In vitro synthesis of uniform poly(dG)-poly(dC) by Klenow exo- fragment of polymerase I

In this paper, we describe a production procedure of the one-to-one double helical complex of poly(dG)–poly(dC), characterized by a well-defined length (up to 10 kb) and narrow size distribution of molecules. Direct evidence of strands slippage during poly(dG)–poly(dC) synthesis by Klenow exo⁻ fragment of polymerase I is obtained by fluorescence resonance energy transfer (FRET). We show that the polymer extension results in an increase in the separation distance between fluorescent dyes attached to 5′ ends of the strands in time and, as a result, losing communication between the dyes via FRET. Analysis of the products of the early steps of the synthesis by high-performance liquid chromatography and mass spectroscopy suggest that only one nucleotide is added to each of the strand composing poly(dG)–poly(dC) in the elementary step of the polymer extension. We show that proper pairing of a base at the 3′ end of the primer strand with a base in sequence of the template strand is required for initiation of the synthesis. If the 3′ end nucleotide in either poly(dG) or poly(dC) strand is substituted for A, the polymer does not grow. Introduction of the T-nucleotide into the complementary strand to permit pairing with A-nucleotide results in the restoration of the synthesis. The data reported here correspond with a slippage model of replication, which includes the formation of loops on the 3′ ends of both strands composing poly(dG)–poly(dC) and their migration over long-molecular distances (μm) to 5′ ends of the strands.     

Binding mode of 4',6-diamidino-2-phenylindole and ethidium to poly(dG).poly(dC).poly(dC)(+) triplex and poly(dG).poly(dC) duplex

Optical spectroscopic properties of 4',6-diamidino-2-phenylindole (DAPI) and ethidium bromide complexed with poly(dG).poly(dC).poly(dC)(+) triplex and poly(dG).poly(dC) duplex were compared in this study. When complexed with both duplex and triplex, ethidium is characterized by hypochromism and a red shift in the absorption spectrum, a complicate induced circular dichroism (CD) band in the polynucleotide absorption region, and a negative reduced linear dichroism signal in both polynucleotide and drug absorption regions. The spectral properties for both duplex- and triplex-bound ethidium are identical and both can be understood by the intercalation binding mode. In contrast, the absorption and CD spectra of DAPI complexed with triplex differ from those of the DAPI-duplex complex, although both complexes can be understood by the intercalation binding mode. Considering that the third strand runs along the major groove of the template duplex, we conclude that the DAPI molecule partially intercalates near the major groove of the duplex, where the third strand can affect its spectroscopic properties.     

Detection of triple-helix related structures adopted by poly(dG)-poly(dC) sequences in supercoiled plasmid DNA.

Negative superhelical strain induces the poly(dG)-poly(dC) sequence to adopt two totally different types of triple-helices, either a dG.dG.dC triplex in the presence of Mg(+)+ at both neutral and acidic pHs or a protonated dC+.dG.dC triplex in the absence of Mg(+)+ ions at acidic pH (1). To examine whether there are still other types of non-B DNA structures formed by the same sequence, we constructed supercoiled plasmid DNAs harboring varying lengths of the poly(dG) tract, and the structures adopted by each supercoiled plasmid DNA were studied with a chemical probe, chloroacetaldehyde. The potential of a poly(dG)-poly(dC) sequence to adopt non-B DNA structures depends critically on the length of the tract. Furthermore, in the presence of Mg(+)+ and at a mildly acidic pH, in addition to the expected dG.dG.dC triplex detected for the poly(dG) tracts of 14 to 30 base pairs (bp), new structures were also detected for the tracts longer than 35 bp. The structure formed by a poly(dG) tract of 45 bp revealed chemical reaction patterns consistent with a dG.dG.dC triplex and protonated dC+.dG.dC triple-helices fused together. This structure lacks single-stranded stretches typical of intramolecular triplexes.     

