Synthesis and properties of oligonucleotides containing 2''-deoxynebularine and 2''-deoxyxanthosine.

The preparation of synthetic oligonucleotides containing 2'-deoxynebularine (dN) and 2'-deoxyxanthosine (dX) is described. The thermal stabilities of duplexes containing dX, dN, and 2'-deoxyinosine (dI) base-paired with the four natural bases have been measured. Xanthine base pairs have stabilities at pH 5.5 that are similar to those of dI-containing duplexes at neutral pH. When xanthine is paired with adenine or cytosine an unusual stabilization of the duplex structure is observed at acid pH. Incorporation of base mispairs opposite template xanthine sites were measured using Drosophila DNA polymerase alpha. The relative nucleoside incorporation rates are in the order: T greater than C much greater than A approximately equal to G. These rates do not correlate with relative thermodynamic stabilities of base mispairs with xanthine obtained from Tm measurements: T greater than G greater than A approximately equal to C. We suggest that DNA polymerase misinsertion rates are greatest when the base mispair can be formed in accordance with Watson-Crick as opposed to other base pairing geometries even though other geometries, e.g. wobble, may result in a more stable final DNA product.     

DNA polymerase insertion fidelity. Gel assay for site-specific kinetics

A quantitative assay based on gel electrophoresis is described to measure nucleotide insertion kinetics at an arbitrary DNA template site. The assay is used to investigate kinetic mechanisms governing the fidelity of DNA synthesis using highly purified Drosophila DNA polymerase alpha holoenzyme complex and M13 primer-template DNA. Km and Vmax values are reported for correct insertion of A and misinsertion of G, C, and T opposite a single template T site. The misinsertion frequencies are 2 X 10(-4) for G-T and 5 X 10(-5) for both C-T and T-T relative to normal A-T base pairs. The dissociation constant of the polymerase-DNA-dNTP complex, as measured by Km, plays a dominant role in determining the rates of forming right and wrong base pairs. Compared with Km for insertion of A opposite T (3.7 +/- 0.7 microM), the Km value is 1100-fold greater for misinsertion of G opposite T (4.2 +/- 0.4 mM), and 2600-fold greater for misinsertion of either C or T opposite T (9.8 +/- 4.2 mM). These Km differences indicate that in the enzyme binding site the stability of A-T base pairs is 4.3 kcal/mol greater than G-T pairs and 4.9 kcal/mol greater than C-T or T-T pairs. In contrast to the large differences in Km, differences in Vmax are relatively small. There is only a 4-fold reduction in Vmax for insertion of G opposite T and an 8-fold reduction for C or T opposite T, compared with the correct insertion of A. For the specific template T site investigated, the nucleotide insertion fidelity for Drosophila polymerase alpha seems to be governed primarily by a Km discrimination mechanism.     

Deoxyuridine misincorporation causes site-specific mutational lesions in the lacI gene of Escherichia coli

Spontaneous forward mutation in lacI was analyzed by DNA sequencing in a Dut- strain of E. coli. Hyperuracil incorporation into DNA due to the defect in deoxyuridinetriphosphatase caused a 5-fold increase in mutation frequency. Deletion, duplication and base-substitution frequencies were all enhanced in the Dut- strain. However, the analysis of the specificity of mutation revealed a remarkable site- and class-specificity. For example, base substitutions at a single site, a G:C = greater than A:T transition (Ochre 34) accounted for 55% of the base substitutions recovered. The spontaneous A:T = greater than G:C hotspot at position +6 at the lac operator was also recovered at an enhanced frequency in the Dut- strain where it accounted for 25% of the base substitutions. Many of the deletion and duplication events were recovered more than once; most had endpoints in A/T rich regions. The spontaneous frameshift hotspot involving the gain or loss of 5'-CTGG-3' in a region where this tetramer is tandemly repeated 3 times, was also greatly enhanced. No frameshifts involving a single base pair nor IS1 insertions were identified among the 86 lacI mutants sequenced. The analysis of these events reveals them to be generally consistent with a mechanism involving AP sites generated by the removal of misincorporated uracil by uracil-N-glycosylase. Considering the number of potential AP sites (approximately 1 per 170 base pairs) E. coli is remarkably refractory to mutational consequences of deoxyuridine misincorporation in place of thymidine.     

