Table of Contents Author Guidelines Submit a Manuscript
Journal of Nucleic Acids
Volume 2010, Article ID 871939, 11 pages
http://dx.doi.org/10.4061/2010/871939
Research Article

Pre-Steady-State Kinetic Analysis of Truncated and Full-Length Saccharomyces cerevisiae DNA Polymerase Eta

1Department of Biochemistry, The Ohio State University, Columbus, OH 43210, USA
2Department of Chemistry, Washington University, St. Louis, MO 63130, USA
3Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA

Received 11 March 2010; Accepted 30 April 2010

Academic Editor: Ashis Basu

Copyright © 2010 Jessica A. Brown et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Understanding polymerase fidelity is an important objective towards ascertaining the overall stability of an organism's genome. Saccharomyces cerevisiae DNA polymerase (yPol), a Y-family DNA polymerase, is known to efficiently bypass DNA lesions (e.g., pyrimidine dimers) in vivo. Using pre-steady-state kinetic methods, we examined both full-length and a truncated version of yPol which contains only the polymerase domain. In the absence of yPol's C-terminal residues 514–632, the DNA binding affinity was weakened by 2-fold and the base substitution fidelity dropped by 3-fold. Thus, the C-terminus of yPol may interact with DNA and slightly alter the conformation of the polymerase domain during catalysis. In general, yPol discriminated between a correct and incorrect nucleotide more during the incorporation step (50-fold on average) than the ground-state binding step (18-fold on average). Blunt-end additions of dATP or pyrene nucleotide -triphosphate revealed the importance of base stacking during the binding of incorrect incoming nucleotides.

1. Introduction

DNA polymerases are organized into seven families: A, B, C, D, X, Y, and reverse transcriptase [1, 2]. Among these families, DNA polymerases are involved in DNA replication, DNA repair, DNA lesion bypass, antibody generation, and sister chromatid cohesion [3]. Despite these diverse roles, DNA polymerases catalyze the nucleotidyl transfer reaction using a two divalent metal ion mechanism [4] with at least one positively charged residue [5] that functions as a general acid [6] at their active site, follow a similar minimal kinetic pathway [7], and share a similar structural architecture consisting of the fingers, palm, and thumb subdomains [8, 9]. Surprisingly, the polymerization fidelity of eukaryotic DNA polymerases spans a wide range: one error per one to one billion nucleotide incorporations (100 to) [10].

The Y-family DNA polymerases are known for catalyzing nucleotide incorporation with low fidelity and poor processivity. These enzymes are specialized for translesion DNA synthesis which involves nucleotide incorporation opposite and downstream of a damaged DNA site. Lesion bypass can be either error-free or error-prone depending on the DNA polymerase and DNA lesion combination. To accommodate a distorted DNA substrate, Y-family DNA polymerases utilize several features: a solvent-accessible [11] and conformationally flexible active site [12], smaller fingers and thumb subdomains [11], an additional subdomain known as the little finger [11], the little finger and polymerase core domains move in opposite directions during a catalytic cycle [13], and a lack of exonuclease activity [14]. Unfortunately, these features, which facilitate lesion bypass, may also contribute to the low fidelity of a Y-family DNA polymerase during replication of a damaged or undamaged DNA template. Thus, it is important to understand the mechanism and fidelity of the Y-family DNA polymerases.

Saccharomyces cerevisiae DNA polymerase (yPol), a Y-family DNA polymerase, is critical for the error-free bypass of UV-induced DNA damage such as a cis-syn thymine-thymine dimer [1519]. To date, Pol remains the only Y-family DNA polymerase with a confirmed biological function [20]. yPol is organized into a polymerase domain, ubiquitin-binding zinc finger (UBZ) domain, and proliferating cell nuclear antigen- (PCNA) interacting peptide (PIP) motif (Figure 1). X-ray crystal structures of yPol’s catalytic core have been solved alone [21] as well as in complex with a cisplatin-DNA adduct and an incoming nucleotide [22]. Due to a lack of structures for full-length yPol, it is unclear if the C-terminal residues 514–632 interact with DNA and contribute to the polymerase function of yPol. Using pre-steady-state kinetic techniques, we have measured the base-substitution fidelity of full-length and truncated yPol (Figure 1) catalyzing nucleotide incorporation into undamaged DNA. In addition, we have determined the DNA binding affinity of both full-length and truncated yPol. Our results show that the C-terminus of yPol has a minor effect on the DNA binding affinity and the base substitution fidelity of this lesion bypass DNA polymerase.

871939.fig.001
Figure 1: Schematic illustration of yPol. The polymerase domain of yPol is at the N-terminus while a ubiquitin-binding zinc finger (UBZ) domain and PCNA-interacting peptide (PIP) motif is at the C-terminus. Residue numbers are denoted above each region. For this study, the truncated construct contains only the polymerase domain.

2. Materials and Methods

2.1. Materials

Materials were purchased from the following companies: [-32P] ATP, MP Biomedicals (Solon, OH); Biospin columns, Bio-Rad Laboratories (Herclues, CA); dNTPs, GE Healthcare (Piscataway, NJ); oligodeoxyribonucleotides, Integrated DNA Technologies, Inc. (Coralville, IA); and OptiKinase, USB (Cleveland, OH).

2.2. Preparation of Substrates and Enzymes

The synthetic oligodeoxyribonucleotides listed in Table 1 were purified as described previously [23]. The primer strand 21-mer or blunt-end 16-mer was -radiolabeled with [-32P]ATP and OptiKinase. Then, the 21-mer was annealed to the appropriate 41?mer template (Table 1) and the palindromic blunt-end substrates were annealed as described previously [23]. The catalytic core of yPol (1–513) containing an N-terminal MGSSH6SSGLVPRGSH tag was purified as described previously [24]. The full-length yPol (1–632) was expressed and purified from yeast [25]. Pyrene -triphosphate (dPTP) was synthesized as described previously [26].

tab1
Table 1: Sequences of DNA substrate
2.3. Pre-Steady-State Kinetic Assays

All experiments were performed in reaction buffer A which contained 40?mM Tris-HCl pH 7.5 at 23°C, 5?mM MgCl2, 1?mM DTT, 10?g/mL BSA, and 10% glycerol. A rapid chemical-quench flow apparatus (KinTek, PA, USA) was used for fast reactions. For burst assays, a preincubated solution of yPol (320?nM) and -[32P]-labeled D-1 DNA (480?nM) was mixed with dTTPM (100?M). To measure the dissociation rate of the yPolDNA binary complex, a preincubated solution of yPol (50?nM) and -[32P]-labeled D-1 DNA (100?nM) was mixed with a molar excess of unlabeled D-1 DNA (2.5?M) for various time intervals prior to initiating the polymerization reaction with dTTPMg2+ (150 and 400?M for truncated and full-length yPol, resp.) for 15?s. For single-turnover kinetic assays, a preincubated solution of yPol (150?nM) and -[32P]-labeled DNA (30?nM) was mixed with an incoming dNTPMg2+ (0.4–800?M). Reactions were quenched at the designated time by adding 0.37?M EDTA. Reaction products were analyzed by sequencing gel electrophoresis (17% acrylamide, 8?M urea, 1 TBE running buffer), visualized using a Typhoon TRIO (GE Healthcare), and quantitated with ImageQuant software (Molecular Dynamics).

