Evidence for a Watson-Crick Hydrogen Bonding Requirement in DNA Synthesis by Human DNA Polymerase {kappa}

The efficiency and fidelity of nucleotide incorporation by high-fidelityreplicative DNA polymerases (Pols) are governed by the geometricconstraints imposed upon the nascent base pair by the activesite. Consequently, these polymerases can efficiently and accuratelyreplicate through the template bases which are isosteric tonatural DNA bases but which lack the ability to engage in Watson-Crick(W-C) hydrogen bonding. DNA synthesis by Pol ${eta}$ , a low-fidelitypolymerase able to replicate through DNA lesions, however, isinhibited in the presence of such an analog, suggesting a dependenceof this polymerase upon W-C hydrogen bonding. Here we examinewhether human Pol ${kappa}$ , which differs from Pol ${eta}$ in having a higherfidelity and which, unlike Pol ${eta}$ , is inhibited at inserting nucleotidesopposite DNA lesions, shows less of a dependence upon W-C hydrogenbonding than does Pol ${eta}$ . We find that an isosteric thymidine analogis replicated with low efficiency by Pol ${kappa}$ , whereas a nucleobaseanalog lacking minor-groove H bonding potential is replicatedwith high efficiency. These observations suggest that both Pol ${eta}$ and Pol ${kappa}$ rely on W-C hydrogen bonding for localizing the nascentbase pair in the active site for the polymerization reactionto occur, thus overcoming these enzymes' low geometric selectivity.

INTRODUCTION

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Introduction

Classical DNA polymerases (Pols) replicate DNA with a high fidelityand are unable to replicate through DNA-distorting lesions.From studies with nonpolar, isosteric analogs, which lack theability to form canonical Watson-Crick (W-C) hydrogen bonds,it has been concluded that W-C H bonding is not the determiningfactor for the efficiency and accuracy of nucleotide incorporationin high-fidelity polymerases such as T7 and Escherichia coliPol I (20, 23, 24); rather, it is apparently the geometric fitwithin the active site of the incoming nucleoside triphosphatewith the templating nucleotide that governs polymerase efficiencyand accuracy (3, 5, 13, 15).

Members of the Y family of DNA polymerases replicate DNA witha low fidelity, and unlike the classical polymerases, they areable to replicate through DNA lesions (28, 29). Humans havefour Y family Pols: ${eta}$ , ${iota}$ , ${kappa}$ , and Rev1. Of these, Rev1 is a highlyspecialized polymerase which predominantly incorporates a Copposite template G and also opposite an abasic site (8, 26),and genetic studies of Saccharomyces cerevisiae have suggestedthat a major role of Rev1 is to act as an assembly factor inPol ${zeta}$ -dependent lesion bypass (28). Although not as extreme inits nucleotide insertion specificity as Rev1, Pol ${iota}$ incorporatesnucleotides opposite the four different template bases withvery different efficiencies and fidelities, and it incorporatesnucleotides opposite template purines with a much higher efficiencyand fidelity than opposite template pyrimidines (6, 11, 34,39, 46). From the ternary crystal structure of Pol ${iota}$ , with a templatingA and an incoming dTTP, it has been determined that unlike allother DNA polymerases, which impose Watson-Crick base pairingin their active site, Pol ${iota}$ uses Hoogsteen base pairing for DNAsynthesis (25).

In contrast to the specificity of Rev1 and Pol ${iota}$ for nucleotideincorporation opposite template bases, all other known DNA polymerases,including the high-fidelity replicative/repair DNA polymerases,as well as the two other members of the Y family, Pol ${eta}$ and Pol ${kappa}$ (10, 12, 41), form the four possible correct base pairs withnearly equivalent catalytic efficiencies. Pols ${eta}$ and ${kappa}$ , however,differ from the high-fidelity polymerases in their low fidelityof nucleotide incorporation and in their ability to replicatethrough DNA lesions. For example, both yeast and human Pol ${eta}$ misincorporatenucleotides with a frequency of $~$ 10^–2 to 10^–3, andboth of these enzymes replicate through a cis-syn TT dimer byinserting two A’s opposite the two T’s of the dimerwith the same efficiency and fidelity as opposite the undamagedT’s (12, 40, 41, 43). The structure of yeast Pol ${eta}$ modeledwith a templating TT dimer and an incoming dATP has indicatedthat it can accommodate both of the residues of the dimer inits active site (35); in this respect, Pol ${eta}$ differs from otherpolymerases, including the other Y family polymerases, as theycan accommodate only the templating residue in their activesite, while the next 5' template base and the rest of the unpairedtemplate are pushed out of the active site at a 90^o angle (28).

