Using 2-aminopurine fluorescence to detect base unstacking in the template strand during nucleotide incorporation by the bacteriophage T4 DNA polymerase

The fluorescence of the base analogue 2-aminopurine (2AP) was used to detect physical changes in the template strand during nucleotide incorporation by the bacteriophage T4 DNA polymerase. Fluorescent enzyme-DNA complexes were formed with 2AP placed in the template strand opposite the primer terminus (the n position) and placed one template position 5' to the primer terminus (the n + 1 position). The fluorescence enhancement for 2AP at the n position was shown to be due to formation of the editing complex, which indicates that the 2AP-T terminal base pair is recognized primarily as a mismatch. 2AP fluorescence at the n + 1 position, however, was a reporter for DNA interactions in the polymerase active center that induce intrastrand base unstacking. T4 DNA polymerase produced base unstacking at the n + 1 position following formation of the phosphodiester bond. Thus, the increase in fluorescence intensity for 2AP at the n + 1 position could be used to measure the nucleotide incorporation rate in primer extension reactions in which 2AP was placed initially at the n + 2 position. Primer extension occurred at the rate of about 314 s(-1). The amount of base unstacking at the template n + 1 position was sensitive to the local DNA sequence. More base unstacking was detected for DNA substrates with an A-T base pair at the primer terminus compared to C-G or G-C base pairs. Since proofreading is also increased by A-T base pairs compared to G-C base pairs at the primer terminus, we propose that base unstacking may provide an opportunity for the DNA polymerase to reexamine the primer terminus.     

Using 2-aminopurine fluorescence to detect bacteriophage T4 DNA polymerase-DNA complexes that are important for primer extension and proofreading reactions

The fluorescence of the base analogue 2-aminopurine (2AP) was used to probe bacteriophage T4 DNA polymerase-induced conformational changes in the template strand produced during the nucleotide incorporation and proofreading reactions. 2AP fluorescence in DNA is quenched by 2AP interactions with neighboring bases, but T4 DNA polymerase binding to DNA substrates labeled with 2AP in the templating position produces large increases in fluorescence intensity. Fluorescence lifetime studies were performed to characterize the fluorescent complexes. Three fluorescence lifetime components were observed for unbound DNA substrates as reported previously, but T4 DNA polymerase binding modulated the amplitudes of these components and created a new, highly fluorescent 10.5 ns component. Experimental evidence for correlation of fluorescence lifetimes with functionally distinct complexes was obtained by forming complexes under different reaction conditions. T4 DNA polymerase complexes were formed with DNA substrates with matched and mismatched primer ends and with A+T- or G+C-rich primer-terminal regions. dTTP was added to binary complexes to form ternary DNA polymerase-DNA-nucleotide complexes. The effect of temperature on complex formation was studied, and complexes were formed with proofreading-defective T4 DNA polymerases. Complexes characterized by the 10.5 ns lifetime were demonstrated to be formed at the crossroads of the primer-extension and proofreading pathways.     

DNA Polymerase �� Substrate Specificity: SIDE CHAIN MODULATION OF THE ��A-RULE��*

Apurinic/apyrimidinic (AP) sites are continuously generated in genomic DNA. Left unrepaired, AP sites represent noninstructional premutagenic lesions that are impediments to DNA synthesis. When DNA polymerases encounter an AP site, they generally insert dAMP. This preferential insertion is referred to as the A-rule. Crystallographic structures of DNA polymerase (pol) β, a family X polymerase, with active site mismatched nascent base pairs indicate that the templating (i.e. coding) base is repositioned outside of the template binding pocket thereby diminishing interactions with the incorrect incoming nucleotide. This effectively produces an abasic site because the template pocket is devoid of an instructional base. However, the template pocket is not empty; an arginine residue (Arg-283) occupies the space vacated by the templating nucleotide. In this study, we analyze the kinetics of pol β insertion opposite an AP site and show that the preferential incorporation of dAMP is lost with the R283A mutant. The crystallographic structures of pol β bound to gapped DNA with an AP site analog (tertrahydrofuran) in the gap (binary complex) and with an incoming nonhydrolyzable dATP analog (ternary complex) were solved. These structures reveal that binding of the dATP analog induces a closed polymerase conformation, an unstable primer terminus, and an upstream shift of the templating residue even in the absence of a template base. Thus, dATP insertion opposite an abasic site and dATP misinsertions have common features.     

