Mispair Formation Research Articles

DNA polymerase ε leading strand signature mutations result from defects in its proofreading activity

The evidence that purified pol2-M644G DNA polymerase (Pol)ε exhibits a highly elevated bias for forming T:dTTP mispairs over A:dATP mispairs and that yeast cells harboring this Polε mutation accumulate A > T signature mutations in the leading strand have been used to assign a role for Polε in replicating the leading strand. Here, we determine whether A > T signature mutations result from defects in Polε proofreading activity by analyzing their rate in Polε proofreading defective pol2-4 and pol2-M644Gcells. Since purified pol2-4 Polε exhibits no bias for T:dTTP mispair formation, A > T mutations are expected to occur at a much lower rate in pol2-4 than in pol2-M644Gcells if Polε replicated the leading strand. Instead, we find that the rate of A > T signature mutations are as highly elevated in pol2-4cells as in pol2-M644Gcells; furthermore, the highly elevated rate of A > T signature mutations is severely curtailed in the absence of PCNA ubiquitination or Polζ in both the pol2-M644G and pol2-4 strains. Altogether, our evidence supports the conclusion that the leading strand A > T signature mutations derive from defects in Polε proofreading activity and not from the role of Polε as a leading strand replicase, and it conforms with the genetic evidence for a major role of Polδ in replication of both the DNA strands.

Human DNA Polymerase ν Catalyzes Correct and Incorrect DNA Synthesis with High Catalytic Efficiency

DNA polymerase ν (pol ν) is a low fidelity A-family polymerase with a putative role in interstrand cross-link repair and homologous recombination. We carried out pre-steady-state kinetic analysis to elucidate the kinetic mechanism of this enzyme. We found that the mechanism consists of seven steps, similar that of other A-family polymerases. pol ν binds to DNA with a Kd for DNA of 9.2 nm, with an off-rate constant of 0.013 s(-1)and an on-rate constant of 14 μm(-1) s(-1). dNTP binding is rapid with Kd values of 20 and 476 μm for the correct and incorrect dNTP, respectively. Pyrophosphorylation occurs with a Kd value for PPi of 3.7 mm and a maximal rate constant of 11 s(-1). Pre-steady-state kinetics, examination of the elemental effect using dNTPαS, and pulse-chase experiments indicate that a rapid phosphodiester bond formation step is flanked by slow conformational changes for both correct and incorrect base pair formation. These experiments in combination with computer simulations indicate that the first conformational change occurs with rate constants of 75 and 20 s(-1); rapid phosphodiester bond formation occurs with a Keq of 2.2 and 1.7, and the second conformational change occurs with rate constants of 2.1 and 0.5 s(-1), for correct and incorrect base pair formation, respectively. The presence of a mispair does not induce the polymerase to adopt a low catalytic conformation. pol ν catalyzes both correct and mispair formation with high catalytic efficiency.

Enhancement of Human DNA Polymerase η Activity and Fidelity Is Dependent Upon a Bipartite Interaction with the Werner Syndrome Protein

We have investigated the interaction between human DNA polymerase η (hpol η) and the Werner syndrome protein (WRN). Functional assays revealed that the WRN exonuclease and RecQ C-terminal (RQC) domains are necessary for full stimulation of hpol η-catalyzed formation of correct base pairs. We find that WRN does not stimulate hpol η-catalyzed formation of mispairs. Moreover, the exonuclease activity of WRN prevents stable mispair formation by hpol η. These results are consistent with a proofreading activity for WRN during single-nucleotide additions. ATP hydrolysis by WRN appears to attenuate stimulation of hpol η. Pre-steady-state kinetic results show that k(pol) is increased 4-fold by WRN. Finally, pulldown assays reveal a bipartite physical interaction between hpol η and WRN that is mediated by the exonuclease and RQC domains. Taken together, these results are consistent with alteration of the rate-limiting step in polymerase catalysis by direct protein-protein interactions between WRN and hpol η. In summary, WRN improves the efficiency and fidelity of hpol η to promote more effective replication of DNA.