One possible consequence of the proficient ability of Pol ${eta}$ tohold two templating residues in its active site instead of oneis that its active site may not be as closely juxtaposed tothe templating base and the incoming deoxynucleoside triphosphate(dNTP) as occurs in the high-fidelity polymerases, which exercisea high degree of geometric selection because of the tight fitof the correct base pair in their active sites (3, 5, 14, 15).We have shown previously that Pol ${eta}$ differs from high-fidelityreplication/repair polymerases in its inability to replicatenon-hydrogen-bonding nucleotide analogs. We hypothesized thatthis enzyme, lacking a tight geometric constraint, may relyinstead upon Watson-Crick hydrogen bonding for the efficiencyand fidelity of DNA synthesis (37).

Pol ${kappa}$ differs from Pol ${eta}$ in several respects, including its higherfidelity and its inability to incorporate nucleotides oppositeDNA lesions. Pol ${kappa}$ exhibits the highest fidelity among the Y familypolymerases, as it misincorporates nucleotides with a frequencyof $~$ 10^–3 to 10^–4 opposite all four template bases(10). Also, in contrast to Pol ${eta}$ , Pol ${kappa}$ is unable to replicate througha cis-syn TT dimer, which results primarily from its inabilityto incorporate nucleotides opposite the 3' T of the dimer (10,38). Pol ${kappa}$ is also highly inefficient at incorporating nucleotidesopposite various other lesions, such as a (6-4) TT photoproduct,an N²-acetyl-aminofluorene adduct, and others (9, 10, 17, 33,38). For some DNA lesions, however, Pol ${kappa}$ can promote the extensionreaction wherein, following the incorporation of a nucleotideopposite the lesion site by another DNA polymerase, it performsthe further extension of the primer terminus (7, 38, 42).

The higher fidelity and the inability to incorporate nucleotidesopposite DNA lesions could result if the Pol ${kappa}$ active site weremore geometrically constrained than that of Pol ${eta}$ . The structureof the Pol ${kappa}$ apoenzyme and the modeling of Pol ${kappa}$ in a ternary complexwith DNA and an incoming nucleotide have suggested this to bethe case, as Pol ${kappa}$ 's active site appears to be more constrainedin the vicinity of the templating base than that of Pol ${eta}$ (36).Since the more enclosed active site of Pol ${kappa}$ could provide fora higher degree of geometric selection than would be possiblefor a polymerase with a more open active site, the dependenceof Pol ${kappa}$ upon other factors such as Watson-Crick or minor-groovehydrogen bonding may be lessened.

Here we examine the effects of difluorotoluene (F), which isidentical in shape, size, and conformation to thymine but lacksthe ability to form W-C hydrogen bonds with adenine (A) (30,31), on DNA synthesis by human Pol ${kappa}$ (hPol ${kappa}$ ). Interestingly, wefind that F is highly inhibitory to DNA synthesis by Pol ${kappa}$ , regardlessof whether it is used as a templating residue or as an incomingnucleotide. This is consistent with the notion that W-C hydrogenbonding makes a paramount contribution to DNA synthesis by Pol ${kappa}$ .The implications of this observation are discussed.

MATERIALS AND METHODS

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Materials and Methods

Purification of DNA polymerases. S. cerevisiae strain BJ5464 was transformed with plasmid pPol42,which carries the gene encoding wild-type hPol ${kappa}$ fused in framewith glutathione S-transferase. Cells were grown and hPol ${kappa}$ waspurified as described previously (10). Cleavage of the glutathioneS-transferase tag from Pol ${kappa}$ resulted in a seven-amino-acid peptideleader sequence attached to the N terminus of the polymerase.The protein concentration was determined by a Bio-Rad proteinassay (Bio-Rad) and by UV absorbance at 280 nm under denaturingconditions (8 M urea), using the molar extinction coefficientcalculated from the amino acid composition. The purified hPol ${kappa}$ was stored in 5-µl aliquots at –80°C. Taq waspurchased from Gene Choice, Inc.

