Requirement of Watson-Crick Hydrogen Bonding for DNA Synthesis by Yeast DNA Polymerase {eta}

Classical high-fidelity DNA polymerases discriminate betweenthe correct and incorrect nucleotides by using geometric constraintsimposed by the tight fit of the active site with the incipientbase pair. Consequently, Watson-Crick (W-C) hydrogen bondingbetween the bases is not required for the efficiency and accuracyof DNA synthesis by these polymerases. DNA polymerase ${eta}$ (Pol ${eta}$ )is a low-fidelity enzyme able to replicate through DNA lesions.Using difluorotoluene, a nonpolar isosteric analog of thymineunable to form W-C hydrogen bonds with adenine, we found thatthe efficiency and accuracy of nucleotide incorporation by Pol ${eta}$ are severely impaired. From these observations, we suggest thatW-C hydrogen bonding is required for DNA synthesis by Pol ${eta}$ ; inthis regard, Pol ${eta}$ differs strikingly from classical high-fidelityDNA polymerases.

INTRODUCTION

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Introduction

Classical DNA polymerases, such as T7 and Escherichia coli Klenow,synthesize DNA with high fidelity, and they are unable to replicatethrough DNA lesions (7). By contrast, the eukaryotic polymerase ${eta}$ (Pol ${eta}$ ) is a low-fidelity enzyme (16, 28, 45) with the abilityto replicate through DNA lesions. Pol ${eta}$ is unique among eukaryoticDNA polymerases in its proficient ability to replicate throughUV-induced cyclobutane pyrimidine dimers (CPDs) (15). Remarkably,both yeast and human Pol ${eta}$ insert A's opposite the two T's ofa cis-syn thymine-thymine (TT) dimer with the same efficiencyand accuracy as they insert A's opposite undamaged templatebases (16, 44). In addition to TT dimers, UV light also inducesthe formation of CPDs at 5'-TC-3' and 5'-CC-3' sites, and geneticstudies with Saccharomyces cerevisiae have indicated a roleof Pol ${eta}$ in the accurate bypass of these lesions as well (48).Because of its role in promoting the error-free bypass of CPDs,inactivation of Pol ${eta}$ in humans causes UV hypermutability (43)and results in the cancer-prone syndrome, the variant form ofxeroderma pigmentosum (14, 27). Yeast Pol ${eta}$ also replicates throughan 8-oxoguanine (8-oxoG) lesion efficiently and accurately,and genetic studies with S. cerevisiae have corroborated therequirement of Pol ${eta}$ in the error-free bypass of this DNA lesion(11).

From studies done with nonpolar isosteric analogs of naturalDNA bases which lack the ability to form Watson-Crick (W-C)hydrogen (H) bonds, it has been concluded that W-C H bondingis not needed for DNA synthesis by classical high-fidelity DNApolymerases (30, 32, 33); rather, the geometric fit of the incomingnucleotide with the templating base within the polymerase activesite is the principal determinant of the efficiency and fidelityof DNA synthesis in these polymerases (7, 9, 22). Here we examinethe effects of difluorotoluene (F), which is virtually identicalin shape, size, and conformation to thymine (T) but lacks theability to form W-C H bonds with adenine (A) (Fig. 1A), on DNAsynthesis by yeast Pol ${eta}$ . Because of the inability of F to formW-C H bonds in water (37, 38), this compound is ideal for examiningthe relative importance of hydrogen bonds and steric effectson DNA synthesis. Previously, it has been shown that high-fidelityDNA polymerases such as T7 and E. coli Klenow efficiently replicateDNA containing the F analog; in fact, these DNA polymerasesincorporate an A opposite an F in the template with nearly thesame efficiency and fidelity as they incorporate an A oppositetemplate T (30, 32, 33).