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.     

Probing DNA polymerase-DNA interactions: examining the template strand in exonuclease complexes using 2-aminopurine fluorescence and acrylamide quenching

The bacteriophage T4 DNA polymerase forms fluorescent complexes with DNA substrates labeled with 2-aminopurine (2AP) in the template strand; the fluorescence intensity depends on the position of 2AP. When preexonuclease complexes are formed, complexes at the crossroads between polymerase and exonuclease complexes, 2AP in the +1 position in the template strand is fully free of contacts with the adjacent bases as indicated by high fluorescence intensity and a long fluorescence lifetime of about 10.9 ns. Fluorescence intensity decreases for 2AP in the template strand when the primer end is transferred to the exonuclease active center to form exonuclease complexes, which indicates a change in DNA conformation; 2AP can now interact with adjacent bases, which quenches fluorescence emission. Some polymerase-induced base unstacking for 2AP in the template strand in exonuclease complexes is observed but is restricted primarily to the n and +1 positions, which indicates that the DNA polymerase holds the template strand in a way that forces base unstacking only in a small region near the primer terminus. A hold on the template strand will help to maintain the correct alignment of the template and primer strands during proofreading. Acrylamide quenches 2AP fluorescence in preexonuclease and in exonuclease complexes formed with DNA labeled with 2AP in the template strand, which indicates that the template strand remains accessible to solvent in both complexes. These studies provide new information about the conformation of the template strand in exonuclease complexes that is not available from structural studies.     

Influence of DNA structure on DNA polymerase beta active site function: extension of mutagenic DNA intermediates

In the ternary substrate complex of DNA polymerase (pol) beta, the nascent base pair (templating and incoming nucleotides) is sandwiched between the duplex DNA terminus and polymerase. To probe molecular interactions in the dNTP-binding pocket, we analyzed the kinetic behavior of wild-type pol beta on modified DNA substrates that alter the structure of the DNA terminus and represent mutagenic intermediates. The DNA substrates were modified to 1) alter the sequence of the duplex terminus (matched and mismatched), 2) introduce abasic sites near the nascent base pair, and 3) insert extra bases in the primer or template strands to mimic frameshift intermediates. The results indicate that the nucleotide insertion efficiency (k(cat)/K(m), dGTP-dC) is highly dependent on the sequence identity of the matched (i.e. Watson-Crick base pair) DNA terminus (template/primer, G/C approximately A/T > T/A approximately C/G). Mismatches at the primer terminus strongly diminish correct nucleotide insertion efficiency but do not affect DNA binding affinity. Transition intermediates are generally extended more easily than transversions. Most mismatched primer termini decrease the rate of insertion and binding affinity of the incoming nucleotide. In contrast, the loss of catalytic efficiency with homopurine mismatches at the duplex DNA terminus is entirely due to the inability to insert the incoming nucleotide, since K(d)((dGTP)) is not affected. Abasic sites and extra nucleotides in and around the duplex terminus decrease catalytic efficiency and are more detrimental to the nascent base pair binding pocket when situated in the primer strand than the equivalent position in the template strand.     

Using 2-aminopurine fluorescence to measure incorporation of incorrect nucleotides by wild type and mutant bacteriophage T4 DNA polymerases

The ability of wild type and mutant T4 DNA polymerases to discriminate in the utilization of the base analog 2-aminopurine (2AP) and the fluorescence of 2AP were used to determine how DNA polymerases distinguish between correct and incorrect nucleotides. Because T4 DNA polymerase incorporates dTMP opposite 2AP under single-turnover conditions, it was possible to compare directly the kinetic parameters for incorporation of dTMP opposite template 2AP to the parameters for incorporation of dTMP opposite template A without the complication of enzyme dissociation. The most significant difference detected was in the K(d) for dTTP, which was 10-fold higher for incorporation of dTMP opposite template 2AP (approximately 367 microm) than for incorporation of dTMP opposite template A (approximately 31 microm). In contrast, the dTMP incorporation rate was reduced only about 2-fold from about 318 s(-1) with template A to about 165 s(-1) for template 2AP. Discrimination is due to the high selectivity in the initial nucleotide-binding step. T4 DNA polymerase binding to DNA with 2AP in the template position induces formation of a nucleotide binding pocket that is preshaped to bind dTTP and to exclude other nucleotides. If nucleotide binding is hindered, initiation of the proofreading pathway acts as an error avoidance mechanism to prevent incorporation of incorrect nucleotides.     