Nucleotides and DNA substrates. 5'-dNTPs (100 mM) were purchased from Roche Diagnostics andstored in aliquots at –20°C. Synthetic oliogodeoxynucleotidetemplate and primers were used to prepare the nondamaged DNAsubstrates. The 3-deazaguanine (3DG)- and F-containing DNA substrateswere synthesized and purified as described previously (37, 45).For the 3DG-containing substrates, the following template/primerpairs were used. The incoming nucleotide (P₀) template/primerwas 5'-CTGCGACTGCTGCGTCTGCGGTGC-3'/5'-GCACCGCAGACGCAGCA. Forthe P₀ substrate, the bold and underlined nucleotide is thetemplating base, and either a dGTP or a 3DG nucleoside triphosphate(d3DGTP) was used as the incoming dNTP. The templating residue(T₀) template/primer was 5'-CTGCGACTXCTGCGTCTGCGGTGC-3'/5'-GCACCGCAGACGCAG.For the T₀ substrate, the bold and underlined nucleotide representsa G or a 3DG. The 2,4-difluorotoluene-containing template sequencewas 5'-ACTGXTCTCCCTATAGTGAGTCGTATTA-3', where the bold and underlinedX represents either an A, a T, or an F. The standing-start andrunning-start primer sequences were 5'-TAATACGACTCACTATAGGGAGA-3'and 5'-TAATACGACTCACTATAG-3', respectively. For the polymeraseassays, ³²P-5'-end-labeled primers (1.0 µM) were annealedto templates (1.5 µM) in 50 mM Tris HCl (pH 7.5) and 100mM NaCl by being heated to 95°C for 2 min, followed by slowcooling to room temperature. The resulting DNA substrates wereused to perform DNA synthesis reactions and to determine thesteady-state kinetics parameters.

DNA polymerase assays. The standard DNA polymerase reaction mixture contained 25 mMTris HCl (pH 7.5), 5 mM dithiothreitol, 0.1 mg/ml bovine serumalbumin, 5 mM MgCl₂, 10 nM DNA, 1 nM polymerase, and dNTPs.Reactions were carried out at 23°C for 3DG or at 30°Cfor F, and reaction mixtures were subsequently quenched with95% formamide loading buffer. Quenched reaction products wereheat denatured at 95°C for 3 min, and product formationwas determined by 15% polyacrylamide gel electrophoresis (8M urea) and quantified by PhosphorImager analysis (MolecularDynamics).

For the steady-state kinetic analyses of the 3DG-containingsubstrates, standard DNA polymerase reaction conditions wereused, with the addition of a single dNTP complementary to thetemplating base (e.g., dCTP opposite G and 3DG, dATP oppositeT, etc.) in a concentration range appropriate for K_m determinations.Deoxynucleotide incorporation was measured at multiple dNTPconcentrations for a single time point, and the steady-statekinetic parameters k_cat and K_m were determined by the best fitto the Michaelis-Menten equation (Sigma Plot 7.0).

For the steady-state kinetic analyses of the 2,4-difluorotoluene-containingsubstrates, standard DNA polymerase reaction conditions wereused, with the addition of a single dNTP complementary to thetemplating base (e.g., dATP opposite T and F, dFTP oppositeA, or dTTP opposite A) in a concentration range appropriatefor K_m determinations. Deoxynucleotide incorporation was measuredat multiple time points for each respective dNTP concentration,and the observed rate of nucleotide incorporation was determinedby linear regression. Values of the steady-state parametersk_cat and K_m for nucleotide incorporation were then determinedby the best fit to the Michaelis-Menten equation (Sigma Plot7.0).