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FIG. 1. (A) Structures of thymine (left) and difluorotoluene (right). (B) The running-start and standing-start DNA substrates. The 28-mer templates have a G, A, T, C, or F base at the N shown underlined and in boldface (position 24). The running-start substrate has an 18-mer primer, and the standing-start substrate has a 23-mer primer. (C) An autoradiogram of the running-start (lanes 1 to 3) and standing-start (lanes 4 to 6) assays with E. coli Klenow (exo^-) and yeast Pol ${eta}$ . Lanes 1 and 4 contain no protein, lanes 2 and 5 contain a template T residue, and lanes 3 and 6 contain a template F residue.

Here we show that Pol ${eta}$ is ineffective at replicating DNA whenF is present in the template or is used as the incoming nucleotide,and steady-state kinetic analyses indicate that the efficiencyof incorporating an A opposite template F or the efficiencyof incorporating an F opposite template A is reduced to almostthe same level as the efficiency for incorporating the wrongnucleotides. We discuss these observations together with theother properties of Pol ${eta}$ and conclude that W-C H bonding playsan important role in the efficiency and accuracy of DNA synthesisby Pol ${eta}$ .

MATERIALS AND METHODS

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Materials and Methods

Proteins and other materials. Yeast Pol ${eta}$ was expressed and purified as described previously(13), except that after binding of the glutathione S-transferase(GST)-Pol ${eta}$ to a glutathione-Sepharose 4B column (Amersham) andwashing, Pol ${eta}$ was eluted by treatment with Precision protease(Amersham) for 16 h at 4°C to cleave off the GST tag. Theresulting full-length Pol ${eta}$ protein with a 7-amino-acid leaderpeptide was stored at -80°C.

Klenow fragment (exo^-; 5 U/ml) was purchased from New EnglandBioLabs. Solutions of all four deoxynucleoside triphosphates(dNTPs) (100 µM) were purchased from Roche Molecular Biochemicals.The difluorotoluene deoxynucleoside 5'-triphosphate (dFTP) andthe F phosphoramidite were synthesized and purified as previouslydescribed (33). Two oligodeoxynucleotides were used as primers,and five oligodeoxynucleotides were used as templates (Fig.1B), where N is a G, A, T, C, or F residue. Primer strands (1µM) were 5' ³²P end labeled by using polynucleotide kinase(Boehringer Mannheim) and [ ${gamma}$ -³²P]ATP (Amersham) at 37°C for1 h and were purified by using a Sephadex G25 spin column (Pharmacia).5' ³²P-end-labeled primer strands (250 nM) were annealed tothe template strands (350 nM) by incubating them in 50 mM TrisHCl (pH 7.5)-100 mM NaCl at 90°C for 2 min, followed byslow cooling to room temperature for several hours.

DNA polymerase assays. All DNA polymerase reactions were carried out in 35 mM TrisHCl (pH 7.5), 20 mM NaCl, 5 mM MgCl₂, 5 mM dithiothreitol, 100µg of bovine serum albumin/ml, and 10% glycerol at 22°C.Running- and standing-start reactions contained all four dNTPs(2 µM each), 50 nM of 5' ³²P-end-labeled DNA substrates,and either 1 nM yeast Pol ${eta}$ or E. coli Klenow (exo^-). Reactionmixtures were incubated for 30 min, quenched with 10 volumesof formamide loading buffer, and run on a 15% polyacrylamidesequencing gel containing 8 M urea. Single-nucleotide incorporationreaction mixtures contained only one dNTP (2 µM), a 50nM concentration of 5' ³²P-end-labeled standing-start DNA substrate,and either 1 nM yeast Pol ${eta}$ or Klenow fragment (exo^-). Reactionmixtures were incubated for 15 min, quenched with 10 volumesof formamide loading buffer, and run on a 15% sequencing gel.