The effect of ionization and deprotonation of guanine on the formation of base pairs

The effect of the ionization of the guanine on the formation of base pairs is investigated using the B3LYP method with 6-31++G ∗∗ basis set. The base pairing with neutral DNA base lowers the adiabatic ionization potential of guanine, while the base pairing with protonated DNA base increases the ionization potential of guanine. The proton transfer can occur in the Watson–Crick GC and GA base pairs when an electron is lost. The effect of the deprotonation of the guanine cation on the formation of base pairs is also explored. From an energy point of view, the ionization of guanine cannot result in the Watson–Crick mispair formation, but the deprotonation from the guanine cation is in favor of the combination of the G(−H) and guanine. In the Hoogsteen base pairs, the ionization of guanine slightly affects the interaction mode, but it may result in the dissociation of the Hoogsteen base pairs if the DNA bases pairing with guanine are the protonated A, T and C. The deprotonation from guanine cation slightly affects the base pairing energies of the Hoogsteen base pairs.

Tautomeric Equilibria of 5-Fluorouracil Anionic Species in Water

It has long been postulated that rare tautomeric or ionized forms of nucleic acid bases may play a role in mispair formation. Therefore, ab initio quantum chemical investigations on the tautomeric equilibrium in 5-fluorouracil (5FU) and its anions (deprotonated from N1, AN1, and from N3, AN3) and their tautomeric forms in water were performed. The effect of the water as solvent was introduced using solute-solvent clusters (four water molecules). The influence of the water molecules on the tautomeric reactions between different forms was considered by multiple proton transfer mechanisms. We show that when a water dimer is located in the reaction site between the two pairs of N-H and C═O groups, the assistive effect of the water molecules is strengthened. All calculations of the solute-water complexes were carried out at an MP2 level of theory and supplemented with correction for higher order correlation terms at CCSD(T) level, using the 6-31+G(d,p) basis set. The ab initio calculated frequencies and Raman intensities of 5FU and its anions AN1, AN3, and dianion are in good agreement with the experimental Raman frequencies in aqueous solution at different pH. In order to establish the pH-induced structural transformation in the molecule of 5FU, further (1)H, (19)F, and (13)C NMR spectra in water solution for pH = 6.9-13.8 were acquired and the chemical shift alterations were determined as a function of pH. On the basis of NMR spectroscopic data obtained for 5FU in aqueous solution at alkaline pH, we suggest the existence of a mixture of the anionic tautomeric forms predicted by our theoretical calculations.

Rapid and sensitive detection of DNA polymerase fidelity by singly labeled smart fluorescent probes

We report here a novel approach to monitor the DNA polymerase fidelity in detailed steps, including mispair extension, mispair formation and 3′ → 5′ proofreading. The method is based on the photo-induced electron transfer between the natural base guanine and the labeled fluorophore. The G:T mispair extension catalyzed by the exonuclease-deficient Klenow fragment DNA polymerase (KF exo −) was easily detected and the effect of the nearest neighbor base pair on the mispair extension rate was clearly observed. More importantly, kinetics of the G:T, G:A and G:G mispair formation and extension under single turnover conditions were measured by continuous fluorescence-based assay for the first time. The probes also showed their applicability to discriminate the 3′ → 5′ proofreading activity of different exonuclease-proficient DNA polymerases. The presented method may greatly simplify the screening and characterization procedures of the increasing number of polymerases that are thought to be potential targets for drug design and cancer treatment. It will also provide important information for deep understanding of the polymerase fidelity mechanism.

DNA duplex stability: the role of preorganized electrostatics.

The insertion of a DNA base moiety at the end of a DNA duplex to form a Watson-Crick or wobble pair during DNA annealing or replication is a step of fundamental biological importance. Therefore, we investigated the energetics of a formation of the terminal G x C, G x T, and G x A base pairs in DNA containing a 5'-dangling G adjacent to the base insertion point using differential scanning calorimetry and computer simulations. The energies calculated along classical molecular dynamics trajectories in aqueous solution were analyzed in the framework of linear-response approximation (LRA) to obtain relative free energies for the base insertion and their electrostatic, van der Waals, and preorganization components. Using the generic set of LRA parameters, the calculated free energies disfavored the mispair formation by 2.5 (G x C --> G x T) and 1.7 (G x C --> G x A) kcal/mol, in reasonable agreement with the experimental free energy differences of 1.8 and 1.4 kcal/mol, respectively. The calculated preorganization components of these free energies of 0.6 (G x C --> G x T) and -0.1 (G x C --> G x A) kcal/mol show that electrostatic preorganization, which is an important source of DNA replication fidelity, plays a lesser role in the mispair destabilization in the absence of DNA polymerase.