Sequence dependence of energy transfer in DNA oligonucleotides

The sequence, temperature, concentration, and solvent dependence of singlet energy transfer from normal DNA bases to the 2-aminopurine base in synthesized DNA oligomers were investigated by optical spectroscopy. Transfer was shown directly by a variable fluorescence excitation band at 260-280 nm. Adenine (A) is the most efficient energy donor by an order of magnitude. Stacks of A adjacent to 2AP act as an antenna for 2AP excitation. An interposed G, C, or T base between A and 2AP effectively blocks transfer from A to 2AP. Base stacking facilitates transfer, while base pairing reduces energy transfer slightly. The efficiency is differentially temperature dependent in single- and double-stranded oligomers and is highest below 0 degrees C in samples measured. An efficiency transition occurs well below the melting transition of a double-stranded decamer. The transfer efficiency in the duplex decamer d(CTGA[2AP]TTCAG)(2) is moderately dependent on the sample and salt concentration and is solvent dependent. Transfer at physiological temperature over more than a few bases is improbable, except along consecutive A's, indicating that singlet energy transfer is not a major factor in the localization of UV damage in DNA. These results have features in common with recently observed electron transfer from 2AP to G in oligonucleotides.     

Effects of bulge composition and flanking sequence on the kinking of DNA by bulged bases

We recently showed that bulged bases kink duplex DNA, with the degree of kinking increasing in roughly equal increments as the number of bases in the bulge increases from one to four [Hsieh, C.-H., & Griffith, J.D. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 4833-4837]. Here we have examined the kinking of DNA by single A, C, G, or T bulges with different neighboring base pairs. Synthetic 30 base pair (bp) duplex DNAs containing 2 single-base bulges spaced by 10 bp were ligated head to tail, and their electrophoretic behavior in highly cross-linked gels was examined. All bulge-containing DNAs showed marked electrophoretic retardations as compared to non-bulge-containing DNA. Regardless of the sequence of the flanking base pairs, purine bulges produced greater retardations than pyrimidine bulges. Furthermore, C and T bulges produced the same retardations as did G and A bulges. Bulged DNA containing different flanking base pairs showed marked differences in electrophoretic mobility. For C-bulged DNA, the greatest retardations were observed with G.C neighbors, the least with T.A neighbors, and an intermediate amount with a mixture of neighboring base pairs. For A-bulged DNA, the retardations were greatest with G.C neighbors, less with T.A neighbors, even less with a mixture of neighboring base pairs, and finally least with C.G neighbors. Thus flanking base pairs affect C-bulged DNA and A-bulged DNA differently, and G.C and C.G flanking base pairs were seen to have very different effects. These results imply an important role of base stacking in determining how neighboring base pairs influence the kinking of DNA by a single-base bulge.     

Mutagen specificity and position effects on mutation in T4rII nonsense sites.

In 14 sites in the T4rII region, spontaneous and induced interconversions of ochre (UAA) and opal (UGA) alleles, as well as the reversion of the nonsense sites to r+, were studied. The mutagens employed were 2-aminopurine (2AP), 5-bromouracil (5BU), N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and hydroxylamine (HA). With the test system employed, mutagen specificity (i.e., the preferential induction of A: T leads to G: C or G: C leads to A: T mutation at a given site) can be studied. Simultaneously, the response of similar base pairs at various locations in the same or in different nucleotide triplets throughout the rII region, to a given mutagen, can be compared. 2-Aminopurine can induce transitions of both A: T and G: C base pairs at high rates. This mutagen shows no preference for either direction. Furthermore, there is a correlation between the response to 2AP of an A: T pair occupying a given site, and that of a G: C pair occupying the same site. NTG and HA induce G: C leads to A: T transitions almost exclusively. However, there is a correlation between the low rates of A: T leads to G: C transition induced in each of the various sites by these mutagens and those of G: C leads to A: T. 5-Bromouracil induces transitions from G: C to A: T more readily than from A: T to G: C. With 5BU-induced mutation, there is no correlation between the rates of G: C leads to A: T transitions and those of A: T leads to G: C. In UAA sites, all three adenine:thymidine paris respond to 2AP mutagenesis in a similar pattern, In each position in the triplet, response to 2 AP is correlated with that to 5BU. In UGA sites, there are correlations among the spontaneous as well as the 2AP-, HA- and NTG-induced transition rates. 5BU-induced transition rates are usually not correlated with those induced by other mutagens or with the sponatneous ones.     

