Analysis of closely related genes by the use of synthetic oligonucleotide probes labeled to a high specific activity

A method for labeling synthetic oligonucleotide probes to high specific activity is described. The method utilizes two partly complementary oligonucleotides that are labeled by a fill-in reaction using the Klenow fragment of DNA polymerase I and four α³²P-nucleoside triphosphates. Such probes can, in combination with Southern blot analysis, be used for routine analysis of individual genes in multigene families.     

A role for DNA polymerase in the specificity of nucleotide incorporation opposite N-acetyl-2-aminofluorene adducts

Escherichia coli DNA polymerase I (Klenow fragment), DNA polymerase α from both calf thymus and human lymphoma cells and DNA polymerase β from calf thymus and Novikoff hepatoma cells can incorporate nucleotides opposite N-guanin-8-yl-acetyl-2-aminofluorene in DNA. The polymerases incorporate dCTP opposite some AAF-dG⁴ lesions when Mg²⁺ is the divalent cation. Substitution of Mn²⁺ for Mg²⁺ broadens the specificity of insertion: E. coli DNA polymerase I (Klenow fragment) also inserts A, and at specific sites G or T; DNA polymerase α inserts any of the four dNTPs with A and C incorporated preferentially to G and T. Polymerase β is specific, inserting mainly C even in the presence of Mn²⁺. The K_m for addition of dATP opposite a lesion by E. coli polymerase I (Klenow fragment) in the presence of Mn²⁺ is about 0.5 mm. dNMPs increase the insertion of nucleotides opposite AAF-dG in the presence of Mg²⁺ and increase both the rate and number of sites at which incorporation occurs in the presence of Mn²⁺. dNTPαS and recA protein increase only the insertion of C.We suppose that the incorporation of dCTP reflects normal base-pairing with the AAF-deoxyguanine in the anti conformation, whereas insertion of the other nucleotides (including some of the C) reflects insertion opposite the AAF adduct in its preferred syn conformation. The fact that the DNA polymerase plays a role in determining the specificity of insertion opposite a lesion terminating DNA synthesis suggests that the spectrum of base substitution mutagenesis seen in vivo may reflect the properties of the protein components, including the polymerase, involved in bypass synthesis.     

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.     

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.     

Chromatin- and nuclei-directed ribonucleic Acid synthesis in sugar beet root

The synthesis of RNA by chromatin-bound RNA polymerase prepared from sugar beet (Beta vulgaris) root tissue is completely dependent on the presence of a divalent metal (Mg²⁺ or Mn²⁺) and the presence of four ribonucleoside triphosphates. Accumulation of labeled acid-insoluble product is inhibited by the addition of RNase and actinomycin D to the reaction. When beet root slices are washed for 25 hours, chromatin-associated RNA polymerase activity increases 7-fold over that of unwashed tissue. This enzyme activity declines with further washing. DNA template availability, as measured by saturating levels of added Escherichia coli RNA polymerase, was also found to follow a pattern similar to that for RNA polymerase. Nearest neighbor frequencies of the RNA synthesized by chromatin isolated from unwashed and washed tissue are different.     

Interactions of replication versus repair DNA substrates with the Pol I DNA polymerases from Escherichia coli and Thermus aquaticus

Different DNA polymerases partition differently between replication and repair pathways. In this study we examine if two Pol I family polymerases from evolutionarily distant organisms also differ in their preferences for replication versus repair substrates. The DNA binding preferences of Klenow and Klentaq DNA polymerases, from Escherichia coli and Thermus aquaticus respectively, have been studied using a fluorescence competition binding assay. Klenow polymerase binds primed-template DNA (the replication substrate) with up to 50× higher affinity than it binds to nicked DNA, DNA with a 2 base single-stranded gap, blunt-ended DNA, or to a DNA end with a 3′ overhang. In contrast, Klentaq binds all of these DNAs almost identically, indicating that Klenow has a stronger ability to discriminate between replication and repair substrates than Klentaq. In contrast, both polymerases bind mismatched primed-template and blunt-ended DNA tighter than they bind matched primed-template DNA, suggesting that these two proteins may share a similar mechanism to identify mismatched DNA, despite the fact that Klentaq has no proofreading ability. In addition, the presence or absence of 5′- or 3′-phosphates has slightly different effects on DNA binding by the two polymerases, but again reinforce Klenow's more effective substrate discrimination capability.     