PH-Dependent Configurations of a 5-Chlorouracil-Guanine Base Pair

Hypochlorous acid (HOCl) from activated neutrophils at sites of inflammation can react with and damage biological molecules, including nucleic acids. The reaction of HOCl with cytosine analogues can generate multiple products, including 5-chlorouracil (ClU). In this paper, we have constructed oligonucleotides containing ClU paired opposite guanine (ClU-G). Melting studies indicate that oligonucleotide duplexes containing the ClU-G mispair are substantially less stable than those containing a ClU-A base pair. The melting temperature of the ClU-G mispair is not experimentally distinguishable from that of a T-G pair. NMR studies indicate that the ClU-G base pair adopts a wobble geometry at neutral pH, similar to a T-G mispair. The exchangeable protons of the ClU-G mispair broaden rapidly with an increase in temperature, indicating that the ClU-G mispair is less stable and opens more easily than the surrounding adjacent base pairs. Unlike the ClU-A base pair studied previously [Theruvathu, J. A., et al. (2009) Biochemistry 48, 7539-7546], the ClU-G mispair undergoes a pH-dependent structural change, assuming an ionized base pair configuration that approximates a Watson-Crick base pair at higher pH. Ionization of ClU in a DNA template could promote mispair formation and mutation, in accord with previous studies on other 5-halouracil analogues. The electron-withdrawing 5-chloro substituent facilitates ionization of the ClU N3 proton, promoting mispair formation, but it also renders the glycosidic bond susceptible to base cleavage by DNA repair glycosylases.

Formation of Purine−Purine Mispairs by Sulfolobus solfataricus DNA Polymerase IV

DNA damage that stalls replicative polymerases can be bypassed with the Y-family polymerases. These polymerases have more open active sites that can accommodate modified nucleotides. The lack of protein-DNA interactions that select for Watson-Crick base pairs correlate with the lowered fidelity of replication. Interstrand hydrogen bonds appear to play a larger role in dNTP selectivity. The mechanism by which purine-purine mispairs are formed and extended was examined with Solfolobus solfataricus DNA polymerase IV, a member of the RAD30A subfamily of the Y-family polymerases, as is pol eta. The structures of the purine-purine mispairs were examined by comparing the kinetics of mispair formation with adenine versus 1-deaza- and 7-deazaadenine and guanine versus 7-deazaguanine at four positions in the DNA, the incoming dNTP, the template base, and both positions of the terminal base pair. The time course of insertion of a single dNTP was examined with a polymerase concentration of 50 nM and a DNA concentration of 25 nM with various concentrations of dNTP. The time courses were fitted to a first-order equation, and the first-order rate constants were plotted against the dNTP concentration to produce k pol and K d (dNTP) values. A decrease in k pol/ K d (dNTP) associated with the deazapurine substitution would indicate that the position is involved in a crucial hydrogen bond. During correct base pair formation, the adenine to 1-deazaadenine substitution in both the incoming dNTP and template base resulted in a >1000-fold decrease in k pol/ K d (dNTP), indicating that interstrand hydrogen bonds are important in correcting base pair formation. During formation of purine-purine mispairs, the k pol/ K d (dNTP) values for the insertion of dATP and dGTP opposite 7-deazaadenine and 7-deazaguanine were decreased >10-fold with respect to those of the unmodified nucleotides. In addition, the rate of incorporation of 1-deaza-dATP opposite guanine was decreased 5-fold. These results suggest that during mispair formation the newly forming base pair is in a Hoogsteen geometry with the incoming dNTP in the anti conformation and the template base in the syn conformation. These results indicate that Dpo4 holds the incoming dNTP in the normal anti conformation while allowing the template nucleotide to change conformations to allow reaction to occur. This result may be functionally relevant in the replication of damaged DNA in that the polymerase may allow the template to adopt multiple configurations.

The Properties of Steric Gate Mutants Reveal Different Constraints within the Active Sites of Y-family and A-family DNA Polymerases

Y-family (lesion-bypass) DNA polymerases show the same overall structural features seen in other members of the polymerase superfamily, yet their active sites are more open, with fewer contacts to the DNA and nucleotide substrates. This raises the question of whether analogous active-site side chains play equivalent roles in the bypass polymerases and their classical DNA polymerase counterparts. In Klenow fragment, an A-family DNA polymerase, the steric gate side chain (Glu710) not only prevents ribonucleotide incorporation but also plays an important role in discrimination against purine-pyrimidine mispairs. In this work we show that the steric gate (Phe12) of the Y-family polymerase Dbh plays a very minor role in fidelity, despite its analogous role in sugar selection. Using ribonucleotide discrimination to report on the positioning of a mispaired dNTP, we found that the pyrimidine of a Pu-dPyTP nascent mispair occupies a similar position to that of a correctly paired dNTP in the Dbh active site, whereas in Klenow fragment the mispaired dNTP sits higher in the active site pocket. If purine-pyrimidine mispairs adopt the expected wobble geometry, the difference between the two polymerases can be attributed to the binding of the templating base, with the looser binding site of Dbh permitting a variety of template conformations with only minimal adjustment at the incoming dNTP. In Klenow fragment the templating base is more rigidly held, so that changes in base pair geometry would affect the dNTP position, allowing the Glu710 side chain to serve as a sensor of nascent mispairs.