Base pairing involving deoxyinosine: implications for probe design.

The thermal stability of oligodeoxyribonucleotide duplexes containing deoxyinosine (I) residues matched with each of the four normal DNA bases were determined by optical melting techniques. The duplexes containing at least one I were obtained by mixing equimolar amounts of an oligonucleotide of sequence dCA3XA3G with one of sequence dCT3YT3G where X and Y were A, C, G, T, or I. Comparison of optical melting curves yielded relative stabilities for the I-containing standard base pairs in an otherwise identical base-pair sequence. I:C pairs were found to be less stable than A:T pairs in these duplexes. Large neighboring-base effects upon stability were observed. For example, when (X,Y) = (I,A), the duplex is eight-fold more stable than when (X,Y) = (A,I). Independent of sequence effects the order of stabilities is: I:C greater than I:A greater than I:T congruent to I:G. This order differs from that of deoxyguanosine which pairs less strongly with dA; otherwise each deoxyinosine base pair is less stable than its deoxyguanosine counterpart in the same sequence environment. Implications of these results for design of DNA oligonucleotide probes are discussed.     

Dynamics of mismatched base pairs in DNA

The structural dynamics of mismatched base pairs in duplex DNA have been studied by time-resolved fluorescence anisotropy decay measurements on a series of duplex oligodeoxynucleotides of the general type d[CGG(AP)GGC].d[GCCXCCG], where AP is the fluorescent adenine analogue 2-aminopurine and X = T, A, G, or C. The anisotropy decay is caused by internal rotations of AP within the duplex, which occur on the picosecond time scale, and by overall rotational diffusion of the duplex. The correlation time and angular range of internal rotation of AP vary among the series of AP.X mismatches, showing that the native DNA bases differ in their ability to influence the motion of AP. These differences are correlated with the strength of base-pairing interactions in the various AP.X mismatches. The interactions are strongest with X = T or C. The ability to discern differences in the strength of base-pairing interactions at a specific site in DNA by observing their effect on the dynamics of base motion is a novel aspect of the present study. The extent of AP stacking within the duplex is also determined in this study since it influences the excited-state quenching of AP. AP is thus shown to be extrahelical in the AP.G mismatch. The association state of the AP-containing oligodeoxynucleotide strand is determined from the temperature-dependent tumbling correlation time. An oligodeoxynucleotide triplex is formed with a particular base sequence in a pH-dependent manner.     

Structures of mismatched base pairs in DNA and their recognition by the Escherichia coli mismatch repair system.

The Escherichia coli mismatch repair system does not recognize and/or repair all mismatched base pairs with equal efficiency: whereas transition mismatches (G X T and A X C) are well repaired, the repair of some transversion mismatches (e.g. A X G or C X T) appears to depend on their position in heteroduplex DNA of phage lambda. Undecamers were synthesized and annealed to form heteroduplexes with a single base-pair mismatch in the centre and with the five base pairs flanking each side corresponding to either repaired or unrepaired heteroduplexes of lambda DNA. Nuclear magnetic resonance (n.m.r.) studies show that a G X A mismatch gives rise to an equilibrium between fully helical and a looped-out structure. In the unrepaired G X A mismatch duplex the latter predominates, while the helical structure is predominant in the case of repaired G X A and G X T mismatches. It appears that the E. coli mismatch repair enzymes recognize and repair intrahelical mismatched bases, but not the extrahelical bases in the looped-out structures.     