Local Conformations and Competitive Binding Affinities of Single- and Double-stranded Primer-Template DNA at the Polymerization and Editing Active Sites of DNA Polymerases

In addition to their capacity for template-directed 5′ → 3′ DNA synthesis at the polymerase (pol) site, DNA polymerases have a separate 3′ → 5′ exonuclease (exo) editing activity that is involved in assuring the fidelity of DNA replication. Upon misincorporation of an incorrect nucleotide residue, the 3′ terminus of the primer strand at the primer-template (P/T) junction is preferentially transferred to the exo site, where the faulty residue is excised, allowing the shortened primer to rebind to the template strand at the pol site and incorporate the correct dNTP. Here we describe the conformational changes that occur in the primer strand as it shuttles between the pol and exo sites of replication-competent Klenow and Klentaq DNA polymerase complexes in solution and use these conformational changes to measure the equilibrium distribution of the primer between these sites for P/T DNA constructs carrying both matched and mismatched primer termini. To this end, we have measured the fluorescence and circular dichroism spectra at wavelengths of >300 nm for conformational probes comprising pairs of 2-aminopurine bases site-specifically replacing adenine bases at various positions in the primer strand of P/T DNA constructs bound to DNA polymerases. Control experiments that compare primer conformations with available x-ray structures confirm the validity of this approach. These distributions and the conformational changes in the P/T DNA that occur during template-directed DNA synthesis in solution illuminate some of the mechanisms used by DNA polymerases to assure the fidelity of DNA synthesis.Escherichia coli DNA polymerase (DNAP)² I is a single subunit polymerase that is organized into three functional domains: an N-terminal domain that is associated with 5′ → 3′ exonuclease activity, an intermediate domain that carries the 3′ → 5′ proofreading activity, and a C-terminal domain that is associated with the 5′ → 3′ template-directed polymerization activity. An important role of DNAP I is to remove the RNA primers of the Okazaki fragments formed during lagging strand DNA synthesis in E. coli replication and to fill in the resulting gaps by template-directed DNA synthesis (1). An N-terminal deletion mutant of DNAP I, known as the “large fragment” or Klenow form of the enzyme, contains only the polymerase (pol) and the 3′ → 5′ exonuclease (exo) domains. The Klenow polymerase has served and continues to serve as an excellent model system for isolating and defining general structure-function relationships in polymerases and in the supporting machinery of DNA replication.The main function of the 3′ → 5′ exonuclease activity of DNAP I is to remove misincorporated nucleotide residues from the 3′-end of the primer (2), thus contributing significantly to the overall fidelity of DNA replication (3). Contrary to initial expectations, crystallographic studies showed that the pol and exo active sites are quite far apart in replication polymerases, about 30 Å in Klenow (4). As a consequence, the ability of polymerases to “shuttle” the 3′-end of the primer strand efficiently between the pol and the exo sites in order to rectify misincorporation events during polymerization is critical to maintaining the overall accuracy of template-directed replication. Elucidation of the mechanisms of this shuttling and determination of the factors that control the rates (and equilibria) of the active site switching reaction will certainly increase our understanding of fidelity control by DNA polymerases.An early crystallographic study of the Klenow polymerase complexed with fully paired primer-template (P/T) DNA revealed that 3–4 nt of the 3′-primer terminus had been unwound from the template stand and partitioned into the exo site and that an extended single-stranded DNA (ssDNA) binding pocket of the exo site appeared to make position-specific hydrophobic contacts with the unstacked bases at the 3′-end of the primer (4). A separate crystallographic study of an editing complex confirmed that an ssDNA fragment 4 nt in length was bound at the exo site in the same conformation as seen for the single-stranded 3′-primer sequence unwound from P/T DNA (5). A structure of Klenow polymerase with the DNA bound at the pol site has not yet been reported, although such structures have been obtained for other homologous polymerases, including Klentaq (the “large fragment” of Thermus aquaticus (Taq) DNAP), Bacillus stearothermophilus (Bst) “large fragment” polymerase, and the T7 DNAP (6–8), all of which are members of the polymerase family that includes Klenow.The amino acid residues involved in the binding of DNA at the pol site in these polymerases (determined from co-crystal structures) and those of Klenow (determined by site-directed mutagenesis studies (9, 10)) are highly conserved, suggesting that a similar DNA binding mode at the pol site may apply to all of the DNAP I polymerases. The crystal structure of Klenow revealed that the polymerization domain has a shape reminiscent of a right hand in which the palm, fingers, and thumb domains form the DNA-binding crevice. Structural studies with various DNAP I polymerases in the presence of P/T DNA constructs yielded an “open” binary complex, whereas the addition of the next correct dNTP (as a chain-terminating dideoxy-NTP) resulted in the formation of a catalytically competent “closed” ternary complex (6–8). In the latter complex, the 3′-primer terminus was base-paired with the template DNA, and the templating base was poised for incorporation of the next correct nucleotide. These structures showed that the conformation of the DNA primer terminus bound at the pol site is markedly different from that of the “frayed open” primer observed at the exo site in Klenow (4, 5).Although crystallographic studies have provided a wealth of information about the conformations of the DNA substrates bound at the active sites of DNAP, replication itself is a dynamic process (reviewed in Ref. 11), and it is critical to be able to distinguish between various forms of DNA-polymerase complexes in solution in order to fully understand the mechanistic details of the replication process. A solution approach used by Millar and co-workers (reviewed in Ref. 12) for studying the conformation of DNA in these complexes involved measuring the time-resolved fluorescence anisotropy properties of a dansyl fluorophore attached to a DNA base located 8 bp upstream of the P/T DNA junction. The changes in the lifetime of the fluorophore, which appeared to depend mostly on the local environment occupied by the probe within the protein (i.e. buried versus partially exposed), were correlated with specific binding conformations of the primer to provide an estimate of the fractional occupancy of the pol and the exo sites. Reha-Krantz and co-workers (13) more recently used a related approach, here involving the monitoring of changes in the fluorescent lifetimes of a single 2-aminopurine (2-AP) base (a fluorescent analogue of adenine) site-specifically substituted in the template strand at the P/T junction, to make similar fractional occupancy measurements. However, we note that structural interpretations of these fluorescence experiments relied heavily on the available crystal structures, and it remained to be shown directly that the 3′-end of the primer in P/T DNA constructs assumes the same distribution of conformations when bound to the protein in solution.To get around this problem, as well as to directly investigate the conformations of the primer DNA in both active sites of the Klenow and Klentaq polymerases, we have used a novel CD spectroscopic approach to characterize the solution conformations of primer DNA bound to Klenow and Klentaq DNAPs. Previously, we had shown that CD spectroscopy, in conjunction with fluorescence measurements, can be used to examine changes in local DNA and RNA conformations at 2-AP dimer probes inserted at specified positions within the nucleic acid frameworks of a variety of macromolecular machines functioning in solution (14–16). 2-AP is a structural isomer of adenine that forms base pairs with thymine in DNA (and uridine in RNA), and the substitution of 2-AP for adenine in such bp does not significantly perturb the structure or stability of the resultant double helix. Furthermore, when these probes are used as dimer pairs, the CD spectrum primarily reflects the interaction of the transition dipoles of the two probes themselves and thus the local conformation of the DNA at those positions within the P/T DNA. The characteristic CD and fluorescence signals for 2-AP probes in nucleic acids occur at wavelengths of >300 nm, a spectral region in which the protein and the canonical nucleic acid components of the “macromolecular machines of gene expression” are otherwise transparent. In this study, we have examined the binding of Klenow and Klentaq polymerases to P/T DNA constructs that were designed to be comparable with the nucleic acid components of functioning replication complexes. By examining the low energy CD spectra of site-specifically placed 2-AP probes, we have been able to characterize base conformations at defined positions within the DNA to reveal conformational features of specific DNA bases bound at and near both the pol and the exo active sites of these polymerases. These measurements, in that they directly reflect the actual conformations of the DNA chains bound within the active sites of the functioning polymerase, have also provided a direct means to estimate the equilibrium distributions of primer ends between the two active sites for various P/T DNA constructs.     