Dynamics of DNA polymerase I (Klenow fragment) under external force

During DNA synthesis, high-fidelity DNA polymerase (DNAP) translocates processively along the template by utilizing the chemical energy from nucleotide incorporation. Thus, understanding the chemomechanical coupling mechanism and the effect of external mechanical force on replication velocity are the most fundamental issues for high-fidelity DNAP. Here, based on our proposed model, we take Klenow fragment as an example to study theoretically the dynamics of high-fidelity DNAPs such as the replication velocity versus different types of external force, i.e., a stretching force on the template, a backward force on the enzyme and a forward force on the enzyme. Replication velocity as a function of the template tension with only one adjustable parameter is in good agreement with the available experimental data. The replication velocity is nearly independent of the forward force, even at very low dNTP concentration. By contrast, the backward force has a large effect on the replication velocity, especially at high dNTP concentration. A small backward force can increase the replication velocity and an optimal backward force exists at which the replication velocity has maximum value; with any further increase in the backward force the velocity decreases rapidly. These results can be tested easily by future experiments and are aid our understanding of the chemomechanical coupling mechanism and polymerization dynamics of high-fidelity DNAP.     

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.     

Minimal kinetic mechanism for misincorporation by DNA polymerase I (Klenow fragment).

The minimal kinetic mechanism for misincorporation of a single nucleotide (dATP) into a short DNA primer/template (9/20-mer) by the Klenow fragment of DNA polymerase I [KF(exo+)] has been previously published [Kuchta, R. D., Benkovic, P., & Benkovic, S.J. (1988) Biochemistry 27, 6716-6725]. In this paper are presented refinements to this mechanism. Pre-steady-state measurements of correct nucleotide incorporation (dTTP) in the presence of a single incorrect nucleotide (dATP) with excess KF-(exo+) demonstrated that dATP binds to the KF(exo+)-9/20-mer complex in two steps preceding chemistry. Substitution of (alpha S)dATP for dATP yielded identical two-step binding kinetics, removing nucleotide binding as a cause of the elemental effect on the rate of misincorporation. Pyrophosphate release from the ternary species [KF'(exo+)-9A/20-mer-PPi] was found to occur following a rate-limiting conformational change, with this species partitioning equally to either nucleotide via internal pyrophosphorolysis or to misincorporated product. The rate of 9A/20-mer dissociation from the central ternary complex (KF'-9A/20-mer-PPi) was shown to be negligible relative to exonucleolytic editing. Pyrophosphorolysis of the misincorporated DNA product (9A/20-mer), in conjunction with measurement of the rate of dATP misincorporation, permitted determination of the overall equilibrium constant for dATP misincorporation and provided a value similar to that measured for correct incorporation. A step by step comparison of the polymerization catalyzed by the Klenow fragment for correct and incorrect nucleotide incorporation emphasizes that the major source of the enzyme's replicative fidelity arises from discrimination in the actual chemical step and from increased exonuclease activity on the ternary misincorporated product complex owing to its slower passage through the turnover sequence.     

Poly(dG).poly(dC) at neutral and alkaline pH: the formation of triple stranded poly(dG).poly(dG).poly(dC).

Alkaline titrations of different samples of poly(dG).poly(dC) and of the constituent homopolymers poly(dG) and poly(dC) have been performed in 0.15 M NaCl and their CD spectra followed. Sample I contained a slight excess of poly(dC) (52% C: 48% G) and showed a single reversible transition (pK = 11.9) due to the dissociation of double stranded poly(dG).poly(dC). Sample II, containing an excess of poly(dG) (43% C: 57% G), showed two transitions (pK1 = 11.4, PK2 = 11.9) the first one being only partially reversible. Examination of the CD spectra along the alkaline titrations indicated the presence of another hydrogen-bonded complex of higher G content. Mixing curves performed at pH 8 have confirmed the presence of a 2G: 1C complex, besides the double stranded complex. It can be formed in amounts up to 30% by mixing the two homopolymers, alkali treatment and heating. The CD spectra of the two complexes have been computed from the CD data of the mixing curves. This permitted the determination of the concentrations of both complexes and homopolymers in all samples. The ratio of triple to double stranded complex is not only dependent on the G/C ratio of the sample, but also a function of the previous physico-chemical conditions. These results explain the variability of many properties of different poly(dG).poly(dC) samples observed by other workers.     

