MOLECULAR AND CELLULAR BIOLOGY, Nov. 2003, p. 8316–8322 0270-7306/03/$08.00⫹0 DOI: 10.1128/MCB.23.22.8316–8322.2003 Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Vol. 23, No. 22
The Mechanism of Nucleotide Incorporation by Human DNA Polymerase Differs from That of the Yeast Enzyme M. Todd Washington, Robert E. Johnson, Louise Prakash, and Satya Prakash* Sealy Center for Molecular Science, University of Texas Medical Branch at Galveston, Galveston, Texas 77555-1061 Received 3 July 2003/Accepted 11 August 2003
DNA polymerase (Pol) catalyzes the efficient and accurate synthesis of DNA opposite cyclobutane pyrimidine dimers, and inactivation of Pol in humans causes the cancer-prone syndrome, the variant form of xeroderma pigmentosum. Pre-steady-state kinetic studies of yeast Pol have indicated that the low level of fidelity of this enzyme results from a poorly discriminating induced-fit mechanism. Here we examine the mechanistic basis of the low level of fidelity of human Pol. Because the human and yeast enzymes behave similarly under steady-state conditions, we expected these enzymes to utilize similar mechanisms of nucleotide incorporation. Surprisingly, however, we find that human Pol differs from the yeast enzyme in several important respects. The human enzyme has a 50-fold-faster rate of nucleotide incorporation than the yeast enzyme but binds the nucleotide with an approximately 50-fold-lower level of affinity. This lower level of binding affinity might provide a means of regulation whereby the human enzyme remains relatively inactive except when the cellular deoxynucleoside triphosphate concentrations are high, as may occur during DNA damage, thereby avoiding the mutagenic consequences arising from the inadvertent action of this enzyme during normal DNA replication. DNA polymerase (Pol) is unique among eukaryotic DNA polymerases in its proficient and accurate ability to replicate through UV-induced cyclobutane pyrimidine dimers (9, 11, 14, 22). Inactivation of Pol in Saccharomyces cerevisiae (10, 16, 27) as well as in humans (21, 25) results in increased UV-induced mutagenesis, and a lack of Pol in humans causes the variant form of xeroderma pigmentosum (8, 14), a syndrome characterized by an elevated incidence of sunlight-induced skin cancers. Thus, Pol acts to reduce the frequency of UV-induced mutagenesis and carcinogenesis. Biochemical studies have shown that both yeast and human Pol replicate through a cis-syn thymine-thymine (TT) dimer efficiently and accurately by incorporating two As opposite the two Ts of the dimer (9, 11, 22). Pol can also efficiently replicate through other DNA lesions, such as 8-oxoguanine and O6-methylguanine (4, 5). We previously suggested (as determined on the basis of the proficient ability of Pol to replicate through distorting DNA lesions) that Pol possesses an active site that is remarkably tolerant of geometric distortions in the DNA, allowing it to incorporate nucleotides opposite DNA lesions (23). Support for this hypothesis came from steadystate kinetic studies, which showed that yeast and human Pol synthesize DNA with a low level of fidelity with error frequencies on the order of 10⫺2 to 10⫺3 (11, 23), and in an in vitro reaction, human Pol was found to synthesize DNA with a low level of fidelity (15). While steady-state kinetics can provide an accurate measure of the fidelity of nucleotide incorporation, it provides little information about the mechanistic basis of fidelity. This is
