Complementary dNTP Research Articles

Modulation of DNA Polymerase Noncovalent Kinetic Transitions by Divalent Cations

Replicative DNA polymerases (DNAPs) require divalent metal cations for phosphodiester bond formation in the polymerase site and for hydrolytic editing in the exonuclease site. Me(2+) ions are intimate architectural components of each active site, where they are coordinated by a conserved set of amino acids and functional groups of the reaction substrates. Therefore Me(2+) ions can influence the noncovalent transitions that occur during each nucleotide addition cycle. Using a nanopore, transitions in individual Φ29 DNAP complexes are resolved with single-nucleotide spatial precision and sub-millisecond temporal resolution. We studied Mg(2+) and Mn(2+), which support catalysis, and Ca(2+), which supports deoxynucleoside triphosphate (dNTP) binding but not catalysis. We examined their effects on translocation, dNTP binding, and primer strand transfer between the polymerase and exonuclease sites. All three metals cause a concentration-dependent shift in the translocation equilibrium, predominantly by decreasing the forward translocation rate. Me(2+) also promotes an increase in the backward translocation rate that is dependent upon the primer terminal 3'-OH group. Me(2+) modulates the translocation rates but not their response to force, suggesting that Me(2+) does not affect the distance to the transition state of translocation. Absent Me(2+), the primer strand transfer pathway between the polymerase and exonuclease sites displays additional kinetic states not observed at >1 mm Me(2+). Complementary dNTP binding is affected by Me(2+) identity, with Ca(2+) affording the highest affinity, followed by Mn(2+), and then Mg(2+). Both Ca(2+) and Mn(2+) substantially decrease the dNTP dissociation rate relative to Mg(2+), while Ca(2+) also increases the dNTP association rate.

Substrate Rescue of DNA Polymerase β Containing a Catastrophic L22P Mutation

DNA polymerase (pol) β is a multidomain enzyme with two enzymatic activities that plays a central role in the overlapping base excision repair and single-strand break repair pathways. The high frequency of pol β variants identified in tumor-derived tissues suggests a possible role in the progression of cancer, making the determination of the functional consequences of these variants of interest. Pol β containing a proline substitution for leucine 22 in the lyase domain (LD), identified in gastric tumors, has been reported to exhibit severe impairment of both lyase and polymerase activities. Nuclear magnetic resonance (NMR) spectroscopic evaluations of both pol β and the isolated LD containing the L22P mutation demonstrate destabilization sufficient to result in LD-selective unfolding with minimal structural perturbations to the polymerase domain. Unexpectedly, addition of single-stranded or hairpin DNA resulted in partial refolding of the mutated lyase domain, both in isolation and for the full-length enzyme. Further, formation of an abortive ternary complex using Ca2+ and a complementary dNTP indicates that the fraction of pol β(L22P) containing the folded LD undergoes conformational activation similar to that of the wild-type enzyme. Kinetic characterization of the polymerase activity of L22P pol β indicates that the L22P mutation compromises DNA binding, but nearly wild-type catalytic rates can be observed at elevated substrate concentrations. The organic osmolyte trimethylamine N-oxide (TMAO) is similarly able to induce folding and kinetic activation of both polymerase and lyase activities of the mutant. Kinetic data indicate synergy between the TMAO cosolvent and substrate binding. NMR data indicate that the effect of the DNA results primarily from interaction with the folded LD(L22P), while the effect of the TMAO results primarily from destabilization of the unfolded LD(L22P). These studies illustrate that substrate-induced catalytic activation of pol β provides an optimal enzyme conformation even in the presence of a strongly destabilizing point mutation. Accordingly, it remains to be determined whether this mutation alters the threshold of cellular repair activity needed for routine genome maintenance or whether the “inactive” variant interferes with DNA repair.