Steady-state kinetics assays. The kinetics of nucleotide incorporation were measured usingthe conditions described above except that 1 nM yeast Pol ${eta}$ , 50nM 5' ³²P-end-labeled standing-start DNA substrate (Fig. 1B),and various concentrations of each nucleotide (0 to 1,000 µM)were incubated for 2.5 min before quenching with 10 volumesof formamide loading buffer. The extended and unextended primerswere separated on a 15% sequencing gel and quantitated by usinga PhosphorImager (Molecular Dynamics). The observed rate ofnucleotide incorporation was graphed as a function of nucleotideconcentration, and the k_cat and K_m steady-state parameters wereobtained from the best fit to the Michaelis-Menten equation.The frequency of incorrect nucleotide incorporation, f_inc, wascalculated using the following equation: f_inc = (k_cat/K_m)_incorrect/(k_cat/K_m)_correct(3).

RESULTS

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Results

DNA synthesis past a template F residue. Because it has been shown previously (33) that H bonds are relativelyunimportant in the case of the exonuclease-deficient Klenowfragment of E. coli DNA polymerase I, we used this enzyme asa basis for comparison in this study. First, we compared theabilities of Klenow fragment (exo^-) and Pol ${eta}$ to synthesize DNAon two substrates containing the template F residue (Fig. 1B).In the running-start substrate, the target T or F residue isthe sixth available template base, while in the standing-startsubstrate, the target T or F residue is the first availabletemplate base. As shown in Fig. 1C, Klenow fragment (exo^-) wasable to synthesize DNA past a template F residue (left panel,lanes 3 and 6). In the running-start assay (Fig. 1C, lanes 3),no stall site was visible at position 23, which is immediatelyprior to the template F residue, but a stall site was visibleat position 24, which is opposite the template F residue. Thisindicates that Klenow fragment (exo^-) was not blocked for nucleotideincorporation opposite the F residue but was hindered in extendingfrom the nucleotide inserted opposite the F residue (31). Bycontrast, Pol ${eta}$ was unable to synthesize DNA past the target templateF residue (Fig. 1C, right panel, lanes 3 and 6). In the running-startassay, a strong stall site was visible at position 23 (Fig.1C, lanes 3), indicating that Pol ${eta}$ was unable to insert nucleotidesopposite the F residue.

Next, we compared the abilities of Klenow fragment (exo^-) andPol ${eta}$ to insert dGTP, dATP, dTTP, dCTP, and dFTP opposite templateG, A, T, C, and F residues. As shown in the upper panels ofFig. 2, Klenow fragment (exo^-) incorporated the correct nucleotideopposite each of the template residues, dFTP was incorporatedopposite both template A and template F residues, and both dATPand dFTP were incorporated opposite the template F residue (33).By contrast, for Pol ${eta}$ (Fig. 2, lower panels), only the correctnucleotides were incorporated opposite each of the templateresidues. Neither the incorporation of dFTP opposite any ofthe template residues nor the incorporation of any dNTP oppositethe template F residue was observed.

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FIG. 2. Incorporation of each dNTP opposite template G, A, T, C, and F residues by E. coli Klenow (exo^-) and yeast Pol ${eta}$ .

Steady-state kinetics of nucleotide incorporation. We used steady-state kinetics assays to quantify the efficiency(k_cat/K_m) and the accuracy (f_inc) of dTTP and dFTP incorporationopposite each of the four template residues for Pol ${eta}$ . dTTP wasincorporated opposite template A with an efficiency which wasapproximately 90-, 70-, and 800-fold greater than that of theincorporation of dTTP opposite G, T, and C template residues,respectively (Table 1). However, dFTP was incorporated oppositetemplate A with an efficiency that was $~$ 220-fold lower than thatof dTTP incorporation opposite template A (Table 1). By contrast,dFTP was incorporated opposite templates G, T, and C with lessthan a 10-fold reduction in efficiency relative to dTTP incorporationopposite templates G, T, and C. Thus, overall, the incorporationof dFTP opposite template A occurred with very low fidelity,being only two- to threefold more efficient than that oppositetemplates G, T, and C (Table 1).