2.4. DNA Binding Assays

The equilibrium dissociation constant of the yPolDNA binary complex was determined using two techniques. First, an electrophoretic mobility shift assay (EMSA) was employed by adding increasing concentrations of yPol (10–450?nM) into a fixed concentration of -[32P]-labeled D-1 DNA (10?nM) in buffer A. The solution established equilibrium during a 20-minute incubation period. Then, the binary complex was separated from unbound DNA using a 4.5% native polyacrylamide gel and running buffer as previously described except the final concentration of Tris was adjusted to 40?mM [27]. Second, a fluorescence titration assay was used. Increasing concentrations of yPol (2–300?nM) were titrated into a fixed concentration of F-8 DNA (25?nM) in buffer A (devoid of BSA). The F-8 DNA substrate (Table 1) was excited at a wavelength of 312?nm with emission and excitation slit widths of 5?nm. The emission spectra were collected at 1 nm intervals from 320 to 500?nm using a Fluoromax-4 (Jobin Jvon Horiba). Emission background from the buffer and intrinsic protein fluorescence were subtracted from each spectrum.

2.5. Data Analysis

For the pre-steady-state burst assay, the product concentration was graphed as a function of time and the data were fit to the burst equation (1) using the nonlinear regression program, KaleidaGraph (Synergy Software): A represents the fraction of active enzyme, represents the observed burst rate constant, and represents the observed steady-state rate constant.

Data for the EMSA were graphed by plotting the concentration of the binary complex as a function of enzyme concentration and fitting it to a quadratic equation (2): is the DNA concentration.

For the fluorescence titration experiments, a modified quadratic equation (3) was applied to a plot of the fluorescence intensity (F) measured at 370?nm versus enzyme concentration: and represent the maximum and minimum fluorescence intensity, respectively.

For the rate of DNA dissociation from the binary complex, a single-exponential equation (4) was applied to a plot of product concentration versus time: A represents the reaction amplitude, is the observed rate constant of DNA dissociation, and C is the concentration of the radiolabeled DNA product in the presence of a DNA trap for unlimited time.

For the single-turnover kinetic assays, a plot of product concentration versus time was fit to a single-exponential equation (5) to extract the observed rate constant of nucleotide incorporation :

To measure the maximum rate constant of incorporation and the apparent equilibrium dissociation constant of an incoming nucleotide, the extracted values were plotted as a function of nucleotide concentration and fit to a hyperbolic equation (6):

The free energy change (G) for a correct and incorrect nucleotide substrate dissociating from the EDNAdNTP complex was calculated according to (7). Here, is the universal gas constant and is the reaction temperature in Kelvin.

3. Results and Discussion

3.1. Truncated and Full-Length yPol Display Biphasic Kinetics

Previously, transient state kinetic techniques have been used to characterize full-length yPol at 30°C [28]. Therefore, we first performed a burst assay (see Section 2) to ensure that our purified proteins, truncated and full-length yPol (Figure 1), behaved in a similar manner at 23°C. Compared to wild-type yPol, the truncated construct contains only the polymerase domain (Figure 1). A preincubated solution of yPol (320?nM) and -[32P]-labeled 21/41?mer D-1 DNA (480?nM) was mixed with dTTPMg2+ (100?M) and quenched with EDTA at various times. Product concentration was plotted as a function of time and was fit to (1), since there were two distinct kinetic phases: a rapid, exponential phase and a slow, linear phase (data not shown). These burst results were similar to those previously published [28]. Biphasic kinetics of nucleotide incorporation indicated that the first turnover rate was the rate of nucleotide incorporation occurring at the enzyme’s active site while subsequent turnovers (i.e., linear phase) were likely limited by the DNA product release step as demonstrated by full-length yPol at 30°C [28] and other DNA polymerases [23, 29, 30].

3.2. The C-Terminal 119 Residues Slightly Enhance DNA Binding Affinity of yPol

The equilibrium dissociation constant for the binary complex of yPolDNA was measured to determine if the C-terminus of yPol affects DNA binding affinity (Scheme 1). First, the was estimated using the EMSA (see Section 2). For example, varying concentrations of full-length yPol (10–450?nM) were incubated with a fixed concentration of -[32P]-labeled D-1 DNA (10?nM) before separating the binary complex from the unbound DNA on a native gel (Figure 2(a)). Then, a quadratic equation (2) was applied to a plot of the binary complex concentration versus yPol concentration which resolved a of ?nM (Figure 2(b) and Table 2). Under similar reaction conditions, the of truncated yPol was estimated to be ?nM, a binding affinity (1/) value that is 2-fold weaker than that of full-length yPol (Table 2).

tab2
Table 2: Rate and equilibrium dissociation constants for the binary complex yPolDNA at 23°C.
871939.sch.001
Scheme 1
fig2
Figure 2: Equilibrium dissociation constant for full-length yPol. (a) Gel image showing binary complex formation at various concentrations of full-length yPol (10–450?nM) in the presence of -[32P]-labeled D-1 DNA (10?nM). (b) The concentration of the binary complex was plotted as a function of full-length yPol concentration and fit to (2) to yield a = 16 ± 1?nM. (c) For the fluorescence titration assay, a plot of fluorescence intensity versus full-length yPol concentration was fit to (3) which resolved a = 7 ± 4?nM.

To corroborate these estimated values, we measured the true for the yPolDNA complex using a fluorescence titration assay. An analog of dA, 2-aminopurine, was embedded into the 41?mer template of F-8 DNA which is identical to 21/41?mer D-8 DNA except that 2-aminopurine flanks the end of the templating dC base (Table 1). The F-8 DNA substrate (25?nM) was excited at 312?nm, and the emission spectrum was collected from 320 to 500?nm. After serial additions of full-length or truncated yPol in independent titrations, a decrease in the fluorescence intensity of F-8 was observed. These changes in fluorescence intensity at 370?nm were plotted as a function of the yPol concentration and were fit to (3) to extract a equal to ?nM for full-length yPol (Figure 2(c)) and ?nM for truncated yPol (Table 2). These measurements were tighter than those determined using EMSA, since the fluorescence titration assay allows yPol to associate and dissociate during data collection. In contrast, EMSA does not maintain a constant equilibrium because dissociated yPol cannot reassociate with DNA during electrophoresis separation. Nonetheless, there was a confirmed ~2-fold difference in the DNA binding affinity between full-length and the catalytic core of yPol which indicates that the C-terminal 119 amino acid residues of yPol slightly enhance the binding of the enzyme to DNA.