because in steady-state kinetics one observes only the slowest step of the nucleotide incorporation reaction, which for Pol and other DNA polymerases occurs after the chemical step of phosphodiester bond formation. Consequently, steady-state kinetics cannot determine the contributions to fidelity of crucial elementary steps such as nucleotide binding to the enzymeDNA binary complex, conformational changes in the enzymeDNA-nucleotide ternary complex, and the chemical step of phosphodiester bond formation, all of which precede the ratedetermining step under steady-state conditions (1, 7, 12, 13, 17, 26). To examine the mechanistic basis of the low level of fidelity of Pol, Washington et al. previously carried out a pre-steadystate kinetic analysis with yeast Pol. They found that relative to classical high-fidelity replicative DNA polymerases, which lack the ability to replicate through distorting DNA lesions, yeast Pol discriminates between the correct and incorrect nucleotide poorly at both the initial nucleotide binding and subsequent nucleotide incorporation steps (24). Washington et al. also showed that the rate-limiting step in the first turnover of nucleotide incorporation was substantially slower than in classical polymerases and presented evidence that this step was a conformational change in the ternary complex immediately preceding the chemical step of phosphodiester bond formation (24). Here we have carried out a pre-steady-state kinetic analysis of human Pol to better understand the mechanistic basis of its low level of fidelity. Because the yeast and human enzymes behave so similarly under steady-state conditions, we expected the pre-steady-state mechanism of nucleotide discrimination by the human enzyme to be the same as that of the yeast enzyme. Quite unexpectedly, we found that the mechanisms of the yeast and human enzymes differ in several important respects. Relative to the yeast enzyme, the human enzyme has a faster intrinsic rate of nucleotide incorporation. In this respect,
* 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) 7478608. E-mail:
[email protected]. 8316
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PRE-STEADY STATE KINETICS OF HUMAN Pol
human Pol is more like a classical DNA polymerase than yeast Pol. However, relative to the yeast enzyme, the human enzyme has a substantially lower level of affinity for the incoming nucleotide. Consequently, unlike the yeast enzyme, the human enzyme could be more sensitive to changes in the cellular deoxynucleoside triphosphate (dNTP) concentrations, and that sensitivity may serve to keep this low-fidelity enzyme relatively inactive except when the cellular dNTP concentrations rise, as might occur upon DNA damage (2). MATERIALS AND METHODS Purification of human Pol. Full-length human Pol and truncated human Pol (1–475) were purified as glutathione-S-transferase fusion proteins from yeast strain BJ5464 carrying plasmids pR30.186 and pR30.233, respectively. The proteins were purified and the glutathione-S-transferase portion of the fusion protein was removed with PreScission protease (Amersham Pharmacia) as described previously (24). Protein concentrations were determined by using molar extinction coefficients of 70,731 M 䡠 cm⫺1 for full-length Pol and 54,318 M 䡠 cm⫺1 for Pol (1-475) measuring UV absorption at 280 nm. Protein concentrations were also determined using a Bio-Rad protein assay with bovine serum albumin as a standard. An active-site titration (see Results) showed that 83% of the total concentration of Pol (1-475) was active, and the concentration of enzyme used in all the experiments was corrected for the amount of active