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TABLE 1. Steady-state kinetics parameters for the incorporation of dTTP and dFTP opposite all four template bases by yeast Pol ${eta}$

We also used steady-state kinetics assays to determine the efficiencyand accuracy of nucleotide incorporation opposite template Tand template F residues (Table 2). dATP was incorporated oppositea template T residue with an efficiency which was 50-, 120-,and 30-fold greater than that of the incorporation of dGTP,dTTP, and dCTP, respectively. The better-than-expected incorporationof dCTP (45) likely reflects the fact that in the standing-startDNA substrate used for these experiments (Fig. 1B), dCTP canbase pair with the next template G residue by "looping out"the T residue. Importantly, dATP was incorporated opposite templateF with an efficiency that was 240-fold lower than that of dATPincorporation opposite template T (Table 2). Relative to theincorporation of dGTP and dTTP opposite template T, the incorporationof these nucleotides opposite F was reduced by 15- and 8-fold,respectively. The incorporation of dCTP opposite F was aboutthe same as that opposite T, further supporting the loopingout notion. Overall, dATP was incorporated opposite templateF with only a three- to fourfold greater efficiency than thatof the incorporation of dGTP and dTTP (Table 2), again demonstratinglow fidelity in the absence of W-C hydrogen bonds.

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TABLE 2. Steady-state kinetics parameters for the incorporation of all four nucleotides opposite templates T and F by yeast Pol ${eta}$

DISCUSSION

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Discussion

Depending on the sequence context and experimental conditions,the measured differences in standard free energy changes ( ${Delta}$ ${Delta}$ G°)for correct versus incorrect base pairs in solution range from0.2 to 4.0 kcal/mol (1, 24, 36). These ${Delta}$ ${Delta}$ G° values can provideas much as a several-hundredfold preference for incorporatingthe correct over the incorrect nucleotide. Since classical DNApolymerases, such as E. coli DNA polymerase I and T7, possessthe ability to discriminate between correct and incorrect nucleotidesby as much as 1 millionfold, they must achieve this discriminationby means other than mere W-C H bonding between the incomingnucleotide and the templating base. The currently accepted paradigmregarding DNA synthesis by classical polymerases holds thatgeometrical alignment of the incoming nucleotide with the templatebase is a major determinant of fidelity, whereas W-C H bondingmakes relatively little contribution to base selectivity, andexperimental support for these conclusions has come from studiesusing isosteric analogs of DNA bases (9, 30, 32, 33).

To determine the importance of W-C H bonding for the efficiencyand fidelity of nucleotide incorporation by Pol ${eta}$ , a low-fidelitylesion bypass polymerase, we examined its ability to utilizeF as either the incoming nucleotide or the template base. Ourresults demonstrate that Pol ${eta}$ is highly inefficient at formingF · A base pairs compared to T · A base pairs.Although the inability of Pol ${eta}$ to efficiently utilize F couldreflect the requirement of W-C H bonding between the incomingnucleotide and the template base, several alternative explanationsneed to be considered first.

Our observations that Pol ${eta}$ poorly incorporates an F oppositetemplate A, or an A opposite template F, could arise from adependence of Pol ${eta}$ on minor-groove H bonding interactions withboth the incoming nucleotide and the templating base. The latterpossibility arises from the fact that F lacks the O2 minor-groovebond acceptor and therefore cannot form H bonds with the protein(31, 37, 38). We examined the contribution of any such proteininteractions with the minor groove of the incipient base pairby using 3-deazaguanine (3DG), which differs from G in havinga carbon instead of a nitrogen at the 3 position, and replacementof a normal G by 3DG in the template or the primer abolishesthe H bonding interaction of the polymerase with the DNA atthe position of 3DG (41). From steady-state kinetics studies,we determined that the placement of 3DG at the position of thetemplating base has no significant effect on the efficiencyor accuracy of C incorporation by yeast Pol ${eta}$ ; however, the incorporationof 3DG opposite a C template is 120-fold less efficient thanthe incorporation of a G opposite C (47). These results indicatethat yeast Pol ${eta}$ makes no functional H-bonding interaction withthe minor groove of the templating residue but needs minor-grooveH-bonding interactions with the incoming nucleotide. Thus, ifthe effects observed with F were due to Pol ${eta}$ 's interactions withthe minor groove, then the efficiency and fidelity of nucleotideincorporation would have been eliminated only when F was usedas the incoming nucleotide but not when F was the templatingbase. Since the efficiency and fidelity of nucleotide incorporationare eliminated regardless of whether F is the incoming nucleotideor the templating base, we consider this explanation to be unlikely.