Exploration of factors driving incorporation of unnatural dNTPS into DNA by Klenow fragment (DNA polymerase I) and DNA polymerase alpha

In order to further understand how DNA polymerases discriminate against incorrect dNTPs, we synthesized two sets of dNTP analogues and tested them as substrates for DNA polymerase α (pol α) and Klenow fragment (exo⁻) of DNA polymerase I (Escherichia coli). One set of analogues was designed to test the importance of the electronic nature of the base. The bases consisted of a benzimidazole ring with one or two exocyclic substituent(s) that are either electron-donating (methyl and methoxy) or electron-withdrawing (trifluoromethyl and dinitro). Both pol α and Klenow fragment exhibit a remarkable inability to discriminate against these analogues as compared to their ability to discriminate against incorrect natural dNTPs. Neither polymerase shows any distinct electronic or steric preferences for analogue incorporation. The other set of analogues, designed to examine the importance of hydrophobicity in dNTP incorporation, consists of a set of four regioisomers of trifluoromethyl benzimidazole. Whereas pol α and Klenow fragment exhibited minimal discrimination against the 5- and 6-regioisomers, they discriminated much more effectively against the 4- and 7-regioisomers. Since all four of these analogues will have similar hydrophobicity and stacking ability, these data indicate that hydrophobicity and stacking ability alone cannot account for the inability of pol α and Klenow fragment to discriminate against unnatural bases. After incorporation, however, both sets of analogues were not efficiently elongated. These results suggest that factors other than hydrophobicity, sterics and electronics govern the incorporation of dNTPs into DNA by pol α and Klenow fragment.     

Kinetic mechanism of DNA polymerase I (Klenow)

The minimal kinetic scheme for DNA polymerization catalyzed by the Klenow fragment of DNA polymerase I (KF) from Escherichia coli has been determined with short DNA oligomers of defined sequence. A key feature of this scheme is a minimal two-step sequence that interconverts the ternary KF.DNAn.dNTP and KF.DNAn+1.PPi complexes. The rate is not limited by the actual polymerization but by a separate step, possibly important in ensuring fidelity [Mizrahi, V., Henrie, R. N., Marlier, J. F., Johnson, K. A., & Benkovic, S. J. (1985) Biochemistry 24, 4010-4018]. Evidence for this sequence is supplied by the observation of biphasic kinetics in single-turnover pyrophosphorolysis experiments (the microscopic reverse of polymerization). Data analysis then provides an estimate of the internal equilibrium constant. The dissociations of DNA, dNTP, and PPi from the various binary and ternary complexes were measured by partitioning (isotope-trapping) experiments. The rate constant for DNA dissociation from KF is sequence dependent and is rate limiting during nonprocessive DNA synthesis. The combination of single-turnover (both directions) and isotope-trapping experiments provides sufficient information to permit a quantitative evaluation of the kinetic scheme for specific DNA sequences.     

Protonated polynucleotide structures, 20. Interaction between poly(dG)-poly(dC) and poly(rC).1.

A study of the interaction between poly(dG)-poly(dC) and poly(rC) demonstrates that, at neutral pH and high ionic strength, there is replacement of the dC strand by poly(rC). At acid pH, formation of a triple-stranded complex which equally may involve the replacement phenomenon is observed. There is no evidence for interaction at neutral pH between poly(dG)-poly(dC) and oligo(rC), while a three-stranded complex is formed at acid pH. These data are consistent with the studies of comparative stabilities of double stranded deoxy or ribo polymers and deoxy-ribo hybrids.     