enzyme. Nucleotides. Ultrapure grade solutions of dATP and dCTP (0.1 M of the sodium salt [pH 7.0]) were obtained from USB and were stored at ⫺70°C. Adenosine 5⬘ [␥-32P]triphosphate (6,000 Ci/mmol) was purchased from Amersham Pharmacia. Oligodeoxynucleotide substrates. The oligodeoxynucleotide used as a primer was a 25mer with the sequence 5⬘GCCTC GCAGC CGTCC AACCA ACTCA. The oligodeoxynucleotide used as a template was a 45mer with the sequence 5⬘GGACG GCATT GGATC GACCT TGAGT TGGTT GGACG GCTGC GAGGC. The primer strand (10 M) was 5⬘ 32P end labeled with [␥-32P]ATP and polynucleotide kinase (Boehringer Mannheim) for 45 min at 37°C. The labeled primer strand (2 M) was annealed to the template strand (2.5 M) in 50 mM TrisCl (pH 7.5)–100 mM NaCl at 90°C for 5 min and slowly cooled at room temperature over several hours. Steady-state kinetics assays. Full-length Pol or Pol (1–475) (1 nM) was incubated with the DNA substrate (200 nM) and various concentrations of either dATP, the correct nucleotide (0 to 10 M), or dCTP, the incorrect nucleotide (0 to 500 M), in 25 mM TrisCl (pH 7.5)–5 mM MgCl2–5 mM dithiothreitol–10% glycerol at room temperature for 5 or 10 min. Reactions were quenched with 10 volumes of formamide-loading buffer (90% deionized formamide, 10 mM EDTA [pH 8.0], 1 mg of xylene cyanol/ml, 1 mg of bromophenol blue/ml), and products were separated on a 15% polyacrylamide sequencing gel. The intensities of the gel bands were quantitated using a PhosphorImager (Molecular Dynamics). The mean and standard error values were determined from three independent experiments. Pre-steady-state kinetics assays. Experiments were carried out (using a Rapid Chemical Quench Flow instrument [KinTek]) in 25 mM TrisCl (pH 7.5)–5 mM MgCl2–5 mM dithiothreitol–10% glycerol at 18°C. Preincubated Pol (50 nM final concentration) and the DNA substrate (0 to 200 nM final concentration) were loaded into one sample loop (15 l), and either dATP (0 to 500 M) or dCTP (0 to 1,500 M) was loaded into the other sample loop. Reactions were quenched with 0.3 M EDTA, and products were separated on a 15% polyacrylamide sequencing gel. Data analysis. For the steady-state experiments, the linear rate of nucleotide incorporation (v) was graphed as a function of [dNTP], and the kcat and Km parameters were obtained by fitting the data by nonlinear regression (using SigmaPlot 7.0 software) to the Michaelis-Menten equation: v ⫽ kcat[Pol][dNTP]/共Km ⫹ [dNTP]兲
(1)
For the pre-steady-state experiments, the amount of product formed (P) was graphed as a function of time (t) and the data were fitted to the burst equation: P ⫽ A共1 ⫺ e⫺kobs t兲 ⫹ v t
(2)
where A is the amplitude of the burst phase, kobs is the rate constant of the burst phase, and v is the rate of the linear phase. The amplitudes of the burst phases
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(A) were graphed as a function of [DNA], and the data were fitted to the quadratic equation: A ⫽ 0.5共KD ⫹ [Pol] ⫹ [DNA]兲 ⫺
冑0.25共KD ⫹ [Pol] ⫹ [DNA]兲2 ⫺ ([Pol][DNA])
(3)
where KD is the dissociation constant for the DNA and [Pol] is the concentration of active Pol. The rate constants of the burst phases (kobs) were graphed as a function of [dNTP], and the data were fitted to the hyperbolic equation: kobs ⫽ kpol[dNTP]/共KD ⫹ [dNTP]兲
(4)
where kpol is the maximum rate constant of the burst phase and KD is the dissociation constant for the dNTP. To ensure reproducibility of the data, two to four independent experiments were performed for each DNA and dNTP concentration; the amplitudes and rates obtained were in close agreement.