Another possible explanation for the inability of Pol ${eta}$ to formF · A base pairs is that the active site of Pol ${eta}$ is verytight along the C1'-C1' axis. Because F does not form W-C Hbonds with A, the F · A base pair is not expected tocontract in the same way that a T · A base pair does.Consequently, the F · A base pair should be slightlylarger than the T · A base pair and might not fit wellwithin the active site of Pol ${eta}$ . We consider this explanationto be highly unlikely given the high-resolution structures ofPol ${eta}$ and several other members of the Y family of DNA polymerases.These structures all reveal active sites that are much moreopen and much less constrained than the active sites of classicalDNA polymerases (25, 40, 42, 49). If the slightly larger sizeof the F · A base pair is not problematic for the highlyconstrained active sites of Klenow or T7 DNA polymerase, thenit should be no more problematic for the relatively unconstrainedactive site of Pol ${eta}$ .

In view of the abovementioned considerations, the inabilityof Pol ${eta}$ to synthesize DNA with F in the template or as the incomingnucleotide can best be explained by its dependence on W-C Hbonding for efficient and accurate nucleotide incorporation.Furthermore, the lesion bypass properties of Pol ${eta}$ also supportsuch an inference. Pol ${eta}$ replicates through a cis-syn TT dimerby inserting A's opposite the two T's of the dimer, and it doesso with the same efficiency and fidelity as when replicatingthrough undamaged T's (16, 44). Although a cis-syn TT dimerdisrupts the DNA helix, bending it by $~$ 30° and unwindingit by $~$ 9°, this distortion does not affect the ability ofthe two T's in the dimer to form normal W-C base pairs withA's (2, 12, 18, 20, 35). Yeast Pol ${eta}$ also replicates through an8-oxoG lesion as efficiently and accurately as through an undamagedG (11). An 8-oxoG in the anti conformation pairs with C involvingthe same three hydrogen bonds as in the G · C base pair;however, the template strand is significantly distorted in thevicinity of the lesion in the 8-oxoG · C pair (23, 26,29, 34). Because of the considerable template distortion inthis base pair, replicative polymerases prefer to insert anA opposite 8-oxoG (11, 39). An 8-oxoG in the syn conformationmimics a T and has the correct geometry to pair with an A viatwo hydrogen bonds (23, 26, 29, 34). Thus, in spite of the greatergeometrical distortion, Pol ${eta}$ prefers to form the 8-oxoG ·C base pair, presumably because of the ability of this basepair to form normal W-C H bonds. In contrast to the efficientand accurate bypass of these two DNA lesions, Pol ${eta}$ is very poorat replicating through an abasic site, a prototypical noninstructionallesion. Classical high-fidelity replicative polymerases fromprokaryotes as well as eukaryotes predominantly insert an Aopposite the abasic site because this nucleotide causes no significantdistortion of the helix, as evidenced from nuclear magneticresonance studies indicating that DNA containing an A oppositethe abasic site retains all aspects of B-form DNA, and the unpairedA and the abasic residue lie inside the helix (4, 5, 17). Pol ${eta}$ ,however, is very inefficient at inserting an A or any othernucleotide opposite an abasic site. Thus, Pol ${eta}$ is $~$ 2,000-foldless efficient in inserting an A opposite an abasic site thanin inserting this nucleotide opposite a T template, and theinsertion of other nucleotides opposite this lesion site isalso highly inefficient (10). We attribute the inefficiencyof Pol ${eta}$ in incorporating a nucleotide opposite an abasic siteto the lack of W-C H bonding.