Mechanism for N-acetyl-2-aminofluorene-induced frameshift mutagenesis by Escherichia coli DNA polymerase I (Klenow fragment)

N-Acetyl-2-aminofluorene (AAF) is a chemical carcinogen that reacts with guanines at the C8 position in DNA to form a structure that interferes with DNA replication. In bacteria, the NarI restriction enzyme recognition sequence (G1G2CG3CC) is a very strong mutational hot spot when an AAF adduct is positioned at G3 of this sequence, causing predominantly a -2 frameshift GC dinucleotide deletion mutation. In this study, templates were constructed that contained an AAF adduct at this position, and primers of different lengths were prepared such that the primer ended one nucleotide before or opposite or one nucleotide after the adduct site. Primer extension and gel shift binding assays were used to study the mechanism of bypass by the Escherichia coli DNA polymerase I (Klenow fragment) in the presence of these templates. Primer extension in the presence of all four dNTPs produced a fully extended product using the unmodified template, while with the AAF-modified template synthesis initially stalled at the adduct site and subsequent synthesis resulted in a product that contained the GC dinucleotide deletion. Extension product and gel shift binding analyses were consistent with the formation of a two-nucleotide bulge structure upstream of the active site of the polymerase after a nucleotide is incorporated across from the adduct. These data support a model in which the AAF adduct in the NarI sequence specifically induces a structure upstream of the polymerase active site that leads to the GC frameshift mutation and that it is this structure that allows synthesis past the adduct to occur.     

Mechanism of DNA replication fidelity for three mutants of DNA polymerase I: Klenow fragment KF(exo+), KF(polA5), and KF(exo-)

Inhibition of the pre-steady-state burst of nucleotide incorporation by a single incorrect nucleotide (nucleotide discrimination) was measured with the Klenow fragment of DNA polymerase I [KF(exo+)]. For the eight mispairs studied on three DNA sequences, only low levels of discrimination ranging from none to 23-fold were found. The kinetics of dNTP incorporation into the 9/20-mer at low nucleotide concentrations was also determined. A limit of greater than or equal to 250 s-1 was placed on the nucleotide off-rate from the KF(exo+)-9/20-dTTP complex in accord with nucleotide binding being at equilibrium in the overall kinetic sequence. The influence of the relatively short length of the 9/20-mer on the mechanism of DNA replication fidelity was determined by remeasuring important kinetic parameters on a 30/M13-mer with high homology to the 9/20-mer. Pre-steady-state data on the nucleotide turnover rates, the dATP(alpha S) elemental effect, and the burst of dAMP misincorporation into the 30/M13-mer demonstrated that the kinetics were not affected by the length of the DNA primer/template. The effects on fidelity of two site-specific mutations, KF(polA5) and KF(exo-), were also examined. KF(polA5) showed an increased rate of DNA dissociation and a decreased rate of polymerization resulting in less processive DNA synthesis. Nevertheless, with at least one misincorporation event, that of dAMP into the 9/20-mer, KF(polA5) displays an increased replication fidelity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of 8-chlorodeoxyadenosine on DNA synthesis by the Klenow fragment of DNA polymerase I

8-chloro-2'-deoxyadenosine (8-Cl-dAdo) was incorporated into synthetic DNA oligonucleotides to determine its effects on DNA synthesis by the 3'-5' exonuclease-free Klenow fragment of Escherichia coli DNA Polymerase I (KF-). Single nucleotide insertion experiments were used to determine the coding potential of 8-Cl-dAdo in a DNA template. KF- inserted TTP opposite 8-Cl-dAdo in the template, but with decreased efficiency relative to natural deoxyadenosine. Running-start primer extensions with KF- resulted in polymerase pausing at 8-Cl-dAdo template sites during DNA synthesis. The 2'-deoxyribonucleoside triphosphate analogue, 8-Cl-dATP, was incorporated opposite thymidine (T) approximately two-fold less efficiently than dATP.     