RESULTS Comparison of full-length and truncated human Pol. We initially purified two forms of human Pol: the full-length protein and Pol (1-475), a truncated version lacking 238 amino acid residues from the C terminus. Pol (1-475) is completely active, efficiently bypasses a TT dimer, and contains all the amino acid residues that comprise the corresponding palm, fingers, thumb, and PAD domains depicted in the highresolution X-ray structure of yeast Pol (20). Since Pol (1475) is expressed about 100-fold better than the full-length protein, we were able to purify this protein in amounts sufficient to carry out a detailed and systematic pre-steady-state kinetic study. To ascertain whether the efficiency of nucleotide incorporation by Pol (1-475) was similar to that by the full-length protein, we compared the nucleotide incorporation activities of these proteins under steady-state conditions. Each form of Pol (1 nM) was incubated with the DNA substrate (200 nM) (Fig. 1A) and various concentrations of either the correct nucleotide dATP (0 to 10 M) or the incorrect nucleotide dCTP (0 to 500 M). The kcat values for the full-length and the truncated forms of Pol were similar, their Km values differed by less than twofold, and the levels of efficiency (kcat/Km) and fidelity of nucleotide incorporation for the two forms of Pol were also quite similar (Table 1). Since both proteins behaved approximately the same, we carried out all further experiments on Pol (1-475), which we will refer to simply as Pol hereafter. Human Pol displays biphasic kinetics. To determine whether human Pol displayed biphasic or “burst” kinetics, we used the Rapid Chemical Quench Flow instrument to examine the incorporation of the correct nucleotide dATP opposite a template T (Fig. 1A) under pre-steady-state conditions. Preincubated Pol (50 nM final concentration) and the DNA substrate (200 nM) in one syringe were rapidly mixed with dATP (500 M) from the other syringe. Figure 1B shows the amount of product formed graphed as a function of time. The kinetics of product formation was indeed biphasic. The fast, pre-steadystate burst phase corresponds to the incorporation of nucleotide during the first enzyme turnover, while the slow, steadystate linear phase corresponds to the incorporation of nucleotide during subsequent enzyme turnovers. Burst kinetics occurs whenever the slowest step of the nucleotide incorporation reaction follows the chemical step. The
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FIG. 1. Pre-steady-state kinetics of nucleotide incorporation by human Pol. (A) The sequences of the DNA substrate used in this study. (B) Preincubated DNA (200 nM) and Pol (50 nM) were mixed with 500 M dATP in a rapid chemical quench flow apparatus for various reaction times at 18°C. The solid line represents the best fit to the burst equation with a burst amplitude equal to 43 ⫾ 1 nM, a burst-rate constant equal to 50 ⫾ 5 s⫺1, and a linear rate equal to 15 ⫾ 2 nM/s.
slowest step, which is likely the dissociation of Pol from the DNA substrate, limits the rate of the subsequent steady-state turnovers but not the rate of the first enzyme turnover. Consequently, the presence of a pre-steady-state burst phase provided an opportunity to examine nucleotide incorporation in the first enzyme turnover and to assess the contributions of the nucleotide binding step and the nucleotide incorporation step toward the efficiency and fidelity of nucleotide incorporation by human Pol. Determination of the KDDNA and the concentration of active human Pol. To find the appropriate conditions under which to carry out subsequent experiments for examining the kinetics of correct and incorrect nucleotide incorporation in the first enzyme turnover, we needed to obtain a KDDNA value for the Pol-DNA complex and to determine the value for a concen-
