Interaction of DNA polymerase I (Klenow fragment) with the single-stranded template beyond the site of synthesis

Cocrystal structures of DNA polymerases from the Pol I (or A) family have provided only limited information about the location of the single-stranded template beyond the site of nucleotide incorporation, revealing contacts with the templating position and its immediate 5' neighbor. No structural information exists for template residues more remote from the polymerase active site. Using a competition binding assay, we have established that Klenow fragment contacts at least the first four unpaired template nucleotides, though the quantitative contribution of any single contact is relatively small. Photochemical cross-linking indicated that the first unpaired template base beyond the primer terminus is close to Y766, as expected, and the two following template bases are close to F771 on the surface of the fingers subdomain. We have constructed point mutations in the region of the fingers subdomain implicated by these experiments. Cocrystal structures of family A DNA polymerases predict contacts between the template strand and S769, F771, and R841, and our DNA binding assays provide evidence for the functional importance of these contacts. Overall, the data are most consistent with the template strand following a path over the fingers subdomain, close to the side chain of R836 and a neighboring cluster of positively charged residues.     

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.     

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.     

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.     

Use of 2-aminopurine fluorescence to examine conformational changes during nucleotide incorporation by DNA polymerase I (Klenow fragment)

We have investigated conformational transitions in the Klenow fragment polymerase reaction by stopped-flow fluorescence using DNA substrates containing the fluorescent reporter 2-aminopurine (2-AP) on the template strand, either at the templating position opposite the incoming nucleotide (designated the 0 position) or 5' to the templating base (the +1 position). By using both deoxy- and dideoxy-terminated primers, we were able to distinguish steps that accompany ternary complex formation from those that occur during nucleotide incorporation. The fluorescence changes revealed two extremely rapid steps that occur early in the pathway for correct nucleotide incorporation. The first, detectable with the 2-AP reporter at the 0 position, occurs within the first few milliseconds and is associated with dNTP binding. This is followed by a rapid step involving relative movement of the +1 base, detectable when the 2-AP reporter is at the +1 position. Finally, when the primer had a 3'-OH, a fluorescence decrease with a rate equal to the rate of nucleotide incorporation was observed with both 0 and +1 position reporters. When the primer was dideoxy-terminated, the only change observed at the rate expected for nucleotide incorporation had a very small amplitude, suggesting that the rate-limiting conformational change does not produce a large fluorescence change, and is therefore unlikely to involve a significant change in the environment of the fluorophore. Fluorescence changes observed during misincorporation were substantially different from those observed during correct nucleotide incorporation, implying that the conformations adopted during correct and incorrect nucleotide incorporation are distinct.     

Interaction of dNTP, pyrophosphate and their analogs with the dNTP-binding sites of E. coli DNA polymerase I Klenow fragment and human DNA polymerase alpha

The 3',5'-exonuclease center of the Klenow fragment of E. coli DNA polymerase I (FK) was selectively blocked by NaF. The latter was shown to forbid the binding of nucleotides and their analogs to the enzyme exonuclease center. In the presence of poly(dT).r(pA)10 template.primer complex and NaF, we observed AMP, ADP, ATP, PPi and dATP to be competitive inhibitors of the FK-catalyzed DNA polymerization. The interactions of the nucleotides with FK and human DNA polymerase alpha were compared to reveal similarity of binding to the DNA polymerizing centers. Structural components of dNTP and PPi playing key roles in forming complexes with pro- and eukaryotic DNA polymerases were identified.     

Production of antibody-tagged enzymes by myeloma cells: application to DNA polymerase I Klenow fragment

Myeloma DNA expression systems can be used for the synthesis and secretion of antibody/enzyme recombinant molecules. Here we describe the construction of a myeloma cell-line that secretes a hapten-specific antibody/enzyme hybrid molecule, in which the antibody Fc portion has been replaced by the Klenow fragment of Escherichia coli DNA polymerase I (PolIk). This Fab-PolIk hybrid molecule is secreted in good yield from the myeloma transfectants, can be purified to homogeneity in a single step on hapten-Sepharose columns, and exhibits PolIk activity as judged by its use in dideoxy nucleotide sequencing. Thus Fab-PolIk can be used for the same purposes as conventional PolIk but has the advantage that it is easily purified to homogeneity in a one-step purification from culture medium.     