Dynamics of the Translocation Step Measured in Individual DNA Polymerase Complexes

Complexes formed between the bacteriophage phi29 DNA polymerase (DNAP) and DNA fluctuate between the pre-translocation and post-translocation states on the millisecond time scale. These fluctuations can be directly observed with single-nucleotide precision in real-time ionic current traces when individual complexes are captured atop the α-hemolysin nanopore in an applied electric field. We recently quantified the equilibrium across the translocation step as a function of applied force (voltage), active-site proximal DNA sequences, and the binding of complementary dNTP. To gain insight into the mechanism of this step in the DNAP catalytic cycle, in this study, we have examined the stochastic dynamics of the translocation step. The survival probability of complexes in each of the two states decayed at a single exponential rate, indicating that the observed fluctuations are between two discrete states. We used a robust mathematical formulation based on the autocorrelation function to extract the forward and reverse rates of the transitions between the pre-translocation state and the post-translocation state from ionic current traces of captured phi29 DNAP-DNA binary complexes. We evaluated each transition rate as a function of applied voltage to examine the energy landscape of the phi29 DNAP translocation step. The analysis reveals that active-site proximal DNA sequences influence the depth of the pre-translocation and post-translocation state energy wells and affect the location of the transition state along the direction of the translocation.

Direct Observation of Translocation in Individual DNA Polymerase Complexes

Complexes of phi29 DNA polymerase and DNA fluctuate on the millisecond time scale between two ionic current amplitude states when captured atop the α-hemolysin nanopore in an applied field. The lower amplitude state is stabilized by complementary dNTP and thus corresponds to complexes in the post-translocation state. We have demonstrated that in the upper amplitude state, the DNA is displaced by a distance of one nucleotide from the post-translocation state. We propose that the upper amplitude state corresponds to complexes in the pre-translocation state. Force exerted on the template strand biases the complexes toward the pre-translocation state. Based on the results of voltage and dNTP titrations, we concluded through mathematical modeling that complementary dNTP binds only to the post-translocation state, and we estimated the binding affinity. The equilibrium between the two states is influenced by active site-proximal DNA sequences. Consistent with the assignment of the upper amplitude state as the pre-translocation state, a DNA substrate that favors the pre-translocation state in complexes on the nanopore is a superior substrate in bulk phase for pyrophosphorolysis. There is also a correlation between DNA sequences that bias complexes toward the pre-translocation state and the rate of exonucleolysis in bulk phase, suggesting that during DNA synthesis the pathway for transfer of the primer strand from the polymerase to exonuclease active site initiates in the pre-translocation state.

Small Mass-Change Detectable Quartz Crystal Microbalance and Its Application to Enzymatic One-Base Elongation on DNA

A highly sensitive 27 MHz quartz crystal microbalance instrument with an automatic flow injection system was developed to obtain realistic minimal frequency noise (±0.05 Hz) and to obtain a stable signal baseline (±1 Hz/h) by controlling the temperature of each part in the quartz crystal microbalance (QCM) system using three Peltier devices with a resolution of ±0.001 °C and by optimizing the flow system to prevent fluctuation of the internal pressure of the QCM. The improved QCM with an automatic flow injection system enabled detection of small mass changes such as binding of biotin to a streptavidin-immobilized QCM with a high signal-to-noise ratio. We also applied this device to enzyme reactions of one-base elongation by DNA polymerase (Klenow fragment, KF). We immobilized dsDNAs including the protruding end of dA, dG, dT, or dC on the QCM electrode and ran complementary dNTP monomers with KF into the QCM flow cell. We could directly detect the enzymatic one-base elongation of DNA as a small mass increase, and we found the difference in the reaction rate for each monomer.

Distinct Complexes of DNA Polymerase I (Klenow Fragment) for Base and Sugar Discrimination during Nucleotide Substrate Selection