Energy barriers and rates of tautomeric transitions in DNA bases: ab initio quantum chemical study

Tautomeric transitions of DNA bases are proton transfer reactions, which are important in biology. These reactions are involved in spontaneous point mutations of the genetic material. In the present study, intrinsic reaction coordinates (IRC) analyses through ab initio quantum chemical calculations have been carried out for the individual DNA bases A, T, G, C and also A:T and G:C base pairs to estimate the kinetic and thermodynamic barriers using MP2/6-31G** method for tautomeric transitions. Relatively higher values of kinetic barriers (about 50-60 kcal/mol) have been observed for the single bases, indicating that tautomeric alterations of isolated single bases are quite unlikely. On the other hand, relatively lower values of the kinetic barriers (about 20-25 kcal/mol) for the DNA base pairs A:T and G:C clearly suggest that the tautomeric shifts are much more favorable in DNA base pairs than in isolated single bases. The unusual base pairing A':C, T':G, C':A or G':T in the daughter DNA molecule, resulting from a parent DNA molecule with tautomeric shifts, is found to be stable enough to result in a mutation. The transition rate constants for the single DNA bases in addition to the base pairs are also calculated by computing the free energy differences between the transition states and the reactants.     

Topography of simian virus 40 A protein-DNA complexes: arrangement of pentanucleotide interaction sites at the origin of replication.

Investigation of the DNA binding properties of the simian virus 40 (SV40) A protein (large T antigen) and the hybrid adenovirus-SV40 D2 protein revealed that both viral proteins protect similar regions of SV40 DNA from digestion by DNase I or methylation by dimethyl sulfate. However, the interaction of D2 protein with DNA was more sensitive to increases of NaCl concentration than was the interaction of wild-type SV40 A protein. Dimethylsulfate footprinting identified 13 DNA pentanucleotide contact sites at the viral origin of replication. The sequences of these sites corresponded to the consensus family 5'-(G greater than T) (A greater than G)GGC-3'. The pentanucleotides were distributed in three regions of origin DNA. Region I contained three pentanucleotide contact sites arranged as direct repetitions encompassing a span of 23 base pairs. In region II, four pentanucleotides were oriented as inverted repetitions that also spanned a total of 23 base pairs. Region III had six recognition pentanucleotides arranged as direct repetitions in a space of 59 base pairs. These fundamental variations in DNA arrangement are likely to determine different patterns of protein binding in each region.     

Changes in DNA Base Sequence Induced by Gamma-Ray Mutagenesis of Lambda Phage and Prophage

Mutations in the cI (repressor) gene were induced by gamma-ray irradiation of lambda phage and of prophage, and 121 mutations were sequenced. Two-thirds of the mutations in irradiated phage assayed in recA host cells (no induction of the SOS response) were G:C to A:T transitions; it is hypothesized that these may arise during DNA replication from adenine mispairing with a cytosine product deaminated by irradiation. For irradiated phage assayed in host cells in which the SOS response had been induced, 85% of the mutations were base substitutions, and in 40 of the 41 base changes, a preexisting base pair had been replaced by an A:T pair; these might come from damaged bases acting as AP (apurinic or apyrimidinic) sites. The remaining mutations were 1 and 2 base deletions. In irradiated prophage, base change mutations involved the substitution of both A:T and of G:C pairs for the preexisting pairs; the substitution of G:C pairs shows that some base substitution mechanism acts on the cell genome but not on the phage. In the irradiated prophage, frameshifts and a significant number of gross rearrangements were also found.     

Base mismatch-specific endonuclease activity in extracts from Saccharomyces cerevisiae.

An endonuclease activity (called MS-nicking) for all possible base mismatches has been detected in the extracts of yeast, Saccharomyces cerevisiae. DNAs with twelve possible base mismatches at one defined position are cleaved at different efficiencies. DNA fragments with A/G, G/A, T/G, G/T, G/G, or A/A mismatches are nicked with greater efficiencies than C/T, T/C, C/A, and C/C. DNA with an A/C or T/T mismatch is nicked with an intermediate efficiency. The MS-nicking is only on one particular DNA strand, and this strand disparity is not controlled by methylation, strand break, or nature of the mismatch. The nicks have been mapped at 2-3 places at second, third, and fourth phosphodiester bonds 5' to the mispaired base; from the time course study, the fourth phosphodiester bond probably is the primary incision site. This activity may be involved in mismatch repair during genetic recombination.     

Thermodynamic studies of base pairing involving 2,6-diaminopurine.