Interaction of dNTP-binding sites of human DNA polymerase alpha and The Klenow fragment of Escherichia coli DNA polymerase I with nucleotides, pyrophosphate and their analogs

AMP and NaF each taken separately were shown to activate DNA polymerization catalyzed by Klenow fragment of DNA polymerase I by means of interaction of AMP or NaF with 3'----5'-exonuclease center of the enzyme. In the presence of NaF which is a selective inhibitor of 3'----5'-exonuclease center, AMP is an inhibitor of polymerization competitive with respect to dATP. Ki values and the pattern of inhibition with respect to dATP were determined for AMP, ADP, ATP, carboxymethylphosphonyl-5'-AMP, Pi, PPi, PPPi, methylenediphosphonic acid and its ethylated esters, phosphonoformic acid, phosphonoacetic acid and its ethylated esters as well as for some bicarbonic acids in the reactions of DNA polymerization catalyzed by Klenow fragment of DNA polymerase I (in the presence of NaF) and DNA polymerase alpha from human placenta in the presence of poly(dT) template and r(pA)10 primer. All nucleotides and their analogs were found to be capable of competing with dATP for the active center of the enzyme. Most of the analogs of PPi and phosphonoacetic acid are inhibitors of Klenow fragment competitive with respect to dATP. Nowever these analogs display a mixed-type inhibition in the case of human DNA polymerase alpha. We postulated a similar mechanism of interaction for dNTP with both DNA-polymerases. It is suggested that each phosphate group of PPi makes equal contribution to the interaction with DNA polymerases and that the distance between the phosphate groups is important for this interaction. beta-phosphate of NTP or dNTP is suggested to make negligible contribution to the efficiency of the formation of enzyme complexes with dNTP. beta-phosphate is likely to be an essential point of PPi interaction with the active center of proteins during the cleavage of the alpha-beta-phosphodiester bond of dNTP in the reaction of DNA polymerization.     

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.     

A domain of the Klenow fragment of Escherichia coli DNA polymerase I has polymerase but no exonuclease activity

The Klenow fragment of DNA polymerase I from Escherichia coli has two enzymatic activities: DNA polymerase and 3'-5' exonuclease. The crystal structure showed that the fragment is folded into two distinct domains. The smaller domain has a binding site for deoxynucleoside monophosphate and a divalent metal ion that is thought to identify the 3'-5' exonuclease active site. The larger C-terminal domain contains a deep cleft that is believed to bind duplex DNA. Several lines of evidence suggested that the large domain also contains the polymerase active site. To test this hypothesis, we have cloned the DNA coding for the large domain into an expression system and purified the protein product. We find that the C-terminal domain has polymerase activity (albeit at a lower specific activity than the native Klenow fragment) but no measurable 3'-5' exonuclease activity. These data are consistent with the hypothesis that each of the three enzymatic activities of DNA polymerase I from E. coli resides on a separate protein structural domain.     

Affinity modification of DNA polymerase I from Escherichia coli and its Klenow fragment with nucleotide imidazolides

Affinity modification of E. coli DNA polymerase I and its Klenow fragment by imidazolides of dNMP (Im-dNMP) and dNTP was studied. DNA polymerase activity of DNA polymerase I was reduced by both Im-dNMP and Im-dNTP. However Im-dNTP does not inactivate of the Klenow fragment. The level of covalent labelling of both enzymes by radioactive Im-dNTP did not exceed 0.01 mol of reagent per mol of enzyme. But the deep inactivation of DNA polymerase I by Im-dNTP was observed. It is likely that this inactivation is due to the formation of intramolecular ether followed by phosphorylation of the carboxyl group. This assumption is strongly supported by the increase of the isoelectrical point of DNA polymerase I after its incubation with Im-dNTP in conditions of enzyme inactivation. All data permit us to suggest that the affinity modification of both enzymes by Im-dNMP and covalent labeling by Im-dNTP takes place without complementary binding of dNTP moiety with the template. However inactivation of DNA polymerase I by Im-dNTP occurs only if the dNTP-moiety is complementary to the template in the template.primer complex. It was shown that His residue was phosphorylated by Im-dNMP and Tyr or Ser residues between Met-802 and Met-848 were phosphorylated by Im-dNTP. We suppose that there are two states of DNA polymerase active site for the binding of dNTPs. One of them is independent on the template, in the other state the dNTP hydrogen bond with the template is formed.     

Crystal structures of the Klenow fragment of Thermus aquaticus DNA polymerase I complexed with deoxyribonucleoside triphosphates.

The crystal structures of the Klenow fragment of the Thermus aquaticus DNA polymerase I (Klentaq1) complexed with four deoxyribonucleoside triphosphates (dNTP) have been determined to 2.5 A resolution. The dNTPs bind adjacent to the O helix of Klentaq1. The triphosphate moieties are at nearly identical positions in all four complexes and are anchored by three positively charged residues, Arg659, Lys663, and Arg587, and by two polar residues, His639 and Gln613. The configuration of the base moieties in the Klentaq1/dNTP complexes demonstrates variability suggesting that dNTP binding is primarily determined by recognition and binding of the phosphate moiety. However, when superimposed on the Taq polymerase/blunt end DNA complex structure (Eom et al., 1996), two of the dNTP/Klentaq1 structures demonstrate appropriate stacking of the nucleotide base with the 3' end of the DNA primer strand, suggesting that at least in these two binary complexes, the observed dNTP conformations are functionally relevant.     