Also consistent with these findings is the recent observationthat the blocking of thymine N3 with a methyl group in the templatelowers the efficiency of dATP insertion by Pol ${eta}$ by a factor of50 (L. Sun, K. Zhang, L. Zhou, P. Hohler, E. T. Kool, X. F.Yuan, Z. Wang, and J. S. Taylor, submitted for publication).It is notable, however, that a nonpolar pyrene nucleotide isefficiently inserted at abasic sites by this enzyme. Thus, hydrogenbonding is not an absolute requirement with this large unnaturallyshaped nucleotide. Presumably, its large size and strong stackingability enable its favorable alignment in the active site.

Although we cannot as yet absolutely rule out the other factorsdescribed above, we propose that the most likely explanationfor the present results with naturally shaped nucleotides isa requirement for W-C hydrogen bonding by this low-fidelityenzyme. Pre-steady-state kinetic analyses have indicated thatyeast Pol ${eta}$ discriminates poorly between the correct and incorrectnucleotide at both the initial nucleotide binding step (K_D^dNTP)and the subsequent nucleotide incorporation step (k_pol) (46).Since Pol ${eta}$ shows a selectivity of only $~$ 5 at the initial nucleotidebinding step (K_D^dNTP), which likely derives from W-C H bonding,the elimination of nucleotide discrimination at the initialbinding step is to be expected with the T-to-F substitution.However, in view of the much greater selectivity ( $~$ 150-fold)at the k_pol step, which likely arises from geometric constraints,the elimination of nucleotide discrimination at this step requiresexplanation. Since the active site of Pol ${eta}$ and related polymerasesis relatively unconstrained and makes few intimate contactswith the incipient base pair (25, 40, 42, 49), we surmise thatin the absence of W-C H bonding between the bases, the incomingnucleotide is able to move about in the active site of Pol ${eta}$ ,and as a result, its base and/or triphosphate are not positionedcorrectly for the subsequent induced-fit conformation changestep or for the subsequent chemistry. By contrast, in the classicalhigh-fidelity DNA polymerases such as T7 and Klenow fragment(exo^-), W-C H bonding is not required at this step (30, 32,33) because the active sites of these enzymes are highly constrained,providing for a tight fit and proper alignment of the incipientbase pair (6, 8, 19).

Although the current paradigm which holds that the geometric-shapecomplementarity of the incoming nucleotide with the templatebase is a major determinant of fidelity and that W-C H bondingmakes a much less important contribution remains valid for classicalhigh-fidelity polymerases (21), Pol ${eta}$ appears not to conform tothis general rule. We attribute the exceptional nature of Pol ${eta}$ to its more open and less highly constrained active site, whichit needs to tolerate the geometric distortions in the DNA whenefficiently bypassing lesions such as the cis-syn TT dimer and8-oxoG. However, the geometry of the incipient base pair isalso likely to be important for Pol ${eta}$ . This is because for certainbase pairs, Pol ${eta}$ preferentially incorporates the correct nucleotideseveral thousandfold more efficiently than the incorrect nucleotide(45) and because Pol ${eta}$ requires minor-groove interactions betweenthe protein and the incoming nucleotide substrate (47). Thelatter observation raises the possibility that this interactionprovides for a "geometry-sensing" mechanism similar to thoseproposed for classical DNA polymerases. Nevertheless, Pol ${eta}$ differsstrikingly from high-fidelity DNA polymerases in its strongdependence on W-C H bonding for efficient and accurate DNA synthesis.

ACKNOWLEDGMENTS

This work was supported by National Institutes of Health grantsGM19261 and GM52956.

FOOTNOTES

* Corresponding author. Mailing address: Sealy Center for Molecular Science, University of Texas Medical Branch at Galveston, 6.104 Blocker Medical Research Building, 11th and Mechanic Streets, Galveston, TX 77555-1061. Phone: (409) 747-8602. Fax: (409) 747-8608. E-mail: s.prakash{at}utmb.edu.

REFERENCES

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