Frameshift Deletion by Sulfolobus solfataricus P2 DNA Polymerase Dpo4 T239W Is Selective for Purines and Involves Normal Conformational Change Followed by Slow Phosphodiester Bond Formation

The human DNA polymerase κ homolog Sulfolobus solfataricus DNA polymerase IV (Dpo4) produces “−1” frameshift deletions while copying unmodified DNA and, more frequently, when bypassing DNA adducts. As judged by steady-state kinetics and mass spectrometry, bypass of purine template bases to produce these deletions occurred rarely but with 10-fold higher frequency than with pyrimidines. The DNA adduct 1,N²-etheno-2′-deoxyguanosine, with a larger stacking surface than canonical purines, showed the highest frequency of formation of −1 frameshift deletions. Dpo4 T239W, a mutant we had previously shown to produce fluorescence changes attributed to conformational change following dNTP binding opposite cognate bases (Beckman, J. W., Wang, Q., and Guengerich, F. P. (2008) J. Biol. Chem. 283, 36711–36723), reported similar conformational changes when the incoming dNTP complemented the base following a templating purine base or bulky adduct (i.e. the “+1” base). However, in all mispairing cases, phosphodiester bond formation was inefficient. The frequency of −1 frameshift events and the associated conformational changes were not dependent on the context of the remainder of the sequence. Collectively, our results support a mechanism for −1 frameshift deletions by Dpo4 that involves formation of active complexes via a favorable conformational change that skips the templating base, without causing slippage or flipping out of the base, to incorporate a complementary residue opposite the +1 base, in a mechanism previously termed “dNTP-stabilized incorporation.” The driving force is attributed to be the stacking potential between the templating base and the incoming dNTP base.     

Altering DNA polymerase incorporation fidelity by distorting the dNTP binding pocket with a bulky carcinogen-damaged template

Fidelity of DNA polymerases is predominantly governed by an induced fit mechanism in which the incoming dNTP in the ternary complex fits tightly into a binding pocket whose geometry is determined by the nature of the templating base. However, modification of the template with a bulky carcinogen may alter the dNTP binding pocket and thereby the polymerase incorporation fidelity. High fidelity DNA polymerases, such as bacteriophage T7 DNA polymerase, are predominantly blocked by bulky chemical lesions on the template strand during DNA replication. However, some mutagenic bypass can occur, which may lead to carcinogenesis. Experimental studies have shown that a DNA covalent adduct derived from (+)-anti-BPDE [(+)-(7R,8S,9S,10R)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene], a carcinogenic metabolite of benzo[a]pyrene (BP), primarily blocks Sequenase 2.0, an exo(-) T7 DNA polymerase; however, a mismatched dATP can be preferentially inserted opposite the damaged adenine templating base within the active site of the polymerase [Chary, P., and Lloyd, R. S. (1995) Nucleic Acids Res. 23, 1398-1405]. The goal of this work is to elucidate structural features that contribute to DNA polymerase incorporation fidelity in the presence of this bulky covalent adduct and to interpret the experimental findings on a molecular level. We have carried out molecular modeling and molecular dynamics simulations with AMBER 6.0, investigating a T7 DNA polymerase primer-template closed ternary complex containing this 10S (+)-trans-anti-[BP]-N(6)-dA adduct in the templating position within the polymerase active site. All four incoming dNTPs were studied. The simulations show that the BP ring system fits well into an open pocket on the major groove side of the modified template adenine with anti glycosidic bond conformation, without disturbing critical polymerase-DNA interactions. However, steric hindrance between the BP ring system and the primer-template DNA causes displacement of the modified template adenine, so that the dNTP base binding pocket is enlarged. This alteration can explain the experimentally observed preference for incorporation of dATP opposite this lesion. These studies also rationalize the observed lower probabilities of incorporation of the other three nucleotides. Our results suggest that the differences in incorporation of dGTP, dCTP, and dTTP are due to the effects of imperfect geometric complementarity. Thus, the simulations suggest that altered DNA polymerase incorporation fidelity can result from adduct-induced changes in the dNTP base binding pocket geometry. Furthermore, plausible structural explanations for the observed effects of [BP]-N(6)-dA adduct stereochemistry on the observed stalling patterns are proposed.     