Poly(dG)-poly(dC) DNA appears shorter than poly(dA)-poly(dT) and possibly adopts an A-related conformation on a mica surface under ambient conditions

Three types of DNA: approximately 2700 bp polydeoxyguanylic olydeoxycytidylic acid [poly(dG)-poly(dC)], approximately 2700 bp polydeoxyadenylic polydeoxythymidylic acid [poly(dA)-poly(dT)] and 2686 bp linear plasmid pUC19 were deposited on a mica surface and imaged by atomic force microscopy. Contour length measurements show that the average length of poly(dG)-poly(dC) is approximately 30% shorter than that of poly(dA)-poly(dT) and the plasmid. This led us to suggest that individual poly(dG)-poly(dC) molecules are immobilized on mica under ambient conditions in a form which is likely related to the A-form of DNA in contrast to poly(dA)-poly(dT) and random sequence DNA which are immobilized in a form that is related to the DNA B-form.     

Monoclonal antibodies specific for poly(dG) X poly(dC) and poly(dG) X poly(dm5C)

Most duplex DNAs that are in the "B" conformation are not immunogenic. One important exception is poly(dG) X poly(dC), which produces a good immune response even though, by many criteria, it adopts a conventional right-handed helix. In order to investigate what features are being recognized, monoclonal antibodies were prepared against poly(dG) X poly(dC) and the related polymer poly(dG) X poly(dm5C). Jel 72, which is an immunoglobulin G, binds only to poly(dG) X poly(dC), while Jel 68, which is an immunoglobulin M, binds approximately 10-fold more strongly to poly(dG) X poly(dm5C) than to poly(dG) X poly(dC). For both antibodies, no significant interaction could be detected with any other synthetic DNA duplexes including poly[d(Gm5C)] X poly[d(Gm5C)] in both the "B" and "Z" forms, poly[d(Tm5Cm5C)] X poly[d(GGA)], and poly[d(TCC)] X poly[d(GGA)], poly(dI) X poly(dC), or poly(dI) X poly(dm5C). The binding to poly(dG) X poly(dC) was inhibited by ethidium and by disruption of the DNA duplex, confirming that the antibodies were not recognizing single-stranded or multistranded structures. Furthermore, Jel 68 binds significantly to phage XP-12 DNA, which contains only m5C residues and will precipitate this DNA in the absence of a second antibody. The results suggest that (dG)n X (dm5C)n sequences in natural DNA exist in recognizably distinct conformations.     

The crystal structure of d(G-G-G-G-C-C-C-C). A model for poly(dG).poly(dC)

The structure of the DNA oligomer d(G-G-G-G-C-C-C-C) has been determined at a resolution of 2.5 A by single-crystal X-ray methods. There are two strands in the asymmetric unit, and these coil about each other to form a right-handed double-helix of the A-type with Watson-Crick hydrogen bonds between base-pairs. The helix has a shallow minor groove and a deep, water-filled major groove; almost all exposed functional groups on the DNA are hydrated, and 106 ordered solvent molecules have been found. The two d(G-G-G-G).d(C-C-C-C) segments in the octamer exhibit similar and uniform structures, but there is a slight discontinuity at the GpC step between them. A recurring feature of the structure is the overlap of adjacent guanine bases in each GpG step, with the five-membered ring of one guanine stacking on the six-membered ring of its neighbour. There is little or no overlap between adjacent cytosine rings. Conformational parameters for these GpG steps are compared with those from other single-crystal X-ray analyses. In general, GpG steps exhibit high slide, low roll and variable twist. Models for poly(dG).poly(dC) were generated by applying a simple rotation and translation to each of the unmodified d(G-G-G-G).d(C-C-C-C) units. Detailed features of these models are shown to be compatible with various assays of poly(dG).poly(dC) in solution, and are useful in understanding the polymorphic behaviour of this sequence under a variety of experimental conditions.     