tration of active Pol molecules. Because the amplitude of the pre-steady-state burst phase is a measure of the amount of active Pol-DNA complex at the start of the reaction, we were able to obtain these values by examining the correspondence of the pre-steady-state burst amplitude to the concentration of the DNA substrate. Preincubated Pol (60 nM total concentration as judged by two methods; see Materials and Methods) and various concentrations (10 to 200 nM) of the DNA substrate were rapidly mixed with 500 M dATP (Fig. 2A). The amplitudes of the pre-steady-state burst phases were graphed as a function of total DNA concentration (Fig. 2B), and a KDDNA value of 38 nM for the Pol-DNA complex and a concentration value of 50 nM for active Pol molecules were obtained from the bestfit curve. Thus, the Pol preparation used in this study was 83% active and the concentration of Pol used in all experiments was corrected for the concentration of active enzyme. Determination of KDdNTP and kpol for nucleotide incorporation by human Pol. To understand the mechanistic basis of the low level of fidelity of Pol, we needed to measure the KDdNTP for the Pol-DNA-dNTP ternary complex and the maximum rate constant of nucleotide incorporation in the first enzyme turnover (kpol) for the correct and the incorrect nucleotides. These values can be obtained by the examination of the variation of the observed rate constant (kobs) of the presteady-state burst phase as a function of dNTP concentration. First, we examined this concentration dependence with the correct nucleotide, dATP (Fig. 3A); we obtained a KDdATP of 110 M and a kpol of 67 s⫺1 from the best-fit curve (Table 2). Next, we examined the concentration dependence with the incorrect nucleotide, dCTP (Fig. 3B); we obtained a KDdCTP of 380 M and a kpol of 0.77 s⫺1 from the best-fit curve (Table 2). Human Pol exhibits only a slight elemental effect. We also examined the effects of substituting a sulfur atom for an oxygen atom at the ␣-phosphate of the incoming nucleotide. These experiments are usually done to ascertain whether the ratelimiting step in the first enzyme turnover (kpol) corresponds to the chemical step of phosphodiester bond formation or to a conformational change step immediately prior to the chemical step. A small (less than fourfold) elemental effect is taken as evidence of a rate-limiting conformational change step, because the sulfur substitution is expected to reduce the rate of the chemical step by a fourfold or greater amount (6); a larger elemental effect, however, would occur if the chemical step were rate limiting. For Pol incubated with 500 M of either
TABLE 1. Determinations of steady-state kinetic parameters for correct and incorrect nucleotide insertion by full-length and truncated (1-475) human Pol Polymerase
Results kcat (s
⫺1
)
Km (M)
(kcat/Km) (M⫺1 s⫺1)
Fidelitya
Full-length Pol Correct Incorrect
0.23 ⫾ 0.009 0.063 ⫾ 0.001
0.53 ⫾ 0.07 26 ⫾ 2
0.43 0.0024
180
Pol (1–475) Correct Incorrect
0.24 ⫾ 0.005 0.055 ⫾ 0.001
0.32 ⫾ 0.03 17 ⫾ 1
0.75 0.0032
230
a
Fidelity was calculated by the following equation: (kcat/Km)correct/(kcat/Km)incorrect.
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FIG. 2. Active-site titration of human Pol. (A) Preincubated Pol (60 nM total protein) and various concentrations of DNA (F, 10 nM; E, 20 nM; ■, 30 nM; 䊐, 40 nM; Œ, 50 nM; ‚, 75 nM; , 100 nM; ƒ, 150 nM; and ⽧, 200 nM) were mixed with 500 M dATP for various reaction times. The solid lines represent the best fits to the burst equation. (B) The burst amplitudes (F) were graphed as a function of [DNA], and the solid line represents the best fit to the quadratic equation with a KDDNA for the Pol-DNA complex equal to 38 ⫾ 4 nM and the active-site concentration of Pol equal to 50 ⫾ 2 nM.
FIG. 3. Concentration dependence of the rate of nucleotide incorporation by human Pol. (A) For correct nucleotide incorporation, the observed pre-steady-state burst-rate constants (kobs) were graphed as a function of [dATP]; the solid line represents the best fit to the hyperbolic equation with a KDdATP equal to 110 ⫾ 20 M and a kpol equal to 67 ⫾ 3 s⫺1. (B) For incorrect nucleotide incorporation, the burstrate constants (kobs) were graphed as a function of [dCTP]; the solid line represents the best fit to the hyperbolic equation with a KDdCTP equal to 380 ⫾ 100 M and a kpol equal to 0.77 ⫾ 0.07 s⫺1.
dATP or dATP␣S (the correct nucleotide), the pre-steadystate burst-rate constants were 45 s⫺1 and 29 s⫺1, respectively, which corresponds to an elemental effect of 1.6. For Pol incubated with 1,500 M of either dCTP or dCTP␣S (the incorrect nucleotide), the pre-steady-state burst-rate constants were 0.52 s⫺1 and 0.21 s⫺1, respectively, which corresponds to an elemental effect of 2.5. These observations support the presence of a rate-limiting conformational change step in the first turnover of human Pol, although they by no means demand it.