Structure of Purine−Purine Mispairs during Misincorporation and Extension by Escherichia coli DNA Polymerase I

The mechanism by which purine-purine mispairs are formed and extended was examined with the high-fidelity Klenow fragment of Escherichia coli DNA polymerase I with the proofreading exonuclease activity inactivated. The structures of the purine-purine mispairs were examined by comparing the kinetics of mispair formation with adenine versus 7-deazaadenine and guanine versus 7-deazaguanine at four positions in the DNA, the incoming dNTP, the template base, and both positions of the terminal base pair. A decrease in rate associated with a 7-deazapurine substitution would suggest that the nucleotide is in a syn conformation in a Hoogsteen base pair with the opposite base. During mispair formation, the k(pol)/K(d) values for the insertion of dATP opposite A (dATP/A) as well as dATP/G and dGTP/G were decreased greater than 10-fold with the deazapurine in the dNTP. These results suggest that during mispair formation the newly forming base pair is in a Hoogsteen geometry with the incoming dNTP in the syn conformation and the template base in the anti conformation. During mispair extension, the only decrease in k(pol)/K(d) was associated with the G/G base pair in which 7-deazaguanine was in the template strand. These results as well as previous results [McCain et al. (2005) Biochemistry 44, 5647-5659] in which a hydrogen bond was found between the 3-position of guanine at the primer terminus and Arg668 during G/A and G/G mispair extension indicate that the conformation of the purine at the primer terminus is in the anti conformation during mispair extension. These results suggest that purine-purine mispairs are formed via a Hoogsteen geometry in which the dNTP is in the syn conformation and the template is in the anti conformation. During extension, however, the conformation of the primer terminus changes to an anti configuration while the template base may be in either the syn or anti conformations.

Cover Picture: Nanoparticle PCR: Nanogold‐Assisted PCR with Enhanced Specificity (Angew. Chem. Int. Ed. 32/2005)

Favorable binding of gold nanoparticles to single‐stranded DNA decreases the likelihood of mispair formation during annealing and clearly increases the specificity and yield of PCR, one of the most important standard methods of molecular biology. In their Communication on pp. 5100 ff., Z. Zhang, C. Fan, J. Hu, and co‐workers report on this promising field of biotechnology. (Xiaobin Wang assisted in the production of this image.)

Monte Carlo study of three-dimensional organization of water molecules around DNA fragments

Interactions with water molecules are important for the stabilization of three-dimensional structures of nucleic acids and for their functioning. The first hydration shells of macromolecules can be considered as structural parts of nucleic acid. We performed a Monte Carlo study of systems containing a nucleic acid base or base pair with water molecules using improved potential functions. These potential functions enable experimental data on both single base–single water interaction energies and enthalpies of base hydration to be reproduced. Hydration shell structures of base pairs are dependent on the pair geometry. Structural elements of hydration shells can contribute to the pair stability and hence to the probability of mispair formation during nucleic acid biosynthesis. The distribution of water molecules around bases and base pairs is essentially nonhomogeneous.

PKaValues of Guanine in Water: Density Functional Theory Calculations Combined with Poisson−Boltzmann Continuum−Solvation Model

It has long been postulated that rare tautomeric or ionized forms of DNA bases may play a role in mispair formation. To investigate the role this phenomenon plays in the mispairing of guanine and to develop a calculation methodology that can be extended to mutagenic DNA damage products, we used first principles quantum mechanics (density functional theory (B3LYP) with the Poisson−Boltzmann continuum−solvation model) to calculate the relative stabilities of tautomers of guanine in various environments and their pKa values in aqueous solution. This method allows us to calculate site specific pKa values information that is experimentally inaccessible as well as overall pKa values for each stage, wherein our numbers are in agreement with experimental values. We find that neutral guanine exists in aqueous phase as a mixture of two major keto tautomers, the N9H form (1) and a N7H form (3). These keto forms are also major species present in the gas phase, as well as the O6H enol tautomer (7a). These results show that tautomeric configurations can be drastically different depending on the environment. Here, we discuss the reasons for this environmental variability and suggest some possible implications. Finally, we estimate that the relative population of deprotonated guanine is 0.2−2% in the range of pH 7−8, a significant enough population to potentially play a role in mispair formation.