RESULTS

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Results

DNA synthesis past a template F residue by Pol ${kappa}$ . To determine whether human Pol ${kappa}$ could synthesize DNA in the absenceof Watson-Crick hydrogen bonds, we examined its ability to polymerizenucleotides past a template F, which is a nonpolar, isostericanalog of thymine. As F lacks the Watson-Crick O² and O⁴ hydrogenbond acceptors and N-H³ donor of thymine (Fig. 1A), it is unableto participate in W-C hydrogen bonding (31). Since W-C hydrogenbonding is not needed for nucleotide incorporation by the Thermusaquaticus (Taq) DNA polymerase (23), we compared the abilitiesof Taq and Pol ${kappa}$ to synthesize DNA on substrates containing anF residue at either the sixth or the first templating positionof the DNA substrate (Fig. 1B). As expected, Taq was able toincorporate a nucleotide opposite the template F residue (Fig.1C, left panel, lanes 3 and 6) and could synthesize DNA pastit, but it was somewhat hindered in extending from the nucleotideincorporated opposite F, as evidenced by the presence of a stallsite opposite the lesion. Pol ${kappa}$ , on the other hand, was unableto synthesize DNA when F was present at either of the templatepositions (Fig. 1C, right panel, lanes 3 and 6). Also, in therunning-start assay, a strong stall site was observed immediatelybefore the F residue, indicating that Pol ${kappa}$ is strongly inhibitedat incorporating a nucleotide opposite the F residue.

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FIG. 1. (A) Overlay of the electrostatic potential and chemical structure of thymine (left) and the nonpolar, isosteric nucleobase analog, 2,4-difluorotoluene (right). (B) Running-start and standing-start DNA substrates. The 28-mer templates have either a T or an F at position 24, shown as a bold N. (C) Running-start and standing-start assays with Taq and human Pol ${kappa}$ . Lanes 1 to 3 are running-start experiments, and lanes 4 to 6 are standing-start experiments. 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. The stalling of synthesis by Pol ${kappa}$ 1 to 2 nucleotides prior to the end of the DNA template is a feature that is characteristic of this polymerase (10).

Steady-state kinetics of nucleotide incorporation by Pol ${kappa}$ . Next, we quantified the effects of an F residue on the efficiencyand accuracy of nucleotide incorporation by Pol ${kappa}$ , by using steady-statekinetic assays on DNA substrates that contained an F residueat the position of the incoming nucleotide or the templatingbase. From the rate of nucleotide incorporation, graphed asa function of [dNTP], and the subsequent best fit to the Michaelis-Mentenequation, the steady-state kinetic parameters k_cat and K_m andthe efficiency (k_cat/K_m) of nucleotide incorporation with respectto F substitution were determined. When F was used as the incomingdNTP opposite template A (i.e., a dFTP · A base pair),nucleotide incorporation was below detectable limits (Fig. 2A).Based upon the estimated minimal detectable levels of incorporation,the dFTP incorporation efficiencies were determined to be $≤$ 1x 10^–5. For comparison, the misincorporation of an A oppositetemplate A occurred with an efficiency of 4 x 10^–3 (Table1). We then determined the efficiency of nucleotide incorporationwhen F is the templating base (Fig. 2B). Pol ${kappa}$ incorporates anA opposite template T with an efficiency of 1.7 (Fig. 2C, leftpanel; Table 1), and it misincorporates a T opposite a templateT with an efficiency of 3.4 x 10^–4 (Table 1). When F occupiesthe position of the templating base, dATP is incorporated withan efficiency of 4.5 x 10^–3 (Fig. 2C, right panel; Table1), suggesting that disrupting the Watson-Crick hydrogen bondinteractions with the incoming dNTP results in a block whichis almost as severe as the block for mispair formation. Thus,the substitution of an F for a T, whether at the templatingposition or the incoming nucleotide position, elicits a largereduction in the efficiency and fidelity of nucleotide incorporationby Pol ${kappa}$ .

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FIG. 2. Effects of F on DNA synthesis by hPol ${kappa}$ . (A) dTTP, dFTP, or dATP incorporation opposite template A. (B) Incorporation of dATP opposite a template T or F. (C) Rates of dATP incorporation opposite a template T (left) and a template F (right), graphed as a function of [dATP]. The solid line represents the best fit to the Michaelis-Menten equation. Steady-state kinetic parameters are listed in Table 1. A portion of the DNA substrates used is shown in panel B.