Role of hoogsteen edge hydrogen bonding at template purines in nucleotide incorporation by human DNA polymerase iota

Human DNA polymerase iota (Pol iota) differs from other DNA polymerases in that it exhibits a marked template specificity, being more efficient and accurate opposite template purines than opposite pyrimidines. The crystal structures of Pol iota with template A and incoming dTTP and with template G and incoming dCTP have revealed that in the Pol iota active site, the templating purine adopts a syn conformation and forms a Hoogsteen base pair with the incoming pyrimidine which remains in the anti conformation. By using 2-aminopurine and purine as the templating residues, which retain the normal N7 position but lack the N(6) of an A or the O(6) of a G, here we provide evidence that whereas hydrogen bonding at N(6) is dispensable for the proficient incorporation of a T opposite template A, hydrogen bonding at O(6) is a prerequisite for C incorporation opposite template G. To further analyze the contributions of O(6) and N7 hydrogen bonding to DNA synthesis by Pol iota, we have examined its proficiency for replicating through the (6)O-methyl guanine and 8-oxoguanine lesions, which affect the O(6) and N7 positions of template G, respectively. We conclude from these studies that for proficient T incorporation opposite template A, only the N7 hydrogen bonding is required, but for proficient C incorporation opposite template G, hydrogen bonding at both the N7 and O(6) is an imperative. The dispensability of N(6) hydrogen bonding for proficient T incorporation opposite template A has important biological implications, as that would endow Pol iota with the ability to replicate through lesions which impair the Watson-Crick hydrogen bonding potential at both the N1 and N(6) positions of templating A.     

Dynamics of nucleotide incorporation: snapshots revealed by 2-aminopurine fluorescence studies

Formation of a noncanonical base pair between dFTP, a dTTP analogue that cannot form H bonds, and the fluorescent base analogue 2-aminopurine (2AP) was studied in order to discover how the bacteriophage T4 DNA polymerase selects nucleotides with high accuracy. Changes in 2AP fluorescence intensity provided a spectroscopic reporter of the nucleotide binding reactions, which were combined with rapid-quench, pre-steady-state reactions to measure product formation. These studies supported and extended previous findings that the T4 DNA polymerase binds nucleotides in multiple steps with increasing selectivity. With 2AP in the template position, initial dTTP binding was rapid but selective: K(d(dTTP)) (first step) = 31 microM; K(d(dCTP)) (first step) approximately 3 mM. In studies with dFTP, this step was revealed to have two components: formation of an initial preinsertion complex in which H bonds between bases in the newly forming base pair were not essential, which was followed by formation of a final preinsertion complex in which H bonds assisted. The second nucleotide binding step was characterized by increased discrimination against dTTP binding opposite template 2AP, K(d) (second step) = 367 microM, and additional conformational changes were detected in ternary enzyme-DNA-dTTP complexes, as expected for forming closed complexes. We demonstrate here that the second binding step occurs before formation of the phosphodiester bond. Thus, the high fidelity of nucleotide insertion by T4 DNA polymerase is accomplished by the sequential application of selectivity in first forming accurate preinsertion complexes, and then additional conformational changes are applied that further increase discrimination against incorrect nucleotides.     