Studies on the Pathway of Incorporation of 2-Aminopurine into the Deoxyribonucleic Acid of Escherichia coli

A pathway for the incorporation of 2-aminopurine into deoxyribonucleic acid (DNA) was studied in cell-free extracts of Escherichia coli. It was demonstrated that the free base can be converted to the deoxynucleoside, and that the deoxynucleotide can be phosphorylated to the di- and triphosphates and then incorporated into the DNA. From a consideration of the individual reactions in crude extracts, it is likely that the rate-limiting step in this pathway is the formation of the deoxynucleotide. Of especial interest is the observation that 2-aminopurine may be viewed as an analogue of either guanine or adenine, depending on which enzymatic step is being considered. On the one hand, it resembles guanine in that it is specifically converted from the mono- to the diphosphate by guanylate kinase and not by adenylate kinase. On the other hand, it replaces adenine rather than guanine in the DNA synthesized with purified DNA polymerases. E. coli DNA polymerase utilizes aminopurine deoxynucleoside triphosphate as a substrate for DNA synthesis much better than does purified phage T5-induced DNA polymerase and is also much less inhibited by this analogue than the T5 enzyme. These experiments in vitro correlate with known differential effects of 2-aminopurine on E. coli and phage in vivo.     

Metal activation of synthetic and degradative activities of phi 29 DNA polymerase, a model enzyme for protein-primed DNA replication.

Analysis of metal activation on the synthetic and degradative activities of phi 29 DNA polymerase was carried out in comparison with T4 DNA polymerase and Escherichia coli DNA polymerase I (Klenow fragment). In the three DNA polymerases studied, both the polymerization and the 3'----5' exonuclease activity had clear differences in their metal ion requirements. The results obtained support the existence of independent metal binding sites for the synthetic and degradative activities of phi 29 DNA polymerase, according with the distant location of catalytic domains (N-terminal for the 3'----5' exonuclease and C-terminal for DNA polymerization) proposed for both Klenow fragment and phi 29 DNA polymerase. Furthermore, DNA competition experiments using phi 29 DNA polymerase suggested that the main differences observed in the metal usage to activate polymerization may be the consequence of metal-induced changes in the enzyme-DNA interactions, whose strength distinguishes processive and nonprocessive DNA polymerases. Interestingly, the initiation of DNA polymerization using a protein as a primer, a special synthetic activity carried out by phi 29 DNA polymerase, exhibited a strong preference for Mn2+ as metal activator. The molecular basis for this preference is mainly the result of a large increase in the affinity for dATP.     

3'-5' Exonucleolytic activity of DNA polymerases: structural features that allow kinetic discrimination between ribo- and deoxyribonucleotide residues

We have determined rates for the excision of nucleotides from the 3' termini of chimeric DNA-RNA oligonucleotides using the Klenow fragment (KF) and two other DNA polymerases, from phages T4 and T7. For these studies, we synthesized DNA-RNA chimeric oligonucleotides with RNA residues in defined positions. When a ribonucleotide residue was placed at the 3' terminus, all three DNA polymerases removed it at the same rate as they did for substrates composed solely of deoxynucleotide residues. There was a decrease in the excision rate, however, when a ribonucleotide residue was located at the second or third position from the 3' terminus. When both the second and third positions were occupied by ribonucleotide residues, the excision rate for the 3' terminal nucleotide was reduced even further and was almost identical to the rate observed when the DNA polymerases encountered single-stranded RNA. The magnitude of the effect of ribonucleotide residues on the excision rate was lower when Mn(2+) replaced Mg(2+) as the essential divalent cation. Two KF mutations, Y423A and N420A, selectively affected the excision rates for the chimeric substrates. Specifically, Y423A totally abolished the rate reduction when there was a single ribonucleotide residue immediately preceding the 3' terminus, whereas N420A diminished, but did not eliminate, the rate reduction relative to that of wild-type KF when the single ribonucleotide residue occupied either the second or third position from the 3' terminus. These results are consistent with the structure of a KF-ss DNA complex from which it can be deduced, by modeling, that a 2' OH group on the second sugar from the 3' terminus would sterically clash with the Tyr 423 side chain, and a 2' OH group on the third sugar would clash with the side chain of Asn 420. The corresponding mutations in T4 DNA polymerase did not affect the rate of hydrolysis of the chimeric oligonucleotides. Thus, there appears to be a major difference in the kinetic behavior of KF and T4 DNA polymerase with respect to the exonuclease reaction. These results are discussed with respect to their possible biological relevance to DNA replication.     