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TABLE 1. Steady-state kinetic parameters for nucleotide incorporation by hPol ${kappa}$ with 2,4-difluorotoluene-containing DNA substrates

DNA synthesis by Pol ${kappa}$ on substrates containing a 3DG substitution. In addition to the geometric constraints imposed by a tightactive site, DNA polymerases can check for the correctness ofa base pair by participating in specific hydrogen bonding interactionswith the minor-groove hydrogen bond acceptors, N3 for purinesand O² for pyrimidines, in DNA (2, 4, 18, 19, 21, 22, 32, 45).Because of the absence of the O² hydrogen bond acceptor in F,we considered the possibility that the reduction in the efficiencyand fidelity of nucleotide incorporation that occurs when Fis used as a templating residue or as an incoming nucleotideoriginates from a possible dependence of Pol ${kappa}$ on minor-groovehydrogen bonding interactions with both the templating baseand the incoming nucleotide. To probe for such effects, we used3DG, which is a base analog of guanine (G) but differs fromG in having a carbon instead of a nitrogen at position 3 ofthe base (Fig. 3A). Because of the absence of an N3 hydrogenbond acceptor, the 3DG analog cannot participate in minor-groovehydrogen bonding interactions with the polymerase.

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FIG. 3. Effects of 3DG on DNA synthesis by hPol ${kappa}$ . (A) Chemical structures of guanine (left) and 3DG (right). (B) Incorporation of dGTP or d3DGTP opposite template C. A portion of the DNA substrate used is shown (top). (C) Rates of dGTP and d3DGTP incorporation opposite a template C, graphed as a function of [dNTP]. The solid line represents the best fit to the Michaelis-Menten equation. Steady-state kinetic parameters are listed in Table 2. (D) Bar graph showing the effect of 3DG substitution on the efficiency (k_cat/K_m) of correct nucleotide incorporation when a G (black) or 3DG (light gray) is used as the incoming nucleotide (P₀) opposite template C or as the templating nucleotide (T₀) for the incoming C. For comparison, the efficiencies of misincorporation of a C (P₀) opposite a template C and of a G opposite template G (T₀) are shown (dark gray).

We examined the effects of a 3DG substitution for G at the positionof the incoming nucleotide, referred to as P₀, or at the templatingposition, referred to as T₀. Compared to the efficiency of Gincorporation opposite template C, Pol ${kappa}$ incorporated 3DG withan $~$ 15-fold reduction in efficiency (Fig. 3B, C, and D; Table2). By contrast, Pol ${kappa}$ misincorporated a C opposite template Cwith an $~$ 5,000-fold reduction in efficiency (Table 2; Fig. 3D).The substitution of 3DG for G at the templating position ledto only an approximately fivefold reduction in the efficiencyof C incorporation, while the efficiency of G misincorporationopposite template G was reduced by $~$ 2,500-fold (Table 2; Fig.3D). Thus, the ablation of any possible minor-groove hydrogenbonding interactions with the P₀ and T₀ positions in DNA confersonly a small impairment in the efficiency of correct nucleotideincorporation by Pol ${kappa}$ . The results therefore indicate stronglythat the large reduction in the efficiency and fidelity of nucleotideincorporation that occurs with analog F must derive from a differentmolecular origin.

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TABLE 2. Steady-state kinetic parameters for nucleotide incorporation by hPol ${kappa}$ with 3DG-containing DNA substrates

DISCUSSION

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Discussion

Based upon thermal denaturation studies of DNA alone, the basepairing free energy differences ( ${Delta}$ ${Delta}$ G°) between correct Watson-Crickbase pairs and mismatched base pairs are as high as 4.0 kcal/mol(1, 16). At the terminus of DNA, where DNA synthesis occurs,however, selectivities are considerably smaller (27). Thus,these free energy differences are far too small to account forthe millionfold stringency manifested by replicative DNA polymerases.Furthermore, the replacement of normal nucleotides with thenucleotide analogs which impair Watson-Crick hydrogen bondinghas very little effect on the efficiency and fidelity of nucleotideincorporation by DNA polymerases such as E. coli Klenow fragmentand T7 (20, 23, 24). From such analyses, it has been inferredthat W-C hydrogen bonding is not needed for base pair synthesisby the high-fidelity polymerases.

In the high-fidelity DNA polymerases, the long ${alpha}$ helices of thefingers domain close tightly on the incoming nucleotide andthe templating residue, and the resulting snug active site mayprovide for a high degree of geometric selectivity that theseenzymes apparently possess (2, 4, 18, 19). Consequently, anyneed for W-C hydrogen bonding is minimized. By contrast, theactive site of Y family polymerases is much more open and stericallyless constrained around the nascent base pair, and this is primarilydue to the fingers domain being very small and stubby (28).As a consequence, the Y family polymerases are not as sensitiveto the geometric distortions conferred upon DNA by the presenceof lesions.