existence of a rate-limiting conformational change step is inferred from the small elemental effects (1.6-fold and 2.5-fold for the incorporation of the correct and incorrect nucleotides, respectively) observed when a sulfur atom was substituted for an oxygen atom on the ␣-phosphate of the incoming nucleotide. If the chemical step of phosphodiester bond formation were rate limiting instead, then we would have expected the kpol to have decreased by at least fourfold upon the sulfur substitution (6). The lack of an elemental effect, however, cannot be considered definitive, and additional experimental evidence is needed to unambiguously assign the rate-limiting step to a conformational change. Further evidence with other DNA polymerases for a rate-limiting conformational change step has come from comparisons of pulse-chase and pulsequench single-turnover experiments (3, 24). The observation of a difference in amplitude between the pulse-chase and pulsequench experiments indicates the accumulation of an intermediate species in which the nucleotide is stably bound to the
DISCUSSION Rate-limiting step in the first turnover of human Pol. We measured the kpol and KDdNTP values for the correct and incorrect nucleotide incorporation by human Pol and have presented evidence consistent with a conformational change being rate limiting in the first enzyme turnover. For human Pol, the
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TABLE 2. Determinations of pre-steady-state kinetics parameters for correct and incorrect nucleotide incorporation by human DNA Pol, yeast DNA Pol, and the Klenow fragment of E. coli DNA polymerase I Results Polymerase
KDdNTP (M)
Human Pol Correct Incorrect
110 380
kpol (s
⫺1
)
67 0.77
kpol/KD
dNTP
KDdNTPincorrect/KDdNTPcorrect
kpol correct/kpol incorrect
Fidelitya
0.61 2.0 ⫻ 10⫺3
3.5
87
300
1.3 8.7 ⫻ 10⫺3
0.54 6.7 ⫻ 10⫺4
5.4
150
810
50 1.0 ⫻ 10⫺2
10 5.9 ⫻ 10⫺4
3.4
5,000
17,000
b
Yeast Pol Correct Incorrect
2.4 13 c
E. coli Klenow fragment Correct Incorrect
5 17
a
Fidelity was calculated by the following equation: (kpol/KDdNTP)correct/(kpol/KDdNTP)incorrect. b Data are from Washington et al. (24). c Data are from Kuchta et al. (12, 13).
enzyme prior to the chemical step. Such experiments have provided compelling evidence for the presence of a rate-limiting conformational change step for the Klenow fragment (3) and also for yeast Pol (24). However, the low level of affinity for nucleotide binding displayed by human Pol makes pulsechase experiments impractical, as the high nucleotide concentrations necessary to perform the chase cannot be readily achieved. Although the evidence for a rate-limiting conformational change in human Pol is not in itself compelling, the case for a rate-limiting conformational change step with yeast Pol is strong. Hence, by analogy with the yeast enzyme and in the absence of any evidence to the contrary, we tentatively assign the rate-limiting step in the first turnover of human Pol to be a conformational change immediately preceding chemistry. The mechanism of nucleotide incorporation by human Pol differs from that of yeast Pol. Yeast Pol and human Pol behave similarly under steady-state conditions, as mentioned earlier, and we anticipated that their pre-steady-state mechanisms would also be similar. Surprisingly, we found that the
mechanism of human Pol differs from that of yeast Pol in several respects. First, the kpol is faster in the case of the human enzyme than in that of the yeast enzyme. In fact, unlike the kpol of the yeast enzyme, the kpol of the human enzyme is comparable in magnitude to that of the Klenow fragment of Escherichia coli DNA polymerase I, a prototypical classical DNA polymerase (Table 2). This can clearly be seen in the free-energy diagrams (Fig. 4), as the activation barrier (⌬G‡) for correct nucleotide incorporation is larger for yeast Pol (17.6 kcal/mol) than it is for either human Pol (14.6 kcal/mol) or for the Klenow fragment (15.0 kcal/mol). However, compared to the results for the Klenow fragment, the differential in kpol for the correct and the incorrect nucleotides is not very large for human Pol. Again, this can be seen in the freeenergy diagrams because the ⌬⌬G‡ (Fig. 4) is much smaller for human (2.6 kcal/mol) and yeast (3.0 kcal/mol) Pol than for the Klenow fragment (5.0 kcal/mol). Another difference between human and yeast Pol involves the binding affinity for the nucleotide. The KD for the nucleotide is much higher in the case of the human enzyme than in
FIG. 4. Gibbs free-energy profiles of yeast Pol, human Pol, and the Klenow fragment of E. coli polymerase (Pol) I. The free-energy changes corresponding to the initial nucleotide binding event and the subsequent rate-limiting nucleotide incorporation event were calculated from the KDdNTP and kpol values, assuming 100 M dNTP concentration. The solid lines represent the incorporation of the correct nucleotide, the dashed lines represent incorporation of the incorrect nucleotide, and the arrows represent the ⌬⌬G‡. The KDdNTP and kpol values for the Klenow fragment are from Kuchta et al. (12, 13), and the values for yeast Pol are from Washington et al. (24).