Wounding-induced Enhancement of the Activity of Chromatin-bound DNA-Dependent RNA Polymerases in Sweet Potato Root

Chromatin fractions were isolated from intact and wounded sweet potato root tissues. The synthesis of RNA by the chromatin fractions was dependent on four ribonucleoside triphosphates and a divalent cation such as Mg²⁺ and Mn²⁺, Mn²⁺ being most effective. Whereas phosphate did not interfere with the polymerase reaction, it was totally blocked by pyrophosphate. The reaction was inhibited by DNase and actinomycin D as well as RNase and trypsin. The RNA polymerases of sweet potato root needed SH-groups for catalysis. Activity of chromatin-bound RNA polymerases (EC 2.7.7.6) promptly increased in the 6 hr after wounding and then decreased gradually up to 24 hr. Under the present experimental conditions it was mostly due to the activity of RNA polymerase I. RNA polymerase II contributed only about 5 to 15% to the total activity. The increase in the activity after wounding was completely inhibited by cycloheximide. Plant hormones such as 2,4-dichlorophenoxyacetic acid, gibberellic acid and dibutyryl cyclic adenosine 3′,5′-monophosphate stimulated the increase in RNA polymerases three to four times after wounding. Ethylene partially suppressed the wound-induced increase of RNA polymerases.     

Family A and family B DNA polymerases are structurally related: evolutionary implications.

In order to establish the evolutionary relationship between the family A and B DNA polymerases, we have closely compared the 3'-->5' exonuclease domains between the Klenow fragment of E.coli DNA polymerase I (a family A DNA polymerase) and the bacteriophage PRD1 DNA polymerase, the smallest member of the DNA polymerase family B. Although the PRD1 DNA polymerase has a smaller 3'-->5' exonuclease domain, its active sites appear to be very similar to those of the Klenow fragment. Site-directed mutagenesis studies revealed that the residues important for the 3'-->5' exonuclease activity, particularly metal binding ligands for the Klenow fragment, are all conserved in the PRD1 DNA polymerase as well. The metal binding ligands are also essential for the strand-displacement activity of the PRD1 DNA polymerase. Based on these results and the studies by others in various systems, we conclude that family A and B DNA polymerases, at least in the 3'-->5' exonuclease domain, are structurally as well as evolutionarily related.     

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.     

Thermodynamics of the DNA Structural Selectivity of the Pol I DNA Polymerases from Escherichia coli and Thermus aquaticus

Understanding the thermodynamics of substrate selection by DNA polymerase I is important for characterizing the balance between replication and repair for this enzyme in vivo. Due to their sequence and structural similarities, Klenow and Klentaq, the large fragments of the Pol I DNA polymerases from Escherichia coli and Thermus aquaticus, are considered functional homologs. Klentaq, however, does not have a functional proofreading site. Examination of the DNA binding thermodynamics of Klenow and Klentaq to different DNA structures: single-stranded DNA (ss-DNA), primer-template DNA (pt-DNA), and blunt-end double-stranded DNA (ds-DNA) show that the binding selectivity pattern is similar when examined across a wide range of salt concentration, but can significantly differ at any individual salt concentration. For both proteins, binding of single-stranded DNA shifts from weakest to tightest binding of the three structures as the salt concentration increases. Both Klenow and Klentaq release two to three more ions when binding to pt-DNA and ds-DNA than when binding to ss-DNA. Klenow exhibits significant differences in the ΔC_p of binding to pt-DNA versus ds-DNA, and a difference in pI for these two complexes, whereas Klentaq does not, suggesting that Klenow and Klentaq discriminate between these two structures differently. Taken together, the data suggest that the two polymerases bind ds-DNA very differently, but that both bind pt-DNA and ss-DNA similarly, despite the absence of a proofreading site in Klentaq.     

Fluorescence of 2-aminopurine reveals rapid conformational changes in the RB69 DNA polymerase-primer/template complexes upon binding and incorporation of matched deoxynucleoside triphosphates