In a previous study, we showed that the substitution of an Ffor a T at the templating site or as an incoming nucleotideis highly inhibitory to DNA synthesis by yeast Pol ${eta}$ (37). Herewe provide evidence that DNA synthesis by Pol ${kappa}$ is severely impairedwhen F is used either as a templating residue or as an incomingnucleotide. From these and other observations reported here,we hypothesize that DNA synthesis by Pol ${kappa}$ is strongly dependentupon W-C hydrogen bonding, and in this respect, it closely resemblesPol ${eta}$ . We suggest that, in a relatively roomy active site, theanalog does not fill the available space and is therefore notfixed in the correct location for proper alignment of the triphosphategroup. In contrast to this, thymine would be constrained byoptimum hydrogen bonding geometry to a fixed location, thusaligning the reactive triphosphate for phosphodiester bond formation.This would also be consistent with the poor activity of mismatchedpairs, which, although hydrogen bonded, would be fixed in anunfavorable position for base pair synthesis.

In making this hypothesis, we considered a number of other possiblereasons for the poor activity of the analog F with Pol ${kappa}$ . Firstis the size of the F · A base pair, which is likely largerthan the natural T · A pair by a small amount (approximately0.3 Å), due to the lack of a hydrogen bonding contraction.However, since the enzyme can replicate through lesions andform mispairs with greater efficiency, this seems an unlikelyexplanation for the present findings. Second is the aforementionedlack of a minor-groove H bond acceptor in F (31); however, ourdata with 3DG show clearly that this lack has only a very smallkinetic effect, both in the template and in the incoming nucleotide.Since the chief chemical difference between F and T is the strongelectrostatic difference along the Watson-Crick edge, we arrivedat the lack of Watson-Crick hydrogen bonding as the best availableexplanation.

The observation that Pol ${kappa}$ shows the same high degree of apparentdependence upon W-C hydrogen bonding as Pol ${eta}$ may seem surprising,in view of the very considerable differences that exist in theirfidelities and damage bypass abilities. Whereas Pol ${eta}$ misincorporatesnucleotides with a frequency of $~$ 10^–2 to 10^–3 andis able to proficiently replicate through cyclobutane pyrimidinedimers as well as through many other sorts of DNA lesions, Pol ${kappa}$ displays a fidelity of $~$ 10^–3 to 10^–4 and is unableto replicate through cyclobutane pyrimidine dimers and otherDNA lesions, which primarily results from its inability to incorporatenucleotides opposite the lesion site. As the lower fidelityand the more proficient ability of Pol ${eta}$ to incorporate nucleotidesopposite lesions such as cyclobutane pyrimidine dimers (andothers) could be ascribed to this polymerase having a more openactive site, for Pol ${kappa}$ , the higher fidelity and the inabilityto incorporate nucleotides opposite lesion sites could suggesta more constrained active site. Consequently, Pol ${kappa}$ 's active sitecould have provided for a higher degree of geometric selectionthan that of Pol ${eta}$ , thereby lessening the dependence of Pol ${kappa}$ uponW-C hydrogen bonding. Our finding that a loss of W-C hydrogenbonding is apparently as detrimental to DNA synthesis by Pol ${kappa}$ as it is to DNA synthesis by Pol ${eta}$ , however, is more in line withthe view that, overall, the active sites of both of these polymerasesshare common structural features which do not allow for a greatdeal of geometric selectivity. That, in turn, puts a strongpremium upon W-C hydrogen bonding for the proper positioningof the nascent base pair in the active site, so that the subsequentinduced fit conformational change (44) and the chemical reactionof phosphodiester bond formation may occur. We presume thenthat the different fidelities and damage bypass abilities ofPol ${eta}$ and Pol ${kappa}$ arise from subtle structural differences in theiractive sites and not from a large global change that could haveallowed for a significantly higher degree of geometric selectivityin one polymerase than in the other.

ACKNOWLEDGMENTS

This work was supported by the National Institute of EnvironmentalHealth Sciences grant ES012411 and by grant GM072705 from NIGMS.

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.

Present address: Department of Biochemistry, University of Iowa,51 Newton Rd., Iowa City, IA 552242-1109.

REFERENCES

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