Interaction of DNA polymerase I (Klenow fragment) with DNA substrates containing extrahelical bases: implications for proofreading of frameshift errors during DNA synthesis

Frameshift mutagenesis occurs through the misalignment of primer and template strands during DNA synthesis and involves DNA intermediates that contain one or more extrahelical bases in either strand of the DNA substrate. To investigate whether these DNA structures are recognized by the proofreading apparatus of DNA polymerases, time-resolved fluorescence spectroscopy was used to examine the interaction between the Klenow fragment of DNA polymerase I and synthetic DNA primer-templates containing extrahelical bases at defined positions within the template strand. A dansyl probe attached to the DNA was used to measure the fractional occupancies of the polymerase and 3'-5' exonuclease sites of the enzyme for DNA substrates with and without the extrahelical bases. The presence of an extrahelical base at the first position from the primer 3' terminus increased the level of partitioning of the DNA substrates into the 3'-5' exonuclease site by 3-7-fold, relative to the perfectly base-paired primer-template, depending on the identity of the extrahelical base. The ability of different extrahelical bases to promote partitioning of DNA into the 3'-5' exonuclease site decreased in the following order: G > A approximately T > C. The results of partitioning measurements for DNA substrates containing a bulged adenine base at different positions within the template showed that an extrahelical base is recognized up to five bases from the primer 3' terminus. The largest effects were observed for the extrahelical base at the third or fourth positions from the primer terminus, which increased the level of partitioning of DNA into the 3'-5' exonuclease site by 8- and 18-fold, respectively, relative to that of the perfectly base-paired substrate. Steady-state fluorescence measurements of analogous primer-templates containing 2-aminopurine (AP) at the primer 3' terminus indicate that extrahelical bases increase the degree of terminus unwinding, especially when close to the terminus. In addition, steady-state kinetic measurements of removal of AP from the primer-templates indicate that the exonucleolytic cleavage activity of Klenow fragment is correlated with the increased level of partitioning of bulged DNA substrates to the 3'-5' exonuclease site relative to that of properly base-paired DNA. The results of this study indicate that misalignment of primer and template strands to generate an extrahelical base strongly promotes transfer of a DNA substrate to the 3'-5' exonuclease site, suggesting that the premutational intermediates in frameshift mutagenesis are subject to proofreading by the polymerase.     

Identification of residues critical for the polymerase activity of the Klenow fragment of DNA polymerase I from Escherichia coli

The Klenow fragment structure, together with many biochemical experiments, has suggested a region of the protein that may contain the polymerase active site. We have changed 7 amino acid residues within this region by site-directed mutagenesis, yielding 12 mutant proteins which have been purified and analyzed in vitro. The results of steady-state kinetic determinations of Km(dNTP) and kcat for the polymerase reaction, together with measurements of DNA binding affinity, suggest strongly that this study has succeeded in targeting important active site residues. Moreover, the in vitro data allow dissection of the proposed active site region into two clusters of residues that are spatially, as well as functionally, fairly distinct. Mutations in Tyr766, Arg841, and Asn845 cause an increase in Km(dNTP), suggesting that contacts with the incoming dNTP are made in this region. Mutations in the second cluster of residues, Gln849, Arg668, and Asp882, cause a large decrease in kcat, suggesting a role for these residues in catalysis of the polymerase reaction. The DNA-binding properties of mutations at positions 849 and 668 may indicate that the catalytic role of these side chains is associated with their interaction with the DNA substrate. Screening of the mutations in vivo for the classical polA-defective phenotype (sensitivity to DNA damage) demonstrated that a genetic screen of this type may be a reasonable predictor or kcat or of DNA binding affinity in future mutational studies.     

Importance of terminal base pair hydrogen-bonding in 3'-end proofreading by the Klenow fragment of DNA polymerase I

We describe studies aimed at evaluating the physical factors governing the rate of 3'-end proofreading by the Klenow fragment of E. coli DNA polymerase I. Two nonpolar deoxynucleoside isosteres containing 2,4-difluorotoluene (F) and 4-methylbenzimidazole (Z), which are non-hydrogen-bonding shape mimics of thymine and adenine, respectively, are used to investigate the effects of base pair geometry and stability on the rate of this exonuclease activity. Steady-state kinetics measurements show that complementary T.A base pairs at the end of a primer-template duplex are edited 14-40-fold more slowly than mismatches. By contrast, a 3'-end T residue in a T. Z pair is edited at a rate equivalent to that of natural base mismatches despite the fact that it resembles a T.A pair in structure. Similarly, the A in an A.F pair is edited as rapidly as a mismatched pair despite its close structural mimicry of an A.T pair. Interestingly, when the base pairs are reversed and F or Z is located at the 3'-end, they are edited more slowly, possibly implicating specific interactions between the exonuclease domain and the base of the nucleotide being edited. Finally, thermal denaturation studies are carried out to investigate the relationship between editing and the ease of unwinding of the duplex. The rapid editing of bases opposite F or Z residues at the duplex terminus seems to correlate well with the stability of these base pairs when placed in a context resembling a primer-template duplex. In general, the rate of 3'-end editing appears to be governed by the rate of fraying of the DNA terminal pair, and base pair geometry appears to have little effect.     

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.     

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.