunt end as a tool enzyme. Its 5′-3′ polymerase activity can extend the 5′ overhanging sticky end to the blunt end, and 3′-5′ exonuclease activity can cleave the 3′ overhanging sticky end to the blunt end. The blunt end is useful for cloning. Here, we for the first time determined that a sticky end can be made by using the 3′-5′ exonuclease activity of KF. We found that KF can cleave the blunt end into certain sticky ends under controlled conditions. We optimized enzyme cleavage conditions, and characterized the cleaved sticky ends to be mainly 2 nt 5′ overhang. By using these sticky ends, we realized ligation reaction in vitro, and accomplished cloning short oligonucleotides directionally with high cloning efficiency. In some cases, this method can provide sticky end fragments in large scale for subsequent convenient cloning at low cost.     

Photoregulation of DNA polymerase I (Klenow) with caged fluorescent oligodeoxynucleotides

The DNA polymerase reaction by Klenow fragment (KF) was efficiently regulated with UV light using a 25-mer caged fluorescent oligodeoxynucleotide (CFO) as the template. The CFO was functionalized with a fluorescein reporter (Fl) and photocleavable DABSYL quencher moiety (Dab). With Fl and Dab at adjacent cytidines in the middle at the template, KF was blocked from extending a complementary 12-mer primer. Upon UV photolysis of the DABSYL blocking group under aerobic conditions, fluorescein emission was restored and 50% of the primers were fully extended by KF.     

Conformation of DNA-DNA polymerase I complex observed by scanning tunneling microscopy.

The conformation of a complex of a 41 mer/31 mer DNA fragment and the Klenow fragment of DNA polymerase I of Escherichia coli was studied by scanning tunnelling microscopy (STM). The results shows that near two turns of double helix of this DNA fragment was outside of enzyme while another part containing more than one turn of helix and 10 nucleotides single strand was combined with enzyme. The dimension and shape of DNA polymerase I (KF) in complex were different from that of free enzyme. The conformation of DNA-DNA polymerase I (KF) complex and the application of STM in studying structure of complex of DNA polymerase with DNA were discussed.     

A mutant of DNA polymerase I (Klenow fragment) with reduced fidelity

The kinetic parameters governing incorporation of correct and incorrect bases into synthetic DNA duplexes have been investigated for Escherichia coli DNA polymerase I [Klenow fragment (KF)] and for two mutants, Tyr766Ser and Tyr766Phe. Tyr766 is located at the C-terminus of helix O in the DNA-binding cleft of KF. The catalytic efficiency for correct incorporation of dNTP is reduced 5-fold for Tyr766Ser. The catalytic efficiencies of all 12 possible misincorporations have been determined for both KF and Tyr766Ser by using single-turnover kinetic conditions and a form of the enzyme that is devoid of the 3'-5' exonuclease activity because of other single amino acid replacements. Tyr766Ser displays an increased efficiency of misincorporation (a reduction in fidelity) for several of the 12 mismatches. The largest increase in efficiency of misincorporation for Tyr766Ser occurs for the misincorporation of TMP opposite template guanosine, a 44-fold increase. In contrast, the efficiencies of misincorporation of dAMP opposite template A, G, or C are little affected by the mutation. A determination of the kinetic parameters associated with a complete kinetic scheme has been made for Tyr766Ser. The rate of addition of the next correct nucleotide onto a preexisting mismatch is decreased for Tyr766Ser. The fidelity of Tyr766Phe was not substantially different from that of KF for the misincorporations examined, indicating that it is the loss of the phenolic ring of the side chain of Tyr766 that leads to the significant decrease in fidelity. The results indicate that KF actively participates in the reduction of misincorporations during the polymerization event and that Tyr766 plays an important role in maintaining the high fidelity of replication by KF.     

Display of functional nucleic acid polymerase on Escherichia coli surface and its application in directed polymerase evolution

We report a first of its kind functional cell surface display of nucleic acid polymerase and its directed evolution to efficiently incorporate 2′-O-methyl nucleotide triphosphates (2′-OMe-NTPs). In the development of polymerase cell surface display, two autotransporter proteins (Escherichia coli adhesin involved in diffuse adherence and Pseudomonas aeruginosa esterase A [EstA]) were employed to transport and anchor the 68-kDa Klenow fragment (KF) of E. coli DNA polymerase I on the surface of E. coli. The localization and function of the displayed KF were verified by analysis of cell outer membrane fractions, immunostaining, and fluorometric detection of synthesized DNA products. The EstA cell surface display system was applied to evolve KF for the incorporation of 2′-OMe-NTPs and a KF variant with a 50.7-fold increased ability to successively incorporate 2′-OMe-NTPs was discovered. Expanding the scope of cell-surface displayable proteins to the realm of polymerases provides a novel screening tool for tailoring polymerases to diverse application demands in a polymerase chain reaction and sequencing-based biotechnological and medical applications. Especially, cell surface display enables novel polymerase screening strategies in which the heat-lysis step is bypassed and thus allows the screening of mesophilic polymerases with broad application potentials ranging from diagnostics and DNA sequencing to replication of synthetic genetic polymers.     