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that of the yeast enzyme, indicating that the human enzyme binds to the nucleotide with weaker affinity than the yeast enzyme. This too can be seen in the free-energy diagrams (Fig. 4), as the level of the energy minima corresponding to the Pol-DNA-dNTP ternary complex (⌬G) is higher in the case of the human enzyme (0.06 kcal/mol) than in that of the yeast enzyme (⫺2.2 kcal/mol). Interestingly, despite the clear differences observed in the pre-steady-state kinetics of the yeast and human enzymes, these mechanisms yield similar steady-state kinetic constants for the two enzymes. The reason for this is that the kpol/KD values for these enzymes are similar (Table 2). If nucleotide binding involves a rapid equilibrium, which is a reasonable assumption, then the kpol/KD value should be numerically equal to the kcat/Km steady-state kinetic parameter (compare Table 1 and Table 2). Thus, if both enzymes had similar rates of DNA dissociation (i.e., similar values for kcat), then the Km values would likewise have to be similar. This represents an important example of two divergent mechanisms yielding precisely the same results under steady-state analysis, highlighting the necessity for pre-steady-state kinetic studies to address these detailed mechanistic questions. While the rate (kcat) and efficiency (kcat/Km) values of singlenucleotide incorporation for yeast and human Pol measured under steady-state conditions are approximately the same (11, 23), we must emphasize that the single-nucleotide incorporation reaction under steady-state conditions is not as biologically relevant as the processive nucleotide incorporation reaction (in which the enzyme incorporates multiple nucleotides prior to dissociating from the DNA). In general, the rate of nucleotide incorporation measured in the first enzyme turnover better reflects the rate of nucleotide incorporation during processive DNA synthesis. Under processive DNA synthesis conditions in the cell, consequently, the yeast and human enzymes would behave quite differently. Under conditions of nucleotide saturation, for instance, the human enzyme would incorporate nucleotides processively at a ⬃50-fold-faster rate than the yeast enzyme. Structural basis of the mechanistic differences. The primary structures of the human and yeast enzymes are quite similar (8, 14); in the region corresponding to the palm, fingers, thumb, and PAD domains of yeast Pol, moreover, the human enzyme shares a high degree of sequence similarity with the yeast enzyme (20). Despite this structural conservation, the significant mechanistic differences between these enzymes must arise from differences in the way they interact with their substrates. For example, the decrease in the affinity of human Pol for the incoming nucleotide compared to that of yeast Pol must have resulted from the relatively fewer contacts between the human enzyme and the nucleotide in the ground state whereas the faster rate of nucleotide incorporation in the first turnover with human Pol must have resulted from additional contacts with the nucleotide and DNA substrates in the transition state for this step. An understanding of the structural basis of the mechanistic differences between these enzymes will require a comparison of the yet-to-be-determined high-resolution structures of the ternary complexes of yeast and human Pol with DNA and dNTP. In addition, studies of site-directed mutant proteins will be necessary to determine the contributions of individual amino acid residues to the stabilization of ground-state nucle-
PRE-STEADY STATE KINETICS OF HUMAN Pol