During each catalytic cycle, DNA polymerases select deoxyribonucleoside triphosphate (dNTP) substrates complementary to a templating base with high fidelity from a pool that includes noncomplementary dNTPs and both complementary and noncomplementary ribonucleoside triphosphates (rNTPs). The Klenow fragment of Escherichia coli DNA polymerase I (KF) achieves this through a series of conformational transitions that precede the chemical step of phosphodiester bond formation. Kinetic evidence from fluorescence and FRET experiments indicates that discrimination of the base and sugar moieties of the incoming nucleotide occurs in distinct, sequential steps during the selection pathway. Here we show that KF-DNA complexes formed with complementary rNTPs or with noncomplementary nucleotides can be distinguished on the basis of their properties when captured in an electric field atop the α-hemolysin nanopore. The average nanopore dwell time of KF-DNA complexes increased as a function of complementary rNTP concentration. The increase was less than that promoted by complementary dNTP, indicating that the rNTP complexes are more stable than KF-DNA binary complexes but less stable than KF-DNA-dNTP ternary complexes. KF-DNA-rNTP complexes could also be distinguished from KF-DNA-dNTP complexes on the basis of ionic current amplitude. In contrast to complementary rNTPs, noncomplementary dNTPs and rNTPs diminished the average nanopore dwell time of KF-DNA complexes in a concentration-dependent manner, suggesting that binding of a noncomplementary nucleotide keeps the KF-DNA complex in a less stable state. These results imply that nucleotide selection proceeds through a series of complexes of increasing stability in which substrates with the correct moiety promote the forward transitions.

Novel Conformational States in Mutator DNA Polymerases Observed Using Single-Molecule FRET

The remarkable fidelity of DNA polymerases depends largely on their efficient rejection of incorrect nucleotides prior to nucleotide addition. Previously, we used single-molecule FRET to examine fidelity-related conformational transitions preceding nucleotide addition by DNA polymerase I (Klenow fragment). Our experiments distinguished the open and closed conformations that predominate in the binary Pol-DNA and ternary Pol-DNA-dNTP(complementary) complexes, and showed that the unliganded polymerase is highly conformationally flexible. We also showed that ternary complexes with mismatched dNTPs or complementary ribonucleotides form novel FRET species perhaps corresponding to partially closed conformations which may act as kinetic checkpoints crucial for fidelity.Here, we studied how amino acids proximal to the polymerase active site contribute to fidelity by examining the conformational states of polymerase derivatives that act as “mutators”, i.e., have decreased fidelity. For example, an E710A substitution (glutamic acid to alanine) reduces the fidelity through a mechanism that remains unclear. Our studies of a doubly labelled E710A polymerase (Pol[E710A]) showed that the binary complex favors the open conformation more than the wild-type. More intriguingly, addition of the complementary dNTP to a binary Pol[E710A]-DNA complex does not form the closed conformation seen in the ternary complex of the wild-type polymerase; in contrast, and at high nucleotide concentrations, Pol[E710A] adopts a FRET state similar to that in ternary complexes of wild-type polymerase with mismatched dNTPs or complementary ribonucleotides; this state may correspond to a partially closed conformation, and is also adopted by Pol[E710A]-DNA-dNTP(mismatched) complexes, offering a possible explanation for the reduced fidelity associated with E710A. Since Pol[E710A] can perform DNA synthesis, our results raise the question whether polymerisation occurs from a partially closed conformation or a transiently formed closed complex; experiments to address this question are in progress, along with studies of additional mutators.

Distinct Roles of the Active-site Mg2+ Ligands, Asp882 and Asp705, of DNA Polymerase I (Klenow Fragment) during the Prechemistry Conformational Transitions

DNA polymerases catalyze the incorporation of deoxynucleoside triphosphates into a growing DNA chain using a pair of Mg(2+) ions, coordinated at the active site by two invariant aspartates, whose removal by mutation typically reduces the polymerase activity to barely detectable levels. Using two stopped-flow fluorescence assays that we developed previously, we have investigated the role of the carboxylate ligands, Asp(705) and Asp(882), of DNA polymerase I (Klenow fragment) in the early prechemistry steps that prepare the active site for catalysis. We find that neither carboxylate is required for an early conformational transition, reported by a 2-aminopurine probe, that takes place in the open ternary complex after binding of the complementary dNTP. However, the subsequent fingers-closing step requires Asp(882); this step converts the open ternary complex into the closed conformation, creating the active-site geometry required for catalysis. Crystal structures indicate that the Asp(882) position changes very little during fingers-closing; this side chain may therefore serve as an anchor point to receive the dNTP-associated metal ion as the nucleotide is delivered into the active site. The Asp(705) carboxylate is not required until after the fingers-closing step, and we suggest that its role is to facilitate the entry of the second Mg(2+) into the active site. The two early prechemistry steps that we have studied take place normally at very low Mg(2+) concentrations, although higher concentrations are needed for covalent nucleotide addition, consistent with the second metal ion entering the ternary complex after fingers-closing.