Novel blunt-end addition reactions catalyzed by DNA polymerase I of Escherichia coli

DNA polymerase I (Klenow fragment) of Escherichia coli catalyzes the addition of deoxynucleotides to 3' hydroxyl termini of blunt-ended DNA fragments. The product of the reaction, which we call +1 addition, is found only in very low yield under conditions that permit editing by the 3'----5' exonuclease activity of the wild-type polymerase. A mutant form of the Klenow fragment that lacks detectable 3'----5' exonuclease activity shows an elevated accumulation of the +1 addition product. The mutant enzyme can use any one of the four dNTPs to carry out the reaction when each precursor is provided individually. However, in the presence of all four dNTPs the addition of dATP is strongly preferred. Suppression of the editing function of the wild-type polymerase through the use of high concentrations of exogenous deoxynucleoside monophosphates also results in a significant increase in the amount of +1 addition product formed. The presence of a high dNMP concentration also alters the specificity of the nucleotide addition reaction carried out by the wild-type enzyme. Thus, in addition to dATP, the dNTP which is complementary to the exogenous deoxynucleoside monophosphate, is also used in the +1 addition reaction. A similar effect of dNMPs on the specificity of nucleotide addition was obtained with the mutant Klenow fragment. These results define two pathways for the +1 addition reaction: one that does not require coding information from the DNA template and a second in which coding information is provided by the exogenous dNMP.     

Kinetic mechanism whereby DNA polymerase I (Klenow) replicates DNA with high fidelity

A complete kinetic scheme describing the polymerization of correct and incorrect dNTPs by the Klenow fragment (KF) of DNA polymerase I has been developed by using short DNA oligomers of defined sequence. The high fidelity arises from a three-stage mechanism. The first stage of discrimination [(1.1 X 10(4-) greater than 1.2 X 10(6]-fold] comes primarily from a dramatically reduced rate of phosphodiester bond formation for incorrect nucleotides, but it also gains a smaller contribution from selective dNTP binding. After phosphodiester bond formation, a conformational change slows dissociation of the incorrect DNA products from KF and, in conjunction with editing by the 3'----5'-exonuclease, increases fidelity 4- greater than 61-fold. Finally, KF polymerizes the next correct dNTP onto a mismatch very slowly, providing a further 6- greater than 340-fold increase in fidelity. Surprisingly, the 3'----5'-exonuclease did not in its hydrolysis reaction differentiate between correctly and incorrectly base-paired nucleotides; rather, an increased lifetime of the enzyme-DNA complex containing the misincorporated base is responsible for discrimination.     

The effect of the 3'',5'' thiophosphoryl linkage on the exonuclease activities of T4 polymerase and the Klenow fragment.

The 3'----5' exonuclease activities of T4 DNA polymerase and the Klenow fragment of Polymerase I towards the phosphoryl and thiophosphoryl 3',5' linkage were examined under comparable conditions of idling-turnover, duplex hydrolysis and turnover during polymerization. With the T4 enzyme there is a negligible effect of thiosubstitution on these activities; with the Klenow fragment there is a greater than one hundred-fold reduction in rate with the thiolinkage for the exonuclease but not polymerization activities. This inability to hydrolyze rapidly the thiophosphoryl linkage extends to the hydrolytic activity of Exonuclease III. The quantitation of the exonuclease activities of these three proteins under various conditions should aid in the successful employment of thiophosphoryl nucleoside triphosphates for their incorporation into DNA.     

Helix formation by the phospholipase A2 38–59 fragment: Influence of chain shortening and dimerization monitored by nmr chemical shifts