Next, we directly measured the rate of DNA dissociation from the yPolDNA complex (see Section 2). A preincubated solution of yPol (50?nM) and -radiolabeled D-1 DNA (100?nM) was combined with a 50-fold molar excess of unlabeled D-1 DNA for various time intervals before dTTP was added for 15 s to allow ample extension of the labeled D-1 DNA that remained in complex with yPol. A plot of product concentration versus the incubation time with the unlabeled DNA trap (data not shown) was fit to (4) which yielded DNA dissociation rates of 0.00.001? and 0.004.0008? for truncated and full-length yPol, respectively (Table 2 and Scheme 1). Interestingly, the rate of DNA dissociation from full-length yPol is 2-fold slower than that from truncated yPol, which indicated that the C-terminus of yPol may slightly contribute to this polymerase’s DNA binding affinity.

Based on the measured from Figure 2(c) and values, the apparent second-order association rate constant of the binary complex yPolDNA was calculated to be 0.62 and 0.59? for truncated and full-length yPol, respectively (Table 2). These similar values indicate that the slightly stronger DNA binding affinity of full-length yPol is mainly due to a slightly slower rate of DNA dissociation (). Taken together, the data in Table 2 suggest that the C-terminal 119 amino acid residues of yPol slightly hinder the dissociation of DNA from the binary complex yPolDNA. This hindrance is through either direct physical interactions between the C-terminus of yPol and DNA, modulation of the conformation of the polymerase domain by the C-terminus of yPol, or both.

3.3. Base Substitution Fidelity of Truncated yPol

Since a pre-steady-state burst was observed for truncated yPol, we continued to investigate the nucleotide incorporation efficiency by measuring the maximum rate of nucleotide incorporation () and the apparent equilibrium dissociation constant () of an incoming nucleotide under single-turnover conditions [31]. By performing these experiments with yPol in molar excess over DNA, the conversion of D- to D- (Scheme 1) was directly observed in a single pass through the enzymatic pathway [32]. A preincubated solution of truncated yPol (150?nM) and -[32P]-labeled D-7 DNA (30?nM) was mixed with varying concentrations of dATPMg2+ (0.4–80?M) and quenched with EDTA at various times (see Section 2). A plot of product concentration versus time was fit to (5) to extract the observed rate constant for dATP incorporation (Figure 3(a)). Then, the values were plotted as a function of dATP concentration and fit to a hyperbolic equation (6) which resolved a of 6..4? and an apparent of 1?M (Figure 3(b)). The pre-steady-state kinetic parameters for the remaining 15 possible dNTP:dN base pair combinations were determined under single-turnover conditions and were used to calculate the substrate specificity constant discrimination factor (, and fidelity (/[ + ]) of truncated yPol (Table 3).

tab3
Table 3: Kinetic parameters of nucleotide incorporation into D-DNA catalyzed by truncated yPol at 23°C.
fig3
Figure 3: Concentration dependence on the pre-steady-state rate constant of nucleotide incorporation catalyzed by truncated yPol. (a) A preincubated solution of truncated yPol (150?nM) and -[32P]-labeled D-7 DNA (30?nM) was mixed with dATPMg2+ (0.4?M, ?; 0.8?M, ?; 2?M, ¦; 4?M, ?; 8?M, ?; 16?M, ?; 40?M, ?; 80?M, ?) and quenched with EDTA at various time intervals. The solid lines are the best fits to a single-exponential equation which determined the observed rate constant, . (b) The values were plotted as a function of dATP concentration. The data (?) were then fit to a hyperbolic equation, yielding a of 6.9 ± 0.4? and a Kd of 17 ± 3?M.

Overall, the base substitution fidelity of truncated yPol was in the range of to which translates into 1 misincorporation per 100 to 10,000 nucleotide incorporations (Table 3). Depending on the mispair, truncated yPol catalyzed a misincorporation with 30- to 2,700-fold (640-fold on average) lower efficiency than the corresponding correct base pair. To better understand the mechanistic basis of truncated yPol’s fidelity, the equation for polymerase fidelity can be simplified as follows: Thus, fidelity is inversely proportional to the rate difference and apparent binding affinity difference between correct and incorrect nucleotide incorporation. In general, the mechanistic basis of yPol’s discrimination was due to a 3- to 68-fold (18-fold on average) weaker apparent binding affinity (1/Kd) and 5- to 220-fold (50-fold on average) slower rate constant of incorporation for a mismatched dNTP.