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otide binding as well as to the stabilization of the transition state of the nucleotide incorporation steps. Regulatory implications of the mechanism of human Pol. A role of a low level of substrate affinity in the regulation of enzymatic activity is a common phenomenon. Enzymes with a low level of substrate affinity are able to respond rapidly to variations in the concentrations of their substrates by increasing their reaction rates in a directly proportional manner. The classic example of this phenomenon is liver glucokinase. When blood glucose levels are low, glucokinase is relatively inactive so that the glucose can be utilized by the other cells of the body. When blood glucose levels are high, however, glucokinase becomes very active and phosphorylates the excess glucose, initiating its conversion to glycogen. A direct consequence of the lower level of nucleotide binding affinity of human Pol is that its activity would be more sensitive to cellular dNTP concentrations than that of the yeast enzyme, and as the cellular dNTP concentrations rise, so would the activity of the human enzyme. It is an intriguing possibility that this provides for a significant degree of regulation in humans of the activity of this rather low-fidelity polymerase. Thus, at low dNTP concentrations such as one would find in resting cells (18, 19), human Pol would be relatively inactive, whereas at the high dNTP concentrations that are likely to occur in dividing cells with DNA damage, its activity would rise. Yeast cells possess higher dNTP levels during the DNA synthesis (S) phase than at other cell cycle stages, and DNA damage elicits a manyfold increase in their levels (2). Although a similar DNA damage-dependent elevation of dNTP concentrations has not yet been documented in human cells, it is quite likely that a similar phenomenon exists. In that case, by primarily restricting the activity of Pol to cells with DNA damage, humans can avoid the mutagenic consequences arising from the inadvertent action of this enzyme in undamaged cells. ACKNOWLEDGMENTS This work was supported by National Institutes of Health grant GM19261 and NIEHS grant ES012411. REFERENCES 1. Carroll, S. S., and S. J. Benkovic. 1990. Mechanistic aspects of DNA polymerases: Escherichia coli DNA polymerase I (Klenow fragment) as a paradigm. Chem. Rev. 90:1291–1307. 2. Chabes, A., B. Georgieva, V. Domkin, X. Zhao, R. Rothstein, and L. Thelander. 2003. Survival of DNA damage in yeast directly depends on increased dNTP levels allowed by relaxed feedback inhibition of ribonucleotide reductase. Cell 112:391–401. 3. Dahlberg, M. E., and S. J. Benkovic. 1991. Kinetic mechanism of DNA polymerase I (Klenow fragment): Identification of a second conformational change and evaluation of the internal equilibrium constant. Biochemistry 30:4835–4843. 4. Haracska, L., S. Prakash, and L. Prakash. 2000. Replication past O6-methylguanine by yeast and human DNA polymerase . Mol. Cell. Biol. 20:8001– 8007. 5. Haracska, L., S.-L. Yu, R. E. Johnson, L. Prakash, and S. Prakash. 2000. Efficient and accurate replication in the presence of 7,8-dihydro-8-oxoguanine by DNA polymerase . Nat. Genet. 25:458–461. 6. Herschlag, D., J. A. Piccirilli, and T. R. Cech. 1991. Ribozyme-catalyzed and nonenzymatic reactions of phosphate diesters: rate effects upon substitution of sulfur for a nonbridging phosphoryl oxygen atom. Biochemistry 30:4844– 4854. 7. Johnson, K. A. 1993. Conformational coupling in DNA polymerase fidelity. Annu. Rev. Biochem. 62:685–713. 8. Johnson, R. E., C. M. Kondratick, S. Prakash, and L. Prakash. 1999. hRAD30 mutations in the variant form of xeroderma pigmentosum. Science 285:263–265. 9. Johnson, R. E., S. Prakash, and L. Prakash. 1999. Efficient bypass of a
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