The solution structure of a peptide fragment corresponding to the 38–59 region of porcine phospholipase A₂ has been investigated using CD, nmr chemical shifts, and nuclear over-hauser effects (NOEs). This isolated fragment of phospholipase forms an α-helix spanning residues 38–55, very similar to the one found in the native protein, except for residues 56–58, which were helical in the crystal but found random in solution. Addition of triflouro-ethanol (TFE) merely increased helix population but it did not redefine helix limits. To investigate how the folding information, in particular that concerning eventual helix start and stop signals, was coded in this particular amino acid sequence, the helices formed by synthetic peptides reproducing sections of this phospholipase 38–59 fragment, namely 40–59, 42–59, 38–50, and 45–57, were characterized using NOEs and helix populations quantitatively evaluated on different peptide chain segments using nmr chemical shifts in two solvents (H₂O and 30% TFE/H₂O). A set of nmr spectra was also recorded and assigned under denaturing conditions (6Murea) to obtain reliable values for the chemical shifts of each peptide in the random state. Based on chemical shift data, it was concluded that the helix formed by the phospholipase 38–59 fragment was not abruptly, but progressively, destabilized all along its length by successive elimination of residues at the N end, while the removal of residues at the C end affected helix stability more locally and to a lesser extent. These results are consistent with the idea that there are not single residues responsible for helix initiation or helix stability, and they also evidence an asymmetry for contributions to helix stability by residues located at the two chain ends. The restriction of molecular mobility caused by linking with a disulphide bridge at Cys 51 two identical 38–59 peptide chains did not increase helix stability. The helix formed by the covalently formed homodimer was very similar in length and population to that formed by the monomer. © 1994 John Wiley & Sons, Inc.     

Direct determination of DNA nucleotide sequences: structure of a fragment of bacteriophage phiX172 DNA

Methods for the direct determination of nucleotide sequences in DNA have been developed and used to determine the complete primary structure of a fragment of bacteriophage φX174 DNA which is 48 residues in length. This fragment was liberated from φX DNA by digestion at low temperature and high ionic strength with the T4 phage-induced endonuclease IV. The fragment was redigested with endonuclease IV under vigorous conditions and the products fractionated two-dimensionally providing a characteristic endonuclease IV “fingerprint” of the fragment. The Burton (Burton &; Petersen, 1960) depurination reaction was used to characterize the redigestion products and identify the pyrimidine residues at their 5′ and 3′ termini. These oligonucleotide products were then fully sequenced by partial exonuclease digestion with spleen and snake venom phosphodiesterase and analysis of the fractionated digests by base composition, depurination, and 5′ end-group analysis using exonuclease I. Rules for the interpretation of two-dimensional fingerprints of partial exonuclease digests, which rapidly provide sequence information by simple inspection, were also deduced. To derive the complete structure of the fragment, the fully sequenced oligonucleotides were ordered by characterizing large, overlapping, partial endonuclease IV digestion products by means of the depurination reaction. The sequencing methods described are general and may be used for the direct determination of the primary structures of other fragments of DNA.     

A unified kinetic mechanism applicable to multiple DNA polymerases

After extensive studies spanning over half a century, there is little consensus on the kinetic mechanism of DNA polymerases. Using stopped-flow fluorescence assays for mammalian DNA polymerase beta (Pol beta), we have previously identified a fast fluorescence transition corresponding to conformational closing, and a slow fluorescence transition matching the rate of single-nucleotide incorporation. Here, by varying pH and buffer viscosity, we have decoupled the rate of single-nucleotide incorporation from the rate of the slow fluorescence transition, thus confirming our previous hypothesis that this transition represents a conformational event after chemistry, likely subdomain reopening. Analysis of an R258A mutant indicates that rotation of the Arg258 side chain is not rate-limiting in the overall kinetic pathway of Pol beta, yet is kinetically significant in subdomain reopening. We have extended our kinetic analyses to a high-fidelity polymerase, Klenow fragment (KF), and a low-fidelity polymerase, African swine fever virus DNA polymerase X (Pol X), and showed that they follow the same kinetic mechanism as Pol beta, while differing in relative rates of single-nucleotide incorporation and the putative conformational reopening. Our data suggest that the kinetic mechanism of Pol beta is not an exception among polymerases, and furthermore, its delineated kinetic mechanism lends itself as a platform for comparison of the kinetic properties of different DNA polymerases and their mutants.     

Kinetic mechanism of DNA polymerase I (Klenow fragment): identification of a second conformational change and evaluation of the internal equilibrium constant.

In a previously determined minimal kinetic scheme for DNA polymerization catalyzed by the Klenow fragment (KF) of Escherichia coli DNA polymerase I, a nonchemical step that interconverted the KF'.DNAn+1.PPi and KF.DNAn+1PPi complexes was not observed in correct incorporation [Kuchta, R. D., Mizrahi, V., Benkovic, P.A., Johnson, K.A., & Benkovic, S.J. (1987) Biochemistry 26, 8410-8417] but was detected in misincorporation [Kuchta, R. D., Benkovic, P.A., & Benkovic, S.J. (1988) Biochemistry 27, 6716-6725]. In a pulse-chase experiment in this study, a burst amplitude of 100% of the enzyme concentration is observed; under pulse-quench conditions, the burst amplitude is 80%, indicative of the accumulation of the KF'.DNA.dNTP species owing to a slow step subsequent to chemical bond formation. This latter step was unequivocally identified by single-turnover pyrophosphorolysis and pyrophosphate-exchange experiments as one interconverting KF'.DNAn+1.PPi and KF.DNAn+1.PPi. The rate constants for this step in both directions were established through the rate constants for processive synthesis and pyrophosphorolysis. Pyrophosphorolysis of a 3'-phosphorothioate DNA duplex confirmed that the large elemental effect observed previously [Mizrahi, V., Henrie, R. N., Marlier, J.F., Johnson, K.A., & Benkovic, S.J. (1985) Biochemistry 24, 4010-4018] in this direction but not in polymerization is due to a marked decrease in the affinity of KF for the phosphorothioate-substituted duplex and not to the chemical step. The combination of the experimentally measured equilibrium constant for the bound KF.DNA species with the collective kinetic measurements further extends previous insights into the dynamics of the polymerization process catalyzed by KF.     