The thermal stabilities of oligodeoxyribonucleotide duplexes containing 2,6-diaminopurine (D) matched with each of the four normal DNA bases were determined by optical melting techniques. Comparison of optical melting curves yielded relative stabilities for the D-containing standard base pairs in an otherwise identical base-pair sequence. The D:T pair was found to be more stable than the A:T pair in dC3DG3:dC3TG3, as stable as the A:T in dCT3DT3G:dCA3TA3G, and less stable than the A:T in dCA3DA3G:dCT7G. The order of stabilities for X:Y in the DNA duplex dCA3XA3G:dCT3YT3G is: (A:T) greater than (T:D) congruent to (D:T) greater than or equal to (T:A) greater than (C:D) congruent to (D:A) congruent to (D:G) greater than or equal to (D:C) congruent to (G:D) congruent to (D:D) greater than or equal to (A:D). Implications of these results for design of DNA oligonucleotide probes are discussed.     

Synthesis of specific diastereomers of a DNA methylphosphonate heptamer, d(CpCpApApApCpA), and stability of base pairing with the normal DNA octamer d(TPGPTPTPTPGPGPC).

DNA methylphosphonates are candidate derivatives for use in antisense DNA therapy. Their efficacy is limited by weak hybridization. One hypothesis to explain this phenomenon holds that one configuration of the chiral methylphosphonate linkage, Rp, permits stronger base pairing than the other configuration, Sp. To test this hypothesis, four specific pairs of Rp and Sp diastereomers of the DNA methylphosphonate heptamer d(CpCpApApApCpA) were prepared by block coupling of different combinations of individual diastereomers of d(CpCpApA) and d(ApCpA). Each pair of the diastereomers of the heptamer was separated into individual diastereomes using affinity chromatography on a Lichrosorb-NH2 silica column with a covalently attached complementary normal DNA octamer, d(pTpGpTpTpTpGpGpC). The stabilities of complementary complexes of phosphodiester d(TpGpTpTpTpGpGpC) with 8 individual diastereomers of methylphosphonate d(CpCpApApApCpA) were studied by measuring their melting temperatures (Tm). A direct correlation of Tm values with the number of Rp methylphosphonate centers in the heptamer was found: the more Rp centers, the higher the stability of the complex. Tm values for the diastereomers with 6 all-Rp or all-Sp methylphosphonate centers were found to be 30.5 degrees and 12.5 degrees C, respectively, in 100 mM NaCl, 10 mM Na2HPO4, 1 mM EDTA, pH 7.0 with 15 microM of each oligomer. On the average, each substitution of one Rp-center to an Sp-center in the heptamer decreased the Tm by 3 degrees C. Under the same conditions, the Tm of the normal DNA heptamer with its complement was 21 degrees C. These results are consistent with the model that all-Rp methylphosphonate DNAs hybridize much more tightly to complementary normal DNA than do racemic methylphosphonate DNAs, and may therefore exhibit greater potency as antisense inhibitors.     

Synthesis and binding properties of new selective ligands for the nucleobase opposite the AP site

DNA is continuously damaged by endogenous and exogenous factors such as oxidative stress or DNA alkylating agents. These damaged nucleobases are removed by DNA N-glycosylase and form apurinic/apyrimidinic sites (AP sites) as intermediates in the base excision repair (BER) pathway. AP sites are also representative DNA damages formed by spontaneous hydrolysis. The AP sites block DNA polymerase and a mismatch nucleobase is inserted opposite the AP sites by polymerization to cause acute toxicities and mutations. Thus, AP site specific compounds have attracted much attention for therapeutic and diagnostic purposes. In this study, we have developed nucleobase-polyamine conjugates as the AP site binding ligand by expecting that the nucleobase part would play a role in the specific recognition of the nucleobase opposite the AP site by the Watson-Crick base pair formation and that the polyamine part should contribute to the access of the ligand to the AP site by a non-specific interaction to the DNA phosphate backbone. The nucleobase conjugated with 3,3'-diaminodipropylamine (A-ligand, G-ligand, C-ligand, T-ligand and U-ligand) showed a specific stabilization of the duplex containing the AP site depending on the complementary combination with the nucleobase opposite the AP site; that is A-ligand to T, G-ligand to C, C-ligand to G, T- and U-ligand to A. The thermodynamic binding parameters clearly indicated that the specific stabilization is due to specific binding of the ligands to the complementary AP site. These results have suggested that the complementary base pairs of the Watson-Crick type are formed at the AP site.     