We have used 2-aminopurine (2AP) as a fluorescent probe in the template strand of a 13/20mer primer/template (D) to detect deoxynucleoside triphosphates (N)-dependent conformational changes exhibited by RB69 DNA polymerase (ED) complexes. The rates and amplitudes of fluorescence quenching depend hyperbolically on the [dTTP] when a dideoxy-primer/template (ddP/T) with 2AP as the templating base (n position) is used. No detectable fluorescence changes occur when a ddP/T with 2AP positioned 5′ to the templating base (n + 1 position) is used. With a deoxy-primer/template (dP/T) with 2AP in the n position, a rapid fluorescence quenching occurs within 2 ms, followed by a second, slower fluorescence quenching with a rate constant similar to base incorporation as determined by chemical quench. With a dP/T having 2AP in the n + 1 position, there is a [dNTP]-dependent fluorescence enhancement that occurs at a rate comparable to dNMP incorporation. Collectively, the results favor a minimal kinetic scheme in which population of two distinct biochemical states of the ternary EDN complex precedes the nucleotidyl transfer reaction. Observed differences between dP/T and ddP/T ternary complexes indicate that the 3′ hydroxyl group of the primer plays a critical role in determining the rate constants of transitions that lead to strong deoxynucleoside triphosphate binding prior to chemistry.     

Participation of active-site carboxylates of Escherichia coli DNA polymerase I (Klenow fragment) in the formation of a prepolymerase ternary complex

We have investigated the roles of four active-site carboxylates in the formation of a prepolymerase ternary complex of Escherichia coli DNA polymerase I (Klenow fragment), containing the template-primer and dNTP. The analysis of nine mutant enzymes with conserved and nonconserved substitutions of Asp(705), Glu(710), Asp(882), and Glu(883) clearly shows that both catalytically essential aspartates, Asp(705) and Asp(882), are required for the formation of a stable ternary complex. Of the two glutamates, only Glu(710) is required for ternary complex formation, while Glu(883) does not participate in this process. This investigation also reveals two interesting properties of the Klenow fragment with regard to enzyme-template-primer binary and enzyme-template-primer-dNTP ternary complex formation. These are (a) the significant resistance of enzyme-template-primer-dNTP ternary complexes to the addition of high salt or template-primer challenge and (b) the ability of the Klenow fragment to form ternary complexes in the presence of noncatalytic divalent cations such as Ca(2+), Co(2+), Ni(2+), and Zn(2+).     

Role of the LEXE Motif of Protein-primed DNA Polymerases in the Interaction with the Incoming Nucleotide

The LEXE motif, conserved in eukaryotic type DNA polymerases, is placed close to the polymerization active site. Previous studies suggested that the second Glu was involved in binding a third noncatalytic ion in bacteriophage RB69 DNA polymerase. In the protein-primed DNA polymerase subgroup, the LEXE motif lacks the first Glu in most cases, but it has a conserved Phe/Trp and a Gly preceding that position. To ascertain the role of those residues, we have analyzed the behavior of mutants at the corresponding φ29 DNA polymerase residues Gly-481, Trp-483, Ala-484, and Glu-486. We show that mutations at Gly-481 and Trp-483 hamper insertion of the incoming dNTP in the presence of Mg²⁺ ions, a reaction highly improved when Mn²⁺ was used as metal activator. These results, together with previous crystallographic resolution of φ29 DNA polymerase ternary complex, allow us to infer that Gly-481 and Trp-483 could form a pocket that orients Val-250 to interact with the dNTP. Mutants at Glu-486 are also defective in polymerization and, as mutants at Gly-481 and Trp-483, in the pyrophosphorolytic activity with Mg²⁺. Recovery of both reactions with Mn²⁺ supports a role for Glu-486 in the interaction with the pyrophosphate moiety of the dNTP.     

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.     

Consecutive incorporation of fluorophore-labeled nucleotides by mammalian DNA polymerase β

In the present study, we investigated mammalian polymerases that consecutively incorporate various fluorophore-labeled nucleotides. We found that rat DNA polymerase β (pol β) consecutively incorporated fluorophore-labeled nucleotides to a greater extent than four bacterial polymerases, Sequenase Version 2.0, Vent_R (exo-), DNA polymerase IIIα and the Klenow fragment, and the mammalian polymerases DNA polymerase α and human DNA polymerase δ, under mesophilic conditions. Furthermore, we investigated the kinetics of correct or mismatched incorporation with labeled nucleotides during synthesis by rat pol β. The kinetic parameters K_m and k_cat were measured and used for evaluating: (i) the discrimination against correct pair incorporation of labeled nucleotides relative to unlabeled nucleotides; and (ii) the fidelity for all nucleotide combinations of mismatched pairs in the presence of labeled or unlabeled nucleotides. We also investigated the effect of fluorophore-labeled nucleotides on terminal deoxynucleotidyl transferase activity of rat pol β. We have demonstrated for the first time that mammalian pol β can consecutively incorporate various fluorophore-labeled dNTPs. These findings suggest that pol β is useful for high-density labeling of DNA probes and single-molecule sequencing for high-speed genome analysis.