The Acyclic 2,4-Diaminopyrimidine Nucleoside Phosphonate Acts as a Purine Mimetic in HIV-1 Reverse Transcriptase DNA Polymerization

The acyclic pyrimidine nucleoside phosphonate (ANP) phosphonylmethoxyethoxydiaminopyrimidine (PMEO-DAPym) differs from other ANPs in that the aliphatic alkyloxy linker is bound to the C-6 of the 2,4-diaminopyrimidine base through an ether bond, instead of the traditional alkyl linkage to the N-1 or N-9 of the pyrimidine or purine base. In this study, we have analyzed the molecular interactions between PMEO-DAPym-diphosphate (PMEO-DAPym-pp) and the active sites of wild-type (WT) and drug-resistant HIV-1 reverse transcriptase (RT). Pre-steady-state kinetic analyses revealed that PMEO-DAPym-pp is a good substrate for WT HIV-1 RT: its catalytic efficiency of incorporation (k(pol)/K(d)) is only 2- to 3-fold less than that of the corresponding prototype purine nucleotide analogs PMEA-pp or (R)PMPA-pp. HIV-1 RT recognizes PMEO-DAPym-pp as a purine base instead of a pyrimidine base and incorporates it opposite to thymine (in DNA) or uracil (in RNA). Molecular modeling demonstrates that PMEO-DAPym-pp fits into the active site of HIV-1 RT without significant perturbation of key amino acid residues and mimics an open incomplete purine ring that allows the canonical Watson-Crick base pairing to be maintained. PMEO-DAPym-pp is incorporated more efficiently than (R)PMPA-pp by mutant K65R HIV-1 RT and is not as efficiently excised as (R)PMPA by HIV-1 RT containing thymidine analog mutations. Overall, the data revealed that PMEO- DAPym represents the prototype compound of a novel class of pyrimidine acyclic nucleoside phosphonates that are recognized as a purine nucleotide and should form the rational basis for the design and development of novel purine nucleo(s)(t)ide mimetics as potential antiviral or antimetabolic agents.

Fingers-Closing and Other Rapid Conformational Changes in DNA Polymerase I (Klenow Fragment) and Their Role in Nucleotide Selectivity

We have developed a FRET-based assay for the fingers-closing conformational transition that occurs when a binary complex of DNA polymerase I (Klenow fragment) with a primer-template binds a complementary dNTP and have used this and other fluorescence assays to place the fingers-closing step within the reaction pathway. Because the rate of fingers-closing was substantially faster than the rate of nucleotide incorporation measured in chemical quench experiments, fingers-closing cannot be the rate-limiting prechemistry step defined by earlier kinetic studies. Experiments using Ca (2+) instead of Mg (2+) as the metal cofactor suggest instead that the prechemistry step may involve a change in metal ion occupancy at the polymerase active site. The use of ribonucleotide substrates shows there is a base discriminating step that precedes fingers-closing. This earlier step, detected by 2-AP fluorescence, is promoted by complementary nucleotides (ribo- as well as deoxyribo-) but is blocked by mismatches. The complementary rNTP blocks the subsequent fingers-closing step. Thus, discrimination against rNTPs occurs during the transition from open to closed conformations, whereas selection against mismatched bases is initiated earlier in the pathway, in the open complex. Mismatched dNTPs accelerate DNA release from the polymerase, suggesting the existence of an early intermediate in which DNA binding is destabilized relative to the binary complex; this could correspond to a conformation that allows an incoming dNTP to preview the template base. The early kinetic checkpoints identified by this study provide an efficient mechanism for the rejection of mismatched bases and ribose sugars and thus enhance polymerase throughput.