3.4. Kinetic Significance of Base Stacking Contributing to the Binding Affinity of an Incoming Nucleotide

Although all four correct dNTPs were bound with similarly high affinity (Table 3), mismatched purine deoxyribonucleotides have 2- to 6-fold lower apparent Kd values than mismatched pyrimidine deoxyribonucleotides. Because -protruding purines have been found to have stronger stacking interactions with a terminal DNA base pair than -protruding pyrimidines [33], the difference in apparent Kd values suggests that base-stacking interactions between an incorrect dNTP and the terminal primer/template base pair dA:dT (Table 1) play a role on the binding of dNTP by truncated yPol. Interestingly, we have previously demonstrated that the preferred nucleotide for template-independent nucleotide incorporation catalyzed by Dpo4, another Y-family DNA polymerase, is dATP mainly due to its strong intrahelical base-stacking ability [26]. To further evaluate the role of base stacking, we first examined if truncated yPol can catalyze template-independent nucleotide incorporation of dATP or dPTP (Figure 4) onto four palindromic, blunt-end DNA substrates (BE1, BE2, BE3, and BE4 in Table 1). The base of dPTP, a dNTP analog, has four conjugated benzene rings but possesses no hydrogen-bonding abilities. The DNA substrates possess all four possible terminal base pairs and each molecule of them can be bound by a single polymerase molecule. Our radioactive experiments showed that truncated yPol was able to incorporate dATP and dPTP (data not shown). Then, we individually measured the kinetic parameters for dATP and dPTP incorporation under single-turnover reaction conditions (Table 4). Interestingly, the apparent Kd values of dATP were 3- to 5-fold smaller with a purine than those with a pyrimidine on the primer’s -base, indicating that base stacking is also important for the binding of dATP to the binary complex of yPolblunt-end DNA. This base-stacking effect is more dramatic for dPTP incorporation onto blunt-end DNA because the apparent Kd values of dPTP are 10- to 80-fold tighter than dATP incorporation onto the same blunt-end DNA substrate (Table 4). Thus, the binding free energy difference between dATP and dPTP is 1.4 to 2.6??kcal/mol. Previously, we have obtained a comparable binding free energy difference of 2.3?kcal/mol for similar blunt-end dATP and dPTP incorporation at 37°C catalyzed by Dpo4 [26]. Although neither dATP nor dPTP forms any hydrogen bonds with a template base when bound by yPolblunt-end DNA, the bases of these two nucleotides should have different base-stacking interactions with a terminal base pair of a blunt-end DNA substrate considering that a dangling pyrene base (1.7?kcal/mol) has previously been found to possess a higher base-stacking free energy than a dangling adenosine (1.0 kcal/mol) [33]. However, the base-stacking free energy difference (0.7?kcal/mol) between pyrene and adenosine is smaller than the aforementioned binding free energy difference (1.4–2.6?kcal/mol) between dPTP and dATP. Thus, other sources likely contribute to the tighter binding of dPTP over dATP. One possible source is favorable van der Waals interactions between pyrene and active site residues of truncated yPol. In addition, the base-stacking effect and van der Waals interactions may stabilize the ternary complex of yPolblunt-end DNAnucleotide and facilitate catalysis, leading to much higher kp values with dPTP than those with dATP (Table 4). Due to the differences in kp and apparent Kd, the substrate specificity values of dPTP are 100- to 1,000-fold higher than those of dATP with blunt-end DNA (Table 4) and 10- to 100-fold higher than mismatched dATP with regular DNA (Table 3).

tab4
Table 4: Kinetic parameters for nucleotide incorporation onto blunt-end DNA catalyzed by truncated yeast Pol at 23°C.
871939.fig.004
Figure 4: Chemical structure of a nonnatural nucleotide analog, dPTP.
3.5. Base Substitution Fidelity of Full-Length yPol

The base substitution fidelities of full-length and truncated yPol may differ because the C-terminal, nonenzymatic regions may alter the polymerization fidelity. For example, the proline-rich domain of human DNA polymerase has been shown to upregulate the polymerase fidelity up to 100-fold [34]. To determine if the C-terminus of yPol influences polymerization fidelity, we measured the pre-steady-state kinetic parameters for dNTP incorporation into D-1 DNA (template dA) catalyzed by full-length yPol (Table 5). The fidelity was calculated to be in the range of (1.4 to 2.6) 10-3 for full-length yPol (Table 5). Relative to the fidelity of truncated yPol with D-1 (Table 3), full-length yPol has a 3-fold higher fidelity. Therefore, the C-terminus of yPol slightly affects the base substitution fidelity. Moreover, truncated yPol discriminated between a correct and incorrect dNTP by ~30-fold on average based on the kp difference while the discrimination for full-length yPol was ~170-fold on average for incorporation into D-1 DNA (Tables 3 and 5). The incorporation rate constant for correct dTTP was 4?s-1 for both yPol enzymes, but the misincorporation rate was 3- to 23-fold faster for truncated yPol. This rate enhancement for truncated yPol is partially offset by a greater discrimination at the apparent ground-state binding level so that the fidelity of truncated yPol was only 3-folder lower than that of full-length yPol.

tab5
Table 5: Kinetic parameters of nucleotide incorporation into D-1 DNA catalyzed by full-length yPol at 23°C.
3.6. Effect of the Nonenzymatic C-Terminus of yPol on Its Polymerase Activity

Our above studies demonstrated that the C-terminus of yPol enhances this enzyme’s DNA binding affinity and base substitution fidelity by 2- and 3-fold, respectively. These results suggest that the nonenzymatic, C-terminal region of yPol (Figure 1) has a mild impact on the N-terminal polymerase domain and its activity. This conclusion is inconsistent with previous studies which have qualitatively demonstrated that mutations or deletions in the UBZ domain or PIP motif do not affect polymerase activity [3537]. However, these reported qualitative assays are not sufficiently sensitive to detect the small perturbation on polymerase activity as described in this paper. The presence of the C-terminal 119 residues of yPol may either interact with DNA, slightly alter the conformation of the polymerase domain, or both (see above discussion), thereby enhancing its DNA binding affinity and polymerase fidelity.

3.7. Kinetic Comparison among Y-Family DNA Polymerases

The fidelity of several Y-family DNA polymerases synthesizing undamaged DNA has been determined by employing steady-state [3848], pre-steady-state [28, 30, 4953], or M13-based mutation assays [39, 41, 42, 45, 54, 55]. From these studies, the fidelity ranges from 100 to 10-4. Under steady-state reaction conditions, the base substitution fidelity of yPol and human Pol has been measured to be in the range from 10-2 to 10-4 and 10-2 to 10-3, respectively [38, 40], which is similar to our pre-steady-state kinetic results. Consistently, Pol displays the highest substrate specificity for the dCTP?:?dG base pair under both steady-state and pre-steady-state reaction conditions (Table 3 and unpublished data, Brown and Suo) [38, 40]. This may seem surprising, since Pol participates in the efficient bypass of UV-induced DNA damage such as a cis-syn thymine-thymine dimer (i.e., a dATP:dT base pair) [1520, 56, 57]. However, Pol has also been shown to be efficient at bypassing guanine-specific damage such as 8-oxo-7,8-dihydro-dG [58, 59], 1,2-cis-diammineplatinum(II)-d(GpG) intrastrand cross-links [6063], and various N2-dG lesions [64, 65].

Among the four eukaryotic Y-family DNA polymerases (i.e., Pol, DNA polymerase , DNA polymerase (Pol), and Rev1), Rev1 exhibits low fidelity on undamaged DNA due to its strong preference for inserting dCTP [46, 52] while Pol has an unusual preference for dGTP:dT mispairs over dATP:dT due to Hoogsteen base pair formation [51, 69]. Interestingly, the lowest fidelity base pair for truncated yPol was dGTP:dT (Table 3). This observation likely results from the formation of a wobble base pair. The two hydrogen bonds established in the wobble base pair may enhance the catalytic efficiency of yPol since hydrogen bonding is important for the efficiency and accuracy of yPol [70]. Also noteworthy, the truncated versions of eukaryotic Y-family DNA polymerases have been used for many biochemical studies in literature. Based on our quantitative kinetic analysis of yPol, these results suggested the nonenzymatic regions of Y-family DNA polymerases do not alter the polymerase activity significantly.