Impact of template overhang-binding region of HIV-1 RT on the binding and orientation of the duplex region of the template-primer

Fingers domain of HIV-1 RT is one of the constituents of the dNTP-binding pocket that is involved in binding of both dNTP and the template-primer. In the ternary complex of HIV-1 RT, two residues Trp-24 and Phe-61 located on the β1 and β3, respectively, are seen interacting with N + 1 to N + 3 nucleotides in the template overhang. We generated nonconservative and conservative mutant derivatives of these residues and examined their impact on the template-primer binding and polymerase function of the enzyme. We noted that W24A, F61A, and F61Y and the double mutant (W24A/F61A) were significantly affected in their ability to bind template-primer and also to catalyze the polymerase reaction while W24F remained unaffected. Using a specially designed template-primer with photoactivatable bromo-dU base in the duplex region at the penultimate position to the primer terminus, we demonstrated that F61A, W24A, F61Y as well as the double mutant were also affected in their cross-linking ability with the duplex region of the template-primer. We also isolated the E–TP covalent complexes of these mutants and examined their ability to catalyze single dNTP incorporation onto the immobilized primer terminus. The E–TP covalent complexes from W24F mutant displayed wild-type activity while those from W24A, F61A, F61Y, and the double mutant (W24A/F61A) were significantly impaired in their ability to catalyze dNTP incorporation onto the immobilized primer terminus. This unusual observation indicated that amino acid residues involved in the positioning of the template overhang may also influence the binding and orientation of the duplex region of the template-primer. Molecular modeling studies based on our biochemical results suggested that conformation of both W24 and F61 are interdependent on their interactions with each other, which together are required for proper positioning of the +1 template nucleotide in the binary and ternary complexes.     

Loss of DNA polymerase beta stacking interactions with templating purines, but not pyrimidines, alters catalytic efficiency and fidelity.

Structures of DNA polymerases bound with DNA reveal that the 5'-trajectory of the template strand is dramatically altered as it exits the polymerase active site. This distortion provides the polymerase access to the nascent base pair to interrogate proper Watson-Crick geometry. Upon binding a correct deoxynucleoside triphosphate, alpha-helix N of DNA polymerase beta is observed to form one face of the binding pocket for the new base pair. Asp-276 and Lys-280 stack with the bases of the incoming nucleotide and template, respectively. To determine the role of Lys-280, site-directed mutants were constructed at this position, and the proteins were expressed and purified, and their catalytic efficiency and fidelity were assessed. The catalytic efficiency for single-nucleotide gap filling with the glycine mutant (K280G) was strongly diminished relative to wild type for templating purines (>15-fold) due to a decreased binding affinity for the incoming nucleotide. In contrast, catalytic efficiency was hardly affected by glycine substitution for templating pyrimidines (<4-fold). The fidelity of the glycine mutant was identical to the wild type enzyme for misinsertion opposite a template thymidine, whereas the fidelity of misinsertion opposite a template guanine was modestly altered. The nature of the Lys-280 side-chain substitution for thymidine triphosphate insertion (templating adenine) indicates that Lys-280 "stabilizes" templating purines through van der Waals interactions.     

Conformational changes during normal and error-prone incorporation of nucleotides by a Y-family DNA polymerase detected by 2-aminopurine fluorescence

Y-family polymerases are specialized to carry out DNA synthesis past sites of DNA damage. Their active sites make fewer contacts to their substrates, consistent with the remarkably low fidelity of these DNA polymerases when copying undamaged DNA. We have used DNA containing the fluorescent reporter 2-aminopurine (2-AP) to study the reaction pathway of the Y-family polymerase Dbh. We detected 3 rapid noncovalent steps between binding of a correctly paired dNTP and the rate-limiting step for dNTP incorporation. These early steps resemble those seen with high-fidelity DNA polymerases, such as Klenow fragment, and include a step that may be related to the unstacking of the 5' neighbor of the templating base that is seen in polymerase ternary complex crystal structures. A significant difference between Dbh and high-fidelity polymerases is that Dbh generates no fluorescence changes subsequent to dNTP binding if the primer lacks a 3'OH, suggesting that the looser active site of Y-family polymerases may enforce reliance on the correct substrate structure in order to assemble the catalytic center. Dbh, like other bypass polymerases of the DinB subgroup, generates single-base deletion errors at an extremely high frequency by skipping over a template base that is part of a repetitive sequence. Using 2-AP as a reporter to study the base-skipping process, we determined that Dbh uses a mechanism in which the templating base slips back to pair with the primer terminus while the base that was originally paired with the primer terminus becomes unpaired.     