Single-molecule and ensemble fluorescence assays for a functionally important conformational change in T7 DNA polymerase

We report fluorescence assays for a functionally important conformational change in bacteriophage T7 DNA polymerase (T7 pol) that use the environmental sensitivity of a Cy3 dye attached to a DNA substrate. An increase in fluorescence intensity of Cy3 is observed at the single-molecule level, reflecting a conformational change within the T7 pol ternary complex upon binding of a dNTP substrate. This fluorescence change is believed to reflect the closing of the T7 pol fingers domain, which is crucial for polymerase function. The rate of the conformational change induced by a complementary dNTP substrate was determined by both conventional stopped-flow and high-time-resolution continuous-flow fluorescence measurements at the ensemble-averaged level. The rate of this conformational change is much faster than that of DNA synthesis but is significantly reduced for noncomplementary dNTPs, as revealed by single-molecule measurements. The high level of selectivity of incoming dNTPs pertinent to this conformational change is a major contributor to replicative fidelity.

Stable Complexes Formed by HIV-1 Reverse Transcriptase at Distinct Positions on the Primer-template Controlled by Binding Deoxynucleoside Triphosphates or Foscarnet

Binding of the next complementary dNTP by the binary complex containing HIV-1 reverse transcriptase (RT) and primer-template induces conformational changes that have been implicated in catalytic function of RT. We have used DNase I footprinting, gel electrophoretic mobility shift, and exonuclease protection assays to characterize the interactions between HIV-1 RT and chain-terminated primer-template in the absence and presence of various ligands. Distinguishable stable complexes were formed in the presence of foscarnet (an analog of pyrophosphate), the dNTP complementary to the first (+1) templating nucleotide or the dNTP complementary to the second (+2) templating nucleotide. The position of HIV-1 RT on the primer-template in each of these complexes is different. RT is located upstream in the foscarnet complex, relative to the +1 complex, and downstream in the +2 complex. These results suggest that HIV-1 RT can translocate along the primer-template in the absence of phosphodiester bond formation. The ability to form a specific foscarnet complex might explain the inhibitory properties of this compound. The ability to recognize the second templating nucleotide has implications for nucleotide misincorporation.

Kinetics of the DNA polymerase pyrococcus kodakaraensis

The polymerase chain reaction is one of the most important reactions in molecular biology. Single stranded DNA is copied in a complex series of steps, at the core of which lies the action of the DNA polymerase. At each nucleotide along the template, the polymerase screens the dNTP pool until it finds the complementary dNTP. The insertion of each dNMP is a balance between high fidelity and rapid elongation. In this study the kinetics of the β type polymerase pyrococcus kodakaraensis (KOD) is analyzed. The kinetics is influenced by reaction conditions such as the dNTP pool composition and temperature. In a previous study by Viljoen et al. [2005, A macroscopic kinetic model for DNA polymerase elongation and high-fidelity nucleotide selection. Computational Biology and Chemistry 29, 101–110], a macroscopic kinetics expression of the polymerase chain reaction has been derived. The model contains four parameters that are intrinsic to a specific polymerase. The experiments to measure the temperature-dependence of the parameters for KOD DNA polymerase are reported. The results indicate that the optimal temperature for an equimolar dNTP pool is 72.5 ∘ C and the optimum temperature shifts to lower temperatures when the dNTP pool composition is biased.

Molecular determinants of multi-nucleoside analogue resistance in HIV-1 reverse transcriptases containing a dipeptide insertion in the fingers subdomain: effect of mutations D67N and T215Y on removal of thymidine nucleotide analogues from blocked DNA primers.