3.8. Fidelity Comparison among Various DNA Polymerase Families

As a Y-family DNA polymerase, yPol displays low fidelity on undamaged DNA (Tables 3 and 5) [38]. In contrast, replicative DNA polymerases in the A- and B-families have a polymerization fidelity that is 1–3 orders of magnitude greater than the Y-family DNA polymerases (Table 6). DNA polymerases with higher fidelity are more proficient at using the ground-state binding affinity to discriminate between a correct and incorrect dNTP. The Y-family DNA polymerases provide little to no discrimination based on the Kd difference while replicative DNA polymerases discriminate up to almost three orders of magnitude. This lack of selection in the ground state by the Y-family DNA polymerases may be due to the relatively loose and solvent-accessible active site which has minimal contacts with the nascent base pair [11, 21, 71]. Moreover, nucleotide selection by the Y-family DNA polymerases in the ground state may be mainly governed by Watson-Crick base pairing, since the calculated G values (0.95–1.7?kcal/mol) are similar to the free energy differences between correct and incorrect base pairs (0.3–1.0?kcal/mol at 37°C) at the primer terminus based on DNA melting studies (Table 6) [72]. However, with G values 3.0?kcal/mol, the replicative DNA polymerases harness the additional 2.0?kcal/mol of energy from other sources such as a tight active site or close contacts with the nascent base pair. One common fidelity checkpoint among DNA polymerases is the varying rate differences between a matched and mismatched base pair. These large differences may correspond to different rate-limiting steps (e.g., protein conformational change, or phosphodiester bond formation) during nucleotide incorporation [9, 30, 71]. For yPol, kinetic data suggest that correct and incorrect dNTPs are limited by a conformational step preceding chemistry, although, additional studies are needed to confirm these results [28].

tab6
Table 6: Comparison of base substitution fidelity for various DNA polymerases.

4. Conclusions

This work presents the mechanistic basis of the base substitution fidelity of yPol on undamaged DNA, which examined all possible dNTP:dN base pair combinations for the first time. yPol discriminates against incorrect nucleotides at both the ground-state nucleotide binding and incorporation steps. Furthermore, base stacking contributes to tighter binding for a misincorporation. Finally, the 119 residues at the C-terminus have a mild impact on the kinetic mechanism of yPol.

Abbreviations

BSA: Bovine serum albumin
dNTP:2'-deoxynucleoside 5'-triphosphate
Dpo4:Sulfolobus solfataricus P2 DNA polymerase IV
dPTP:Pyrene 5'-triphosphate
EMSA:Electrophoretic mobility shift assay
HPol?:Human mitochondrial DNA polymerase gamma
PCNA:Proliferating cell nuclear antigen
PIP:PCNA-interacting peptide
PolB1:Exonuclease-deficient DNA polymerase B1 from
?sulfolobus solfataricus P2
Pol?:DNA polymerase iota
rPolß:Rat DNA polymerase beta
TBETris/boric acid/EDTA
UBZ:Ubiquitin-binding zinc finger
YPol?:Saccharomyces cerevisiae DNA polymerase eta.

Acknowledgments

This work was supported by the National Institutes of Health Grants CA040463 (to Zucai Suo and John-Stephen Taylor) and GM032431 (to Peter M.J. Burgers). Jessica A. Brown was supported by an American Heart Association Pre-doctoral Fellowship (Grant 0815382D). Shanen M. Sherrer was supported by a Predoctoral Fellowship from the National Institutes of Health Chemistry-Biology Interface Program at The Ohio State University (Grant 5 T32 GM008512-13). Brown and Zhang contributed equally to this work.