Potent and selective inhibition of angiotensin AT1 receptor signaling by RGS2: roles of its N-terminal domain

Emerging evidence indicates that R4/B subfamily RGS (regulator of G protein signaling) proteins play roles in functional regulation in the cardiovascular system. In this study, we compared effects of three R4/B subfamily proteins, RGS2, RGS4 and RGS5 on angiotensin AT1 receptor signaling, and investigated roles of the N-terminus of RGS2. In HEK293T cells expressing AT1 receptor stably, intracellular Ca²⁺ responses induced by angiotensin II were much more strongly attenuated by RGS2 than by RGS4 and RGS5. N-terminally deleted RGS2 proteins lost this potent inhibitory effect. Replacement of the N-terminal residues 1-71 of RGS2 with the corresponding residues (1-51) of RGS5 decreased significantly the inhibitory effect. On the other hand, replacement of the residues 1-51 of RGS5 with the residues 1-71 of RGS2 increased the inhibitory effect dramatically. Furthermore, we investigated functional contribution of N-terminal subdomains of RGS2, namely, an N-terminal region (residues 16-55) with an amphipathic α helix domain (the subdomain N1), a probable non-specific membrane-targeting subdomain, and another region (residues 56-71) between the α helix and the RGS box (the subdomain N2), a probable GPCR-recognizing subdomain. RGS2 chimera proteins with the residues 1-33 or 34-52 of RGS5 showed weak inhibitory activity, and either of RGS5 chimera proteins with residues 1-55 or 56-71 of RGS2 showed strong inhibitory effects on AT1 receptor signaling. The present study indicates the essential roles of both N-terminal subdomains for the potent inhibitory activity of RGS2 on AT1 receptor signaling.     

A Structural Model For Sequence-Specific Proflavin-DNA Interactions During In Vitro Frameshift Mutagenesis

Abstract

Molecular models describing intermediates that may lead to proflavin-induced 1 bp deletions during in vitro polymerization by E. coli DNA polymerase I Klenow fragment are proposed. The models provide structural explanations for the fact that the induced frameshifts always occur opposite template bases that are adjacent to 5′ pyrimidines and are based on the underlying hypothesis that the deletions arise because the polymerase passes by a template base without copying it. Because the most frequent mutations are opposite Pu in the template sequence 5′ Py Pu 3′, a single-strand loop-out model was constructed for this sequence and proflavin was added, using structures found in crystalline oligonucleotides and their complexes with proflavin. The model seeks to rationalize the roles of the 5′ pyrimidine and proflavin in facilitating the bypass. Four potential roles for proflavin in mutagenesis are described: 1) stacking on the looped-out base; 2) stacking on the base pair immediately preceding the site of mutation; 3) hydrogen bonding with the 5′ pyrimidine; 4) hydrogen bonding with the phosphate backbone. These models point to the possibility that a number of proflavin-DNA interactions may be involved. In contrast, modeling does not suggest a role for classically intercalated proflavin in frameshift mutagenesis arising during in vitro DNA polymerization.     

Comparison of interactions of 5′-derivatives of deoxyoctathymidylate with human DNA polymerize α and HIV reverse transcriptase

K_m and V_max values for d(pT₈) and its derivatives containing various 5′-end groups were estimated in the reaction of DNA polymerization α catalyzed by DNA polymerase α and HIV-RT. The effect of 5′-end modification of primer is more pronounced in the case of HIV-RT. Strong influence is observed for an intercalating (ethidium) group. The affinity of EtpT₈ is 200-fold higher than that of d(pT₈). Attachment of Phn-, Dnm- and Hem-groups results in the increase of affinity of modified primer from 10 up to 20 times. For DNA polymerase α the influence of modifiers on primer affinity is much weaker. The effect of 5′-end residues on the V_max values is also more pronounced for HIV RT. The way to improve selective interaction of oligonucleotide derivatives with the primer site of HIV RT is suggested.