The interactions of square platinum(II) complexes with guanine and adenine: a quantum-chemical ab initio study of metalated tautomeric forms.

The influence of binding of square planar platinum complexes on tautomeric equilibria of the DNA bases guanine and adenine was investigated using the density functional B3LYP method. Neutral trans-dichloro(amine)-, +1 charged chloro(diamine)-, and +2 charged triamine-platinum(II) species were chosen for coordination to bases. Only the N7 interaction site of the bases was considered. The calculations demonstrate that the neutral platinum adduct does not change the tautomeric equilibria of the bases. Furthermore, N7 binding of the neutral Pt adduct moderately reduces the probability of protonation of the N1 position of adenine. Larger effects can be observed for +1 and mainly +2 adducts, but these can be rationalized by electrostatic effects. Since the electrostatic effects are expected to be efficiently compensated for by a charged backbone of DNA and counterions in a polar solvent, no dramatic increase in mispair formation is predicted for Pt(II) adducts, which is in agreement with experiment. The interaction energies between Pt adducts and the nucleobases were also evaluated. These interaction energies range from ca. 210 kJ/mol for neutral adducts, interacting with both bases and their tautomers, up to 500 kJ/mol for the +2 charged adducts, interacting with the keto-guanine tautomer and the anti-imino-adenine tautomer. The surprisingly large interaction energy for the latter structure is due to the strong H-bond between the NH3 ligand group of the metal adduct and the N6 nitrogen atom of the base.

The fidelity of 3' misinsertion and mispair extension during DNA synthesis exhibited by two drug-resistant mutants of the reverse transcriptase of human immunodeficiency virus type 1 with Leu74-->Val and Glu89-->Gly.

The relatively low fidelity of DNA synthesis characteristic to the reverse transcriptases (RTs) of the AIDS-causing viruses, human immunodeficiency viruses types 1 and 2 (HIV-1 and HIV-2, respectively) was implicated as a dominant factor that contributes to the genetic hypervariability of these viruses. The formation of 3'-mispaired DNA and the subsequent extension of this DNA were shown to be key determinants that lead to the error proneness of these RTs. As part of our goal to study the structure/function relationship in HIV-1 RT, we have conducted mutational studies aimed at identifying amino-acid residues involved in affecting the fidelity of DNA synthesis by the enzyme. We have recently found that two mutants of HIV-1 RT, which show resistance to nucleoside analog inhibitors ([Leu184]RT and [Phe183]RT), exhibit in vitro error proneness of DNA synthesis lower than that of wild-type enzyme [Bakhanshvili, M., Avidan, O. & Hizi, A. (1996) Mutational studies of human immunodeficiency virus type 1 reverse transcriptase: the involvement of residues 183 and 184 in the fidelity of DNA synthesis, FEBS Lett. 391, 257-262]. Using both criteria, the current comparative study suggests that these two mutant RTs display a substantially enhanced fidelity of DNA synthesis relative to the wild-type RT counterpart. In the current study we have analyzed two additional drug-resistant mutants of HIV-1 RT, [Val74]RT and [Gly89]RT, for their in vitro fidelity of DNA synthesis using two parameters of DNA synthesis: 3' mispair formation and elongation of 3'-mismatched DNA. The current comparative study suggests that these two mutant RTs display a substantially enhanced fidelity of DNA synthesis relative to the wild-type RT counterpart, using both criteria. Analysis of the relative frequencies of misinsertion and mispair extension indicates that the overall error proneness of DNA synthesis in HIV-1 RT is wild-type > [Val74]RT > [Gly89]RT mutant. The results further support the possible linkage between the capacity of an enzyme to incorporate a nucleoside analog instead of the correct dNTP (leading to drug sensitivity) and the ability to incorporate and extend a wrong nucleotide (resulting in mutagenesis). Our results may bear on the potential use of selecting and maintaining HIV virions with high fidelity and drug-resistant RTs to suppress the subsequent appearance of virions resistant to other drugs.