References

  1. J. D. Fowler and Z. Suo, “Biochemical, structural, and physiological characterization of terminal deoxynucleotidyl transferase,” Chemical Reviews, vol. 106, no. 6, pp. 2092–2110, 2006. View at Publisher · View at Google Scholar · View at Scopus
  2. M. Garcia-Diaz and K. Bebenek, “Multiple functions of DNA polymerases,” Critical Reviews in Plant Sciences, vol. 26, no. 2, pp. 105–122, 2007. View at Publisher · View at Google Scholar · View at Scopus
  3. P. M. J. Burgers, E. V. Koonin, E. Bruford et al., “Eukaryotic DNA polymerases: proposal for a revised nomenclature,” Journal of Biological Chemistry, vol. 276, no. 47, pp. 43487–43490, 2001. View at Publisher · View at Google Scholar · View at Scopus
  4. T. A. Steitz, “DNA- and RNA-dependent DNA polymerases,” Current Opinion in Structural Biology, vol. 3, no. 1, pp. 31–38, 1993. View at Publisher · View at Google Scholar · View at Scopus
  5. J. D. Fowler, J. A. Brown, M. Kvaratskhelia, and Z. Suo, “Probing conformational changes of human DNA polymerase lambda using mass spectrometry-based protein footprinting,” Journal of Molecular Biology, vol. 390, no. 3, pp. 368–379, 2009. View at Publisher · View at Google Scholar · View at Scopus
  6. C. Castro, E. D. Smidansky, J. J. Arnold et al., “Nucleic acid polymerases use a general acid for nucleotidyl transfer,” Nature Structural and Molecular Biology, vol. 16, no. 2, pp. 212–218, 2009. View at Publisher · View at Google Scholar · View at Scopus
  7. A. A. Johnson, K. A. Fiala, and Z. Suo, “DNA polymerases and their interactions with DNA and nucleotides,” in Nucleoside Triphosphates and Their Analogs: Chemistry, Biotechnology, and Biological Applications, M. M. Vaghefi, Ed., pp. 133–168, Taylor & Francis, Boca Raton, Fla, USA, 2005. View at Google Scholar
  8. T. A. Steitz, “DNA polymerases: structural diversity and common mechanisms,” Journal of Biological Chemistry, vol. 274, no. 25, pp. 17395–17398, 1999. View at Publisher · View at Google Scholar · View at Scopus
  9. C. M. Joyce and S. J. Benkovic, “DNA polymerase fidelity: kinetics, structure, and checkpoints,” Biochemistry, vol. 43, no. 45, pp. 14317–14324, 2004. View at Publisher · View at Google Scholar · View at Scopus
  10. S. D. McCulloch and T. A. Kunkel, “The fidelity of DNA synthesis by eukaryotic replicative and translesion synthesis polymerases,” Cell Research, vol. 18, no. 1, pp. 148–161, 2008. View at Publisher · View at Google Scholar · View at Scopus
  11. H. Ling, F. Boudsocq, R. Woodgate, and W. Yang, “Crystal structure of a Y-family DNA polymerase in action: a mechanism for error-prone and lesion-bypass replication,” Cell, vol. 107, no. 1, pp. 91–102, 2001. View at Publisher · View at Google Scholar · View at Scopus
  12. S. Mizukami, T. W. Kim, S. A. Helquist, and E. T. Kool, “Varying DNA base-pair size in subangstrom increments: evidence for a loose, not large, active site in low-fidelity Dpo4 polymerase,” Biochemistry, vol. 45, no. 9, pp. 2772–2778, 2006. View at Publisher · View at Google Scholar · View at Scopus
  13. C. Xu, B. A. Maxwell, J. A. Brown, L. Zhang, and Z. Suo, “Global conformational dynamics of a Y-family DNA polymerase during catalysis,” PLoS Biology, vol. 7, Article ID e1000225, 2009. View at Google Scholar
  14. H. Ohmori, E. C. Friedberg, R. P. P. Fuchs et al., “The Y-family of DNA Polymerases,” Molecular Cell, vol. 8, no. 1, pp. 7–8, 2001. View at Publisher · View at Google Scholar · View at Scopus
  15. J. P. McDonald, A. S. Levine, and R. Woodgate, “The Saccharomyces cerevisiae RAD30 gene, a homologue of Escherichia coli dinB and umuC, is DNA damage inducible and functions in a novel error-free postreplication repair mechanism,” Genetics, vol. 147, no. 4, pp. 1557–1568, 1997. View at Google Scholar · View at Scopus
  16. R. E. Johnson, S. Prakash, and L. Prakash, “Requirement of DNA polymerase activity of yeast Rad30 protein for its biological function,” Journal of Biological Chemistry, vol. 274, no. 23, pp. 15975–15977, 1999. View at Publisher · View at Google Scholar · View at Scopus
  17. A. A. Roush, M. Suarez, E. C. Friedberg, M. Radman, and W. Siede, “Deletion of the Saccharomyces cerevisiae gene RAD30 encoding an Escherichia coli DinB homolog confers UV radiation sensitivity and altered mutability,” Molecular and General Genetics, vol. 257, no. 6, pp. 686–692, 1998. View at Publisher · View at Google Scholar · View at Scopus
  18. R. E. Johnson, S. Prakash, and L. Prakash, “Efficient bypass of a thymine-thymine dimer by yeast DNA polymerase, Polη,” Science, vol. 283, no. 5404, pp. 1001–1004, 1999. View at Publisher · View at Google Scholar · View at Scopus
  19. S.-L. Yu, R. E. Johnson, S. Prakash, and L. Prakash, “Requirement of DNA polymerase η for error-free bypass of UV-induced CC and TC photoproducts,” Molecular and Cellular Biology, vol. 21, no. 1, pp. 185–188, 2001. View at Publisher · View at Google Scholar · View at Scopus
  20. C. Masutani, R. Kusumoto, A. Yamada et al., “The XPV (xeroderma pigmentosum variant) gene encodes human DNA polymerase η,” Nature, vol. 399, no. 6737, pp. 700–704, 1999. View at Publisher · View at Google Scholar · View at Scopus
  21. J. Trincao, R. E. Johnson, C. R. Escalante, S. Prakash, L. Prakash, and A. K. Aggarwal, “Structure of the catalytic core of S. cerevisiae DNA polymerase η: implications for translesion DNA synthesis,” Molecular Cell, vol. 8, no. 2, pp. 417–426, 2001. View at Publisher · View at Google Scholar · View at Scopus
  22. A. Alt, K. Lammens, C. Chiocchini et al., “Bypass of DNA lesions generated during anticancer treatment with cisplatin by DNA polymerase η,” Science, vol. 318, no. 5852, pp. 967–970, 2007. View at Publisher · View at Google Scholar · View at Scopus
  23. K. A. Fiala and Z. Suo, “Mechanism of DNA polymerization catalyzed by Sulfolobus solfataricus P2 DNA polymerase IV,” Biochemistry, vol. 43, no. 7, pp. 2116–2125, 2004. View at Publisher · View at Google Scholar · View at Scopus
  24. V. J. Cannistraro and J.-S. Taylor, “DNA-thumb interactions and processivity of T7 DNA polymerase in comparison to yeast polymerase η,” Journal of Biological Chemistry, vol. 279, no. 18, pp. 18288–18295, 2004. View at Publisher · View at Google Scholar · View at Scopus
  25. P. Garg, C. M. Stith, J. Majka, and P. M. J. Burgers, “Proliferating cell nuclear antigen promotes translesion synthesis by DNA polymerase ζ,” Journal of Biological Chemistry, vol. 280, no. 25, pp. 23446–23450, 2005. View at Publisher · View at Google Scholar · View at Scopus