A.T and C.C+ base pairs can form simultaneously in a novel multistranded DNA complex

Previous experiments have established that in certain synthetic oligomeric DNA sequences, including mixtures of d(AACC)5 with d(CCTT)5, adenine-thymine (A.T) base pairs form to the exclusion of neighboring protonated cytosine-cytosine (C.C+) base pairs [Edwards, E., Ratliff, R., & Gray, D. (1988) Biochemistry 27, 5166-5174]. In the present work, circular dichroism and other measurements were used to study DNA oligomers that represented two additional classes with respect to the formation of A.T and/or C.C+ base pairs. (1) One class included two sets of repeating pentameric DNA sequences, d(CCAAT)3-6 and d(AATCC)4,5. For both of these sets of oligomers, an increase in the magnitude of the long-wavelength positive CD band centered at about 280 nm occurred as the pH was lowered from 7 to 5 at 0.1 and 0.5 M Na+, indicating that C.C+ base pairs formed. Even though it may have been possible for these oligomers to form duplexes with two antiparallel A.T base pairs per pentamer, no A.T base pairing was detected by monitoring the CD changes at 250 nm. Thus, spectral data showed that as few as 40% C.C+ base pairs were stable in two sets of oligomers in which A.T base pairs did not form adjacent to, or in place of, C.C+ base pairs. (2) Another class of oligomer was represented by d(C4A4T4C4), which was studied by CD, HPLC, and centrifugation experiments. We confirmed previous work that this sequence was able to form both types of base pairs as the pH and temperature were lowered [Gray, D., Cui, T., & Ratliff, R. (1984) Nucleic Acids Res. 12, 7565-7580].(ABSTRACT TRUNCATED AT 250 WORDS)     

Modification of the human thymine-DNA glycosylase by ubiquitin-like proteins facilitates enzymatic turnover

DNA glycosylases initiate base excision repair (BER) through the generation of potentially harmful abasic sites (AP sites) in DNA. Human thymine-DNA glycosylase (TDG) is a mismatch-specific uracil/thymine-DNA glycosylase with an implicated function in the restoration of G*C base pairs at sites of cytosine or 5-methylcytosine deamination. The rate-limiting step in the action of TDG in vitro is its dissociation from the product AP site, suggesting the existence of a specific enzyme release mechanism in vivo. We show here that TDG interacts with and is covalently modified by the ubiquitin-like proteins SUMO-1 and SUMO-2/3. SUMO conjugation dramatically reduces the DNA substrate and AP site binding affinity of TDG, and this is associated with a significant increase in enzymatic turnover in reactions with a G*U substrate and the loss of G*T processing activity. Sumoylation also potentiates the stimulatory effect of APE1 on TDG. These observations implicate a function of sumoylation in the controlled dissociation of TDG from the AP site and open up novel perspectives for the understanding of the molecular mechanisms coordinating the early steps of BER.     

Interaction of phenosafranine with nucleic acids and model polyphosphates. III. Heterogeneity in phenosafranine interactions with DNA base pairs.

Fluorescence and circular dichroism spectral measurements, thermal denaturation studies and binding competition experiments with netropsin and actinomycin D were carried out in systems containing phenosafranine bound to DNA's differing in base composition. The investigated properties exhibit a heterogeneity related to the content of A.T and G.C pairs in DNA and to the nature of phenosafranine binding modes. At low level of saturation of binding sites (r less than 0.1) phenosafranine does not show strong preference for any of the DNA base pairs in the overall binding. However, the strong monomer non-cooperative binding outside the helix (mode I1) occurs predominantly, even though not exclusively in G.C rich regions. The strong binding modes involving intercalated dye molecules (mode I2 and eventually mode II1) prevail in A.T rich regions. These binding modes become the principal types of strong phenosafranine interaction with DNA when the level of saturation of binding sites increases, i.e. at r greater than 0.1.20     

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.