Human immunodeficiency virus type 1 isolates having dipeptide insertions in the fingers subdomain of the reverse transcriptase (RT) show high level resistance to 3 '-azido-3 '-deoxythymidine (AZT) and other nucleoside analogues. Insertions are usually associated with thymidine analogue resistance mutations, such as T215Y. The resistance phenotype correlates with increased ATP-dependent phosphorolytic activity, which facilitates removal of thymidine analogues from inhibitor-terminated primers. In this report, we show that substituting Thr, Ser, or Asn for Tyr-215 in a multidrug-resistant RT, bearing a Ser-Ser insertion between codons 69 and 70, leads to AZT and stavudine resensitization through the loss of the ATP-mediated removal activity. The mutation D67N, which is rarely found in insertion-containing strains, had no effect on excision and a minor influence on resistance. Substituting Tyr-215 had a larger effect than deleting the dipeptide insertion. The presence of both the insertion and mutation T215Y in the wild-type BH10 RT conferred significant ATP-mediated removal activity and moderate resistance to AZT. However, resistance levels and unblocking activities were lower than those observed with the multidrug-resistant enzyme. Removal reactions can be inhibited by the next complementary dNTP. Both Tyr-215 and the dipeptide insertion affect RT-DNA.DNA-dNTP ternary complex formation, an effect that was not detected in the presence of foscarnet. Based on crystal structures of binary and ternary complexes of HIV-1 RT, we propose that Tyr-215 exerts its action by facilitating a proper orientation of the pyrophosphate donor molecule, whereas the effects on dNTP binding are indirect and could be related to significant conformational changes occurring during polymerization.

Structure of DNA Polymerase β with the Mutagenic DNA Lesion 8-Oxodeoxyguanine Reveals Structural Insights into Its Coding Potential

Oxidative damage to DNA generates 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG). During DNA replication and repair synthesis, 8-oxodG can pair with cytosine or adenine. The ability to accurately replicate through this lesion depends on the DNA polymerase. We report the first structure of a polymerase with a promutagenic DNA lesion, 8-oxodG, in the confines of its active site. The modified guanine residue is in an anti conformation and forms Watson-Crick hydrogen bonds with an incoming dCTP. To accommodate the oxygen at C8, the 5′-phosphate backbone of the templating nucleotide flips 180°. Thus, the flexibility of the template sugar-phosphate backbone near the polymerase active site is one parameter that influences the anti-syn equilibrium of 8-oxodG. Our results provide insights into the mechanisms employed by polymerases to select the complementary dNTP.

Multidrug-resistant HIV-1 reverse transcriptase: involvement of ribonucleotide-dependent phosphorolysis in cross-resistance to nucleoside analogue inhibitors.

Human immunodeficiency virus type 1 (HIV-1) strains having a dipeptide insertion between codons 69 and 70 of the viral reverse transcriptase (RT) have been observed in isolates from patients treated with 3'-azido-3'-deoxythymidine (AZT) and other nucleoside analogues. These viruses contain additional mutations related to drug resistance and display reduced susceptibility to most nucleoside analogue inhibitors, including AZT. The mechanism of AZT resistance implies an increased ability of the multidrug-resistant (SS) RT to remove AZT-monophosphate (AZTMP) from blocked primers through a nucleotide-dependent reaction. We show that its higher ATP-dependent phosphorolytic activity is also detectable with primers terminated with 2',3'-didehydro-3'-deoxythymidine-5'-monophosphate (d4TMP) or 2',3'-dideoxythymidine-5'-monophosphate (ddTMP), but is significantly reduced when the dipeptide insertion is deleted. Removal of AZTMP, d4TMP and ddTMP can be inhibited by the next complementary deoxynucleoside triphosphate (dNTP). AZTMP removal reactions catalysed by SS RT were highly resistant to dNTP inhibition (IC(50)>0.25mM), while unblocking of d4TMP- and ddTMP-terminated primers was around tenfold more sensitive to inhibition by the next complementary dNTP. Both SS and mutant 2S0S RTs were able to unblock and extend primers terminated with 2',3'-dideoxycytidine-5'-monophosphate (ddCMP) in the presence of ATP, albeit very poorly. Under these conditions, none of the RTs was able to remove 2',3'-dideoxy-3'-thiacytidine-5'-monophosphate (3TCMP) from a terminated DNA primer. Resistance mediated by ATP-dependent phosphorolysis depends on the intracellular levels of dNTP. High levels as found in transformed cell lines (i.e. H-9, CEM lymphoblasts, SupT1 cells, etc.) may prevent repair of primers terminated with d4TMP. However, ATP-dependent phosphorolysis could be relevant for d4T resistance in cells having low levels of dNTPs. This proposal could explain why insertion-containing HIV-1 variants have been detected in the absence of AZT, during d4T treatment.