  26. K. A. Fiala, J. A. Brown, H. Ling et al., “Mechanism of template-independent nucleotide incorporation catalyzed by a template-dependent DNA polymerase,” Journal of Molecular Biology, vol. 365, no. 3, pp. 590–602, 2007. View at Publisher · View at Google Scholar · View at Scopus
  27. K. A. Fiala, C. D. Hypes, and Z. Suo, “Mechanism of abasic lesion bypass catalyzed by a Y-family DNA polymerase,” Journal of Biological Chemistry, vol. 282, no. 11, pp. 8188–8198, 2007. View at Publisher · View at Google Scholar · View at Scopus
  28. M. T. Washington, L. Prakash, and S. Prakash, “Yeast DNA polymerase η utilizes an induced-fit mechanism of nucleotide incorporation,” Cell, vol. 107, no. 7, pp. 917–927, 2001. View at Publisher · View at Google Scholar · View at Scopus
  29. J. A. Brown and Z. Suo, “Elucidating the kinetic mechanism of DNA polymerization catalyzed by Sulfolobus solfataricus P2 DNA polymerase B1,” Biochemistry, vol. 48, no. 31, pp. 7502–7511, 2009. View at Publisher · View at Google Scholar · View at Scopus
  30. K. A. Fiala, S. M. Sherrer, J. A. Brown, and Z. Suo, “Mechanistic consequences of temperature on DNA polymerization catalyzed by a Y-family DNA polymerase,” Nucleic Acids Research, vol. 36, no. 6, pp. 1990–2001, 2008. View at Publisher · View at Google Scholar · View at Scopus
  31. Y.-C. Tsai and K. A. Johnson, “A new paradigm for DNA polymerase specificity,” Biochemistry, vol. 45, no. 32, pp. 9675–9687, 2006. View at Publisher · View at Google Scholar · View at Scopus
  32. K. A. Johnson, “Transient-state kinetic analysis of enzyme reaction pathways,” Enzymes, vol. 20, pp. 1–61, 1992. View at Google Scholar
  33. K. M. Guckian, B. A. Schweitzer, R. X.-F. Ren, C. J. Sheils, D. C. Tahmassebi, and E. T. Kool, “Factors contributing to aromatic stacking in water: evaluation in the context of DNA,” Journal of the American Chemical Society, vol. 122, no. 10, pp. 2213–2222, 2000. View at Publisher · View at Google Scholar · View at Scopus
  34. K. A. Fiala, W. W. Duym, J. Zhang, and Z. Suo, “Up-regulation of the fidelity of human DNA polymerase λ by its non-enzymatic proline-rich domain,” Journal of Biological Chemistry, vol. 281, no. 28, pp. 19038–19044, 2006. View at Publisher · View at Google Scholar · View at Scopus
  35. L. Haracska, C. M. Kondratick, I. Unk, S. Prakash, and L. Prakash, “Interaction with PCNA is essential for yeast DNA polymerase η function,” Molecular Cell, vol. 8, no. 2, pp. 407–415, 2001. View at Publisher · View at Google Scholar · View at Scopus
  36. N. Acharya, A. Brahma, L. Haracska, L. Prakash, and S. Prakash, “Mutations in the ubiquitin binding UBZ motif of DNA polymerase η do not impair its function in translesion synthesis during replication,” Molecular and Cellular Biology, vol. 27, no. 20, pp. 7266–7272, 2007. View at Publisher · View at Google Scholar · View at Scopus
  37. Z. Zhuang, R. E. Johnson, L. Haracska, L. Prakash, S. Prakash, and S. J. Benkovic, “Regulation of polymerase exchange between Polη and Polδ by monoubiquitination of PCNA and the movement of DNA polymerase holoenzyme,” Proceedings of the National Academy of Sciences of the United States of America, vol. 105, no. 14, pp. 5361–5366, 2008. View at Publisher · View at Google Scholar · View at Scopus
  38. M. T. Washington, R. E. Johnson, S. Prakash, and L. Prakash, “Fidelity and processivity of Saccharomyces cerevisiae DNA polymerase η,” Journal of Biological Chemistry, vol. 274, no. 52, pp. 36835–36838, 1999. View at Publisher · View at Google Scholar · View at Scopus
  39. T. Matsuda, K. Bebenek, C. Masultanl, F. Hanaoka, and T. A. Kunkel, “Low fidelity DNA synthesis by human DNA polymerase-η,” Nature, vol. 404, no. 6781, pp. 1011–1013, 2000. View at Publisher · View at Google Scholar · View at Scopus
  40. R. E. Johnson, M. T. Washington, S. Prakash, and L. Prakash, “Fidelity of human DNA polymerase η,” Journal of Biological Chemistry, vol. 275, no. 11, pp. 7447–7450, 2000. View at Publisher · View at Google Scholar · View at Scopus
  41. Y. Zhang, F. Yuan, H. Xin et al., “Human DNA polymerase κ synthesizes DNA with extraordinarily low fidelity,” Nucleic Acids Research, vol. 28, no. 21, pp. 4147–4156, 2000. View at Google Scholar · View at Scopus
  42. E. Ohashi, K. Bebenek, T. Matsuda et al., “Fidelity and processivity of DNA synthesis by DNA polymerase κ, the product of the human DINB1 gene,” Journal of Biological Chemistry, vol. 275, no. 50, pp. 39678–39684, 2000. View at Publisher · View at Google Scholar · View at Scopus
  43. A. Tissier, J. P. McDonald, E. G. Frank, and R. Woodgate, “polι, a remarkably error-prone human DNA polymerase,” Genes and Development, vol. 14, no. 13, pp. 1642–1650, 2000. View at Google Scholar · View at Scopus
  44. F. Boudsocq, S. Iwai, F. Hanaoka, and R. Woodgate, “Sulfolobus solfataricus P2 DNA polymerase IV (Dp04): an archaeal DinB-like DNA polymerase with lesion-bypass properties akin to eukaryotic polη,” Nucleic Acids Research, vol. 29, no. 22, pp. 4607–4616, 2001. View at Google Scholar · View at Scopus
  45. R. J. Kokoska, K. Bebenek, F. Boudsocq, R. Woodgate, and T. A. Kunkel, “Low fidelity DNA synthesis by a Y family DNA polymerase due to misalignment in the active site,” Journal of Biological Chemistry, vol. 277, no. 22, pp. 19633–19638, 2002. View at Publisher · View at Google Scholar · View at Scopus
  46. L. Haracska, S. Prakash, and L. Prakash, “Yeast Rev1 protein is a G template-specific DNA polymerase,” Journal of Biological Chemistry, vol. 277, no. 18, pp. 15546–15551, 2002. View at Publisher · View at Google Scholar · View at Scopus
  47. A. Vaisman, H. Ling, R. Woodgate, and W. Yang, “Fidelity of Dpo4: effect of metal ions, nucleotide selection and pyrophosphorolysis,” EMBO Journal, vol. 24, no. 17, pp. 2957–2967, 2005. View at Publisher · View at Google Scholar · View at Scopus
  48. Y. Zhang, X. Wu, O. Rechkoblit, N. E. Geacintov, J.-S. Taylor, and Z. Wang, “Response of human REV1 to different DNA damage: preferential dCMP insertion opposite the lesion,” Nucleic Acids Research, vol. 30, no. 7, pp. 1630–1638, 2002. View at Google Scholar · View at Scopus
  49. M. T. Washington, R. E. Johnson, L. Prakash, and S. Prakash, “The mechanism of nucleotide incorporation by human DNA polymerase η differs from that of the yeast enzyme,” Molecular and Cellular Biology, vol. 23, no. 22, pp. 8316–8322, 2003. View at Publisher · View at Google Scholar · View at Scopus