Unwinding of primer-templates by archaeal family-B DNA polymerases in response to template-strand uracil

Archaeal family-B DNA polymerases bind tightly to deaminated bases and stall replication on encountering uracil in template strands, four bases ahead of the primer-template junction. Should the polymerase progress further towards the uracil, for example, to position uracil only two bases in front of the junction, 3′–5′ proof-reading exonuclease activity becomes stimulated, trimming the primer and re-setting uracil to the +4 position. Uracil sensing prevents copying of the deaminated base and permanent mutation in 50% of the progeny. This publication uses both steady-state and time-resolved 2-aminopurine fluorescence to show pronounced unwinding of primer-templates with Pyrococcus furiosus (Pfu) polymerase–DNA complexes containing uracil at +2; much less strand separation is seen with uracil at +4. DNA unwinding has long been recognized as necessary for proof-reading exonuclease activity. The roles of M247 and Y261, amino acids suggested by structural studies to play a role in primer-template unwinding, have been probed. M247 appears to be unimportant, but 2-aminopurine fluorescence measurements show that Y261 plays a role in primer-template strand separation. Y261 is also required for full exonuclease activity and contributes to the fidelity of the polymerase.     

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.     

The 3′–5′ proofreading exonuclease of archaeal family-B DNA polymerase hinders the copying of template strand deaminated bases

Archaeal family B polymerases bind tightly to the deaminated bases uracil and hypoxanthine in single-stranded DNA, stalling replication on encountering these pro-mutagenic deoxynucleosides four steps ahead of the primer–template junction. When uracil is specifically bound, the polymerase–DNA complex exists in the editing rather than the polymerization conformation, despite the duplex region of the primer-template being perfectly base-paired. In this article, the interplay between the 3′–5′ proofreading exonuclease activity and binding of uracil/hypoxanthine is addressed, using the family-B DNA polymerase from Pyrococcus furiosus. When uracil/hypoxanthine is bound four bases ahead of the primer–template junction (+4 position), both the polymerase and the exonuclease are inhibited, profoundly for the polymerase activity. However, if the polymerase approaches closer to the deaminated bases, locating it at +3, +2, +1 or even 0 (paired with the extreme 3′ base in the primer), the exonuclease activity is strongly stimulated. In these situations, the exonuclease activity is actually stronger than that seen with mismatched primer-templates, even though the deaminated base-containing primer-templates are correctly base-paired. The resulting exonucleolytic degradation of the primer serves to move the uracil/hypoxanthine away from the primer–template junction, restoring the stalling position to +4. Thus the 3′–5′ proofreading exonuclease contributes to the inability of the polymerase to replicate beyond deaminated bases.     

SHAMS: Combining chemical modification of RNA with mass spectrometry to examine polypurine tract-containing RNA/DNA hybrids

Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) has gained popularity as a facile method of examining RNA structure both in vitro and in vivo, exploiting accessibility of the ribose 2′-OH to acylation by N-methylisatoic anhydride (NMIA) in unpaired or flexible configurations. Subsequent primer extension terminates at the site of chemical modification, and these products are fractionated by high-resolution gel electrophoresis. When applying SHAPE to investigate structural features associated with the wild-type and analog-substituted polypurine tract (PPT)–containing RNA/DNA hybrids, their size (20–25 base pairs) rendered primer extension impractical. As an alternative method of detection, we reasoned that chemical modification could be combined with tandem mass spectrometry, relying on the mass increment of RNA fragments containing the NMIA adduct (M_r = 133 Da). Using this approach, we demonstrate both specific modification of the HIV-1 PPT RNA primer and variations in its acylation pattern induced by replacing template nucleotides with a non-hydrogen-bonding thymine isostere. Our selective 2′-hydroxyl acylation analyzed by mass spectrometry strategy (SHAMS) should find utility when examining the structure of small RNA fragments or RNA/DNA hybrids where primer extension cannot be performed.