Joining of DNA ends bearing non-matching 3′-overhangs

Using the Xenopus egg extract as an in vitro system for double strand break repair, the joining of DNA ends bearing non-complementary, homopolymeric, 4 nt 3′-protruding single strands (“overhangs”) was examined. Such 3′-overhangs can not be filled-in and cannot align and anneal by canonical base pair interactions, thus presenting a special challenge to the repair machinery. The results indicate that two such non-matching 3′-overhangs typically overlap by 2 nt forming non-canonical base pairs, from which the filling-in of the remaining gaps is primed. The repair reaction is inhibited in Ku-depleted extracts. Unexpectedly, with some of the substrates the predominant repair products were joints with no nucleotide loss, suggesting that the two DNA ends aligned without overlap. However, the additional finding that an activity in the egg extract adds one or a few nucleotides to a fraction of the 3′-ends favors a model in which most or all of the zero-loss joints are the net result of 3′-overhang extension and 2 bp overlap formation. The nucleotide addition reaction is stimulated by increasing the concentration of the complementary dNTP or ddNTP in the extract, suggesting a process templated by free nucleotides and the involvement of a DNA polymerase-like activity.

Differential Influence of Nucleoside Analog-resistance Mutations K65R and L74V on the Overall Mutation Rate and Error Specificity of Human Immunodeficiency Virus Type 1 Reverse Transcriptase

Differential Influence of Nucleoside Analog-resistance Mutations K65R and L74V on the Overall Mutation Rate and Error Specificity of Human Immunodeficiency Virus Type 1 Reverse Transcriptase

A conformational change in E. coli DNA polymerase I (Klenow fragment) is induced in the presence of a dNTP complementary to the template base in the active site.

It is well established that the insertion of a nucleotide into a growing DNA chain requires a conformational change in the structure of a DNA polymerase. These enzymes have been shown to bind a primer-template in the open conformation and then upon binding of a complementary dNTP undergo a conformational rearrangement to the closed ternary complex. This movement results in the positioning of the incoming nucleotide in the proper geometry for the nucleophilic attack by the 3'-hydroxyl of the primer. In this work, tryptic digestion experiments were performed to detect this conformational change in the structure of the exonuclease-deficient DNA polymerase I (Klenow fragment). Three distinct digestion patterns were observed: one for the polymerase alone, one for the binary complex with the primer-template, and one for the ternary polymerase-DNA-dNTP complex. The latter conformational change leads to a stable ternary closed complex formation only when the correct nucleotide is present in the reaction mixture. Positioning of nucleotides with incorrect geometry in the protein active site inhibits or eliminates formation of the closed complex. Similarly, this conformational change is inhibited when the primer terminus of the DNA molecule is altered by the presence of the 2'-hydroxyl.

New Human Immunodeficiency Virus, Type 1 Reverse Transcriptase (HIV-1 RT) Mutants with Increased Fidelity of DNA Synthesis: ACCURACY, TEMPLATE BINDING, AND PROCESSIVITY

Infidelity of DNA synthesis by human immunodeficiency virus, type 1 reverse transcriptase (HIV-1 RT) is a presumptive determinant of HIV-1 hypervariability and is incompletely understood at the mechanistic and structural levels. Amino acid substitution at only three residues, including Asp-76 (Kim, B., Hathaway, T. R., and Loeb, L. A. (1996) Biochemistry 37, 5831-5839), is known to increase fidelity. We report here that substitution at Arg-78 can also increase accuracy. Mutant R78A RT showed reduced primer extension in misincorporation assays lacking a complementary dNTP and exhibited a 9-fold decrease in mutation frequency in the M13mp2 lacZ forward mutation assay. Previous structural studies indicate that Arg-78 and Asp-76 lie in a region that interacts with template nucleotides. Interestingly, R78A RT exhibited 6- to 8-fold decreases in binding affinity (K(d)) for RNA and DNA templates relative to wild type RT. In contrast, D76V RT, which also increases fidelity (Kim et al., 1996), showed a 6- to 7-fold increased affinity. The processivity of R78A RT on both RNA and DNA templates was substantially reduced relative to wild type RT, whereas the processivity of D76V RT was increased. We discuss relationships of fidelity, template binding, and processivity in these and other HIV RT mutants.