Ionization of bromouracil and fluorouracil stimulates base mispairing frequencies with guanine

To test whether ionized base pairs influence polymerase-catalyzed misinsertion rates, we measured the efficiency of forming 5-bromouracil (B), 5-fluorouracil (F), and thymine base pairs with guanine and adenine as a function of pH using avian myeloblastosis reverse transcriptase. When B, F, and T were present as dNTP substrates, misincorporation efficiencies opposite G, normalized to incorporation of C opposite G, increased by about 20-, 13-, and 7-fold, respectively, as reaction pH increased from 7.0 to 9.5. Incorporation efficiencies to form the correct base pairs, B.A and F.A, normalized to T.A, decreased by 4- and 8-fold, respectively, with increasing pH. The effects of pH on misincorporation efficiencies were about 10-fold greater when B, F, and T were present as template bases. The relative misincorporation efficiencies of G opposite template B, F, and T, normalized to incorporation of A opposite B, F, and T, increased by about 430-, 370-, and 70-fold, respectively, as pH was increased from 6.5 to 9.5, while correct incorporation of A opposite template B and F decreased about 10-fold over the same pH range. Plots depicting incorrect and correct incorporation efficiencies versus pH were fit to a pH titration equation giving the fraction of ionized base as a function of pH. We conclude that avian myeloblastosis reverse transcriptase forms B.G and F.G mispairs in an ionized Watson-Crick conformation in preference to a neutral wobble structure containing favored keto tautomers of B or F. Although participation of disfavored enol tautomers in enzyme-catalyzed base mispair formation cannot be ruled out, the results are inconsistent with the "standard" disfavored tautomer model of mutagenesis. Instead, the data support a model in which ionization of halouracil bases is primarily responsible for B- and F-induced mutagenesis.

Computational investigation of the role of hydration in nucleic acid structure and function

AbstractThe results of the Monte Carlo Metropolis simulation of water structure and of the hydration of nucleic acid fragments, complementary base pairs and mispairs, base pair stacks, and duplex fragments have been summarized. Systematic investigations suggest some general conclusions: (1) the hydration shell structure of the major and minor grooves of the duplex depends significantly on DNA conformation (or stack configuration) and nucleotide sequence, while global hydration characteristics (average energy, the number of water–water and water–base H‐bonds) are only slightly dependent on these factors, (2) hydration economy takes place in the B–A transition due to an increase of the number of water molecules forming hydrogen bonds with two or more atoms of bases (water bridging), and (3) the hydration of the duplex could contribute to nucleic acid functioning via water‐bridged mispair formation and stabilization of specific conformations.

Effect of pH on the base-mispairing properties of 5-bromouracil during DNA synthesis.

We have utilized an electrophoretic assay of misincorporation to investigate the possibility that ionization of 5-bromouracil (BU) may play a role in its mispairing during DNA synthesis in vitro. We examined the effects of increasing pH on the relative rates of formation of BU.G and T.G mispairs during chain elongation catalyzed by various DNA polymerases. For the Klenow fragment of Escherichia coli DNA polymerase I, increasing pH facilitated BU.G mispair formation (relative to T.G mispairing) when BU was present in the template strand. This effect showed a strong dependence on sequence context. Increasing pH had little effect on the relative rate of misincorporation of BrdUMP versus dTMP (at template G) by the Klenow polymerase. Misincorporation opposite template BU residues catalyzed by Maloney murine leukemia virus DNA polymerase and DNA polymerase beta (Novikoff hepatoma) also increased with pH, but for these two enzymes, there was no apparent dependence on sequence context. With T4 DNA polymerase and E. coli DNA polymerase III holoenzyme, a similar occurrence of BU.G and T.G mispairing during polymerization was observed, whether BU was present in the template or in the incoming nucleotide, and there was little effect of pH. The results reported here are consistent with a mispairing mechanism for template BU wherein the anionic form of the base mispairs with G.

Mispair formation in DNA can involve rare tautomeric forms in the template.

The formation of pyridine-pyrimidine- and pyrimidine-pyrimidine base pairs after in vitro DNA replication with the large fragment of Escherichia coli DNA polymerase I indicates that Watson-Crick-like base pairing between pyrimidine bases can occur in the enzyme due to the presence of the rare tautomers of deoxycytidylate and thymidylate in the template strand. The implications to mispair formation in DNA, such as the difference between the structures of the mispairs during and after replication, are discussed and the possible action of mutagenic DNA protonating and deprotonating agents in vivo is considered.