Interaction of DNA with the Klenow fragment of DNA polymerase I studied by time-resolved fluorescence spectroscopy

The interaction of a fluorescent duplex DNA oligomer with the Klenow fragment of DNA polymerase I from Escherichia coli has been studied in solution by using time-resolved fluorescence spectroscopy. An aminonaphthalenesulfonate (dansyl) fluorescent probe was linked by a propyl chain to a C5-modified uridine base located at a specific site in the primer strand of the DNA oligomer. The fluorescent oligomer bound tightly to the Klenow fragment (KD = 7.9 nM), and the probe's position within the DNA-protein complex was varied by stepwise elongation of the primer strand upon addition of the appropriate deoxynucleoside triphosphates. The decay of the total fluorescence intensity and the polarization anisotropy were measured with a picosecond laser and a time-correlated single photon counting system. The fluorescence lifetimes, the correlation time for internal rotation, and the angular range of internal rotation varied according to the probe's position within the DNA-protein complex. These results showed that five or six bases of the primer strand upstream of the 3' terminus were in contact with the protein and that within this contact region there were differences in the degree of solvent accessibility and the closeness of contact. Further, a minor binding mode of the DNA-protein complex was identified, on the basis of heterogeneity of the probe environment observed when the probe was positioned seven bases upstream from the primer 3' terminus, which resulted in a distinctive "dip and rise" in the anisotropy decay. Experiments with an epoxy-terminated DNA oligomer and a site-directed mutant protein established that the labeled DNA was binding at the polymerase active site (major form) and at the spatially distinct 3'----5' exonuclease active site (minor form). The abundance of each of these distinct binding modes of the DNA-protein complex was estimated under solution conditions by analyzing the anisotropy decay of the dansyl probe. About 12% of the labeled DNA was bound at the 3'----5' exonuclease site. This method should be useful for investigating the editing mechanism of this important enzyme.     

How DNA travels between the separate polymerase and 3'-5'-exonuclease sites of DNA polymerase I (Klenow fragment)

The polymerase and 3'-5'-exonuclease activities of the Klenow fragment of DNA polymerase I are located on separate structural domains of the protein, separated by about 30 A. To determine whether a DNA primer terminus can move from one active site to the other without dissociation of the enzyme-DNA complex, we carried out reactions on a labeled DNA substrate in the presence of a large excess of unlabeled DNA, to limit observations to a single enzyme-DNA encounter. The results indicated that while Klenow fragment is capable of intramolecular shuttling of a DNA substrate between the two catalytic sites, the intermolecular pathway involving enzyme-DNA dissociation can also be used. Thus, there is nothing in the protein structure or the reaction mechanism that dictates a particular means of moving the DNA substrate. Instead, the use of the intermolecular or the intramolecular pathway is determined by the competition between the polymerase or exonuclease reaction and DNA dissociation. When the substrate has a mispaired primer terminus, DNA dissociation seems generally more rapid than exonucleolytic digestion. Thus, Klenow fragment edits its own polymerase errors by a predominantly intermolecular process, involving dissociation of the enzyme-DNA complex and reassociation of the DNA with the exonuclease site of a second molecule of Klenow fragment.     

Poly(dG)-poly(dC) sequences, under torsional stress, induce an altered DNA conformation upon neighboring DNA sequences

Supercoiled plasmid DNAs (at bacterial superhelical density) harboring the homopurine-homopyrimidine sequence, poly(dG)-poly(dC), were reacted with bromoacetaldehyde (BAA), a reagent that reacts with unpaired DNA bases. Not only did the poly(dG)-poly(dC) sequence react with BAA but, surprisingly, neighboring sequences located 3' to the contiguous G sequences also reacted. The altered conformation in the poly(dG)-poly(dC) sequence and in the neighboring sequence occurred in the same supercoiled plasmid DNA molecule. Furthermore, the occurrence of an "unpaired" conformation in the neighboring sequence is strictly due to a positional effect, since it is observed when the poly(dG)-poly(dC) segment is adjacent to a variety of neighboring sequences.