  50. K. A. Fiala and Z. Suo, “Pre-steady-state kinetic studies of the fidelity of Sulfolobus solfataricus P2 DNA polymerase IV,” Biochemistry, vol. 43, no. 7, pp. 2106–2115, 2004. View at Publisher · View at Google Scholar · View at Scopus
  51. M. T. Washington, R. E. Johnson, L. Prakash, and S. Prakash, “Human DNA polymerase ι utilizes different nucleotide incorporation mechanisms dependent upon the template base,” Molecular and Cellular Biology, vol. 24, no. 2, pp. 936–943, 2004. View at Publisher · View at Google Scholar · View at Scopus
  52. C. A. Howell, S. Prakash, and M. T. Washington, “Pre-steady-state kinetic studies of protein-template-directed nucleotide incorporation by the yeast Rev1 protein,” Biochemistry, vol. 46, no. 46, pp. 13451–13459, 2007. View at Publisher · View at Google Scholar · View at Scopus
  53. S. M. Sherrer, J. A. Brown, L. R. Pack et al., “Mechanistic studies of the bypass of a bulky single-base lesion catalyzed by a Y-family DNA polymerase,” Journal of Biological Chemistry, vol. 284, no. 10, pp. 6379–6388, 2009. View at Publisher · View at Google Scholar · View at Scopus
  54. O. Potapova, N. D. F. Grindley, and C. M. Joyce, “The mutational specificity of the Dbh lesion bypass polymerase and its implications,” Journal of Biological Chemistry, vol. 277, no. 31, pp. 28157–28166, 2002. View at Publisher · View at Google Scholar · View at Scopus
  55. F. Boudsocq, R. J. Kokoska, B. B. Plosky et al., “Investigating the role of the little finger domain of Y-family DNA polymerases in low fidelity synthesis and translesion replication,” Journal of Biological Chemistry, vol. 279, no. 31, pp. 32932–32940, 2004. View at Publisher · View at Google Scholar · View at Scopus
  56. R. E. Johnson, C. M. Kondratick, S. Prakash, and L. Prakash, “hRAD30 mutations in the variant form of xeroderma pigmentosum,” Science, vol. 285, no. 5425, pp. 263–265, 1999. View at Publisher · View at Google Scholar · View at Scopus
  57. M. T. Washington, L. Prakash, and S. Prakash, “Mechanism of nucleotide incorporation opposite a thymine-thymine dimer by yeast DNA polymerase η,” Proceedings of the National Academy of Sciences of the United States of America, vol. 100, no. 21, pp. 12093–12098, 2003. View at Publisher · View at Google Scholar · View at Scopus
  58. L. Haracska, S.-L. Yu, R. E. Johnson, L. Prakash, and S. Prakash, “Efficient and accurate replication in the presence of 7,8-dihydro-8-oxoguanine by DNA polymerase η,” Nature Genetics, vol. 25, no. 4, pp. 458–461, 2000. View at Publisher · View at Google Scholar · View at Scopus
  59. K. D. Carlson and M. T. Washington, “Mechanism of efficient and accurate nucleotide incorporation opposite 7,8-dihydro-8-oxoguanine by Saccharomyces cerevisiae DNA polymerase η,” Molecular and Cellular Biology, vol. 25, no. 6, pp. 2169–2176, 2005. View at Publisher · View at Google Scholar · View at Scopus
  60. A. Vaisman, C. Masutani, F. Hanaoka, and S. G. Chaney, “Efficient translesion replication past oxaliplatin and cisplatin GpG adducts by human DNA polymerase η,” Biochemistry, vol. 39, no. 16, pp. 4575–4580, 2000. View at Publisher · View at Google Scholar · View at Scopus
  61. C. Masutani, R. Kusumoto, S. Iwai, and F. Hanaoka, “Mechanisms of accurate translesion synthesis by human DNA polymerase η,” EMBO Journal, vol. 19, no. 12, pp. 3100–3109, 2000. View at Google Scholar · View at Scopus
  62. E. Bassett, A. Vaisman, K. A. Tropea et al., “Frameshifts and deletions during in vitro translesion synthesis past Pt-DNA adducts by DNA polymerases β and η,” DNA Repair, vol. 1, no. 12, pp. 1003–1016, 2002. View at Publisher · View at Google Scholar · View at Scopus
  63. E. Bassett, A. Vaisman, J. M. Havener, C. Masutani, F. Hanaoka, and S. G. Chaney, “Efficiency of extension of mismatched primer termini across from cisplatin and oxaliplatin adducts by human DNA polymerases β and η in vitro,” Biochemistry, vol. 42, no. 48, pp. 14197–14206, 2003. View at Publisher · View at Google Scholar · View at Scopus
  64. J.-Y. Choi and F. P. Guengerich, “Adduct size limits efficient and error-free bypass across bulky N 2-guanine DNA lesions by human DNA polymerase η,” Journal of Molecular Biology, vol. 352, no. 1, pp. 72–90, 2005. View at Publisher · View at Google Scholar · View at Scopus
  65. J.-Y. Choi, H. Zang, K. C. Angel et al., “Translesion synthesis across 1,N2-ethenoguanine by human DNA polymerases,” Chemical Research in Toxicology, vol. 19, no. 6, pp. 879–886, 2006. View at Publisher · View at Google Scholar · View at Scopus
  66. J. Ahn, V. S. Kraynov, X. Zhong, B. G. Werneburg, and M.-D. Tsai, “DNA polymerase β: effects of gapped DNA substrates on dNTP specificity, fidelity, processivity and conformational changes,” Biochemical Journal, vol. 331, part 1, pp. 79–87, 1998. View at Google Scholar · View at Scopus
  67. L. Zhang, J. A. Brown, S. A. Newmister, and Z. Suo, “Polymerization fidelity of a replicative DNA polymerase from the hyperthermophilic archaeon Sulfolobus solfataricus P2,” Biochemistry, vol. 48, no. 31, pp. 7492–7501, 2009. View at Publisher · View at Google Scholar · View at Scopus
  68. H. R. Lee and K. A. Johnson, “Fidelity of the human mitochondrial DNA polymerase,” Journal of Biological Chemistry, vol. 281, no. 47, pp. 36236–36240, 2006. View at Publisher · View at Google Scholar · View at Scopus
  69. D. T. Nair, R. E. Johnson, S. Prakash, L. Prakash, and A. K. Aggarwal, “Replication by human DNA polymerase-ι occurs by Hoogsteen base-pairing,” Nature, vol. 430, no. 6997, pp. 377–380, 2004. View at Publisher · View at Google Scholar · View at Scopus
  70. M. T. Washington, S. A. Helquist, E. T. Kool, L. Prakash, and S. Prakash, “Requirement of Watson-Crick hydrogen bonding for DNA synthesis by yeast DNA polymerase η,” Molecular and Cellular Biology, vol. 23, no. 14, pp. 5107–5112, 2003. View at Publisher · View at Google Scholar · View at Scopus
  71. J. H. Wong, K. A. Fiala, Z. Suo, and H. Ling, “Snapshots of a Y-family DNA polymerase in replication: substrate-induced conformational transitions and implications for fidelity of Dpo4,” Journal of Molecular Biology, vol. 379, no. 2, pp. 317–330, 2008. View at Publisher · View at Google Scholar · View at Scopus
  72. J. Petruska, M. F. Goodman, M. S. Boosalis, L. C. Sowers, C. Cheong, and I. Tinoco Jr., “Comparison between DNA melting thermodynamics and DNA polymerase fidelity,” Proceedings of the National Academy of Sciences of the United States of America, vol. 85, no. 17, pp. 6252–6256, 1988. View at Google Scholar · View at Scopus