Replication Protein A as a “Fidelity Clamp” for DNA Polymerase α
2001; Elsevier BV; Volume: 276; Issue: 21 Linguagem: Inglês
10.1074/jbc.m009599200
ISSN1083-351X
AutoresGiovanni Maga, Isabelle Frouin, Silvio Spadari, Ulrich Hübscher,
Tópico(s)Genomics and Chromatin Dynamics
ResumoThe current view of DNA replication in eukaryotes predicts that DNA polymerase α (pol α)-primase synthesizes the first 10-ribonucleotide-long RNA primer on the leading strand and at the beginning of each Okazaki fragment on the lagging strand. Subsequently, pol α elongates such an RNA primer by incorporating about 20 deoxynucleotides. pol α displays a low processivity and, because of the lack of an intrinsic or associated 3′→ 5′ exonuclease activity, it is more error-prone than other replicative pols. Synthesis of the RNA/DNA primer catalyzed by pol α-primase is a critical step in the initiation of DNA synthesis, but little is known about the role of the DNA replication accessory proteins in its regulation. In this paper we provide evidences that the single-stranded DNA-binding protein, replication protein A (RP-A), acts as an auxiliary factor for pol α playing a dual role: (i) it stabilizes the pol α/primer complex, thus acting as a pol clamp; and (ii) it significantly reduces the misincorporation efficiency by pol α. Based on these results, we propose a hypothetical model in which RP-A is involved in the regulation of the early events of DNA synthesis by acting as a "fidelity clamp" for pol α. The current view of DNA replication in eukaryotes predicts that DNA polymerase α (pol α)-primase synthesizes the first 10-ribonucleotide-long RNA primer on the leading strand and at the beginning of each Okazaki fragment on the lagging strand. Subsequently, pol α elongates such an RNA primer by incorporating about 20 deoxynucleotides. pol α displays a low processivity and, because of the lack of an intrinsic or associated 3′→ 5′ exonuclease activity, it is more error-prone than other replicative pols. Synthesis of the RNA/DNA primer catalyzed by pol α-primase is a critical step in the initiation of DNA synthesis, but little is known about the role of the DNA replication accessory proteins in its regulation. In this paper we provide evidences that the single-stranded DNA-binding protein, replication protein A (RP-A), acts as an auxiliary factor for pol α playing a dual role: (i) it stabilizes the pol α/primer complex, thus acting as a pol clamp; and (ii) it significantly reduces the misincorporation efficiency by pol α. Based on these results, we propose a hypothetical model in which RP-A is involved in the regulation of the early events of DNA synthesis by acting as a "fidelity clamp" for pol α. DNA polymerases α/δ/ε proliferating cell nuclear antigen replication protein A single-stranded The highly conserved DNA polymerase (pol)1 α-primase complex is the only eukaryotic polymerase able to initiate DNA synthesisde novo, making it of central importance in DNA replication. In fact, it is required both for the initiation of DNA replication at chromosomal origins and for the discontinuous synthesis of Okazaki fragments on the lagging strand of the replication fork (1Bambara R.A. Murante R.S. Henricksen L.A. J. Biol. Chem. 1997; 272: 4647-4650Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar, 2Henricksen L.A. Bambara R.A. Leuk. Res. 1998; 22: 1-5Crossref PubMed Scopus (13) Google Scholar, 3Hubscher U. Nasheuer H.P. Syvaoja J.E. Trends Biochem. Sci. 2000; 25: 143-147Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). The current view of DNA replication in eukaryotes predicts that pol α-primase synthesizes the first RNA/DNA primer on the leading strand. Then, at a critical length of 30 nucleotides, replication factor C binds to the 3′-OH end of the nascent DNA strand and displaces pol α, thereby loading PCNA and pol δ. pol α-primase switches activity to initiate the synthesis of Okazaki fragments on the lagging strand (4Waga S. Stillman B. Nature. 1994; 369: 207-212Crossref PubMed Scopus (497) Google Scholar, 5Maga G. Stucki M. Spadari S. Hubscher U. J. Mol. Biol. 2000; 295: 791-801Crossref PubMed Scopus (70) Google Scholar, 6Mossi R. Keller R.C. Ferrari E. Hubscher U. J. Mol. Biol. 2000; 295: 803-814Crossref PubMed Scopus (48) Google Scholar). pol α-primase has to synthesize short RNA/DNA primers. Thus, its intrinsic low processivity is compatible with its function. However, the switch between primase and polymerase activity, leading to the RNA-to-DNA synthesis transition, occurs through an intramolecular mechanism that does not require dissociation of pol α from the primer (7Copeland W. Wang T. J. Biol. Chem. 1993; 268: 26179-26189Abstract Full Text PDF PubMed Google Scholar). Thus, it would be important to ensure stable binding of pol α to the template until completion of the RNA/DNA primer synthesis. In addition, the lack of any intrinsic proofreading function for pol α could lead to misincorporation events during RNA/DNA primer synthesis. The critical roles of the pol α-primase make it a likely target for mechanisms that control DNA synthesis initiation and progression (8Nasheuer H. Moore A. Wahl A. Wang T. Biol. Chem. 1991; 266: 7893-7903Abstract Full Text PDF Google Scholar). In particular, a mechanism ensuring a transient but stable binding of pol α to the primer and increasing its fidelity could represent an advantage for the cell. The eukaryotic ssDNA-binding protein RP-A is a heterotrimer consisting of three subunits of 70, 32, and 14 kDa (p70, p32, and p14, respectively) (9Hübscher U. Maga G. Podust V.N. DePamphelis M.L. DNA Replication in Eukaryotic Cell. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1996: 525-543Google Scholar, 10Wold M.S. Annu. Rev. Biochem. 1997; 66: 61-92Crossref PubMed Scopus (1188) Google Scholar, 11Iftode C. Daniely Y. Borowiec J.A. Crit. Rev. Biochem. Mol. Biol. 1999; 34: 141-180Crossref PubMed Scopus (400) Google Scholar). RP-A has multiple roles in the cell, being essential for DNA replication initiation and elongation (12Walther A.P. Bjerke M.P. Wold M.S. Nucleic Acids Res. 1999; 27: 656-664Crossref PubMed Scopus (19) Google Scholar, 13Daniely Y. Borowiec J.A. J. Cell Biol. 2000; 149: 799-810Crossref PubMed Scopus (96) Google Scholar), DNA repair (14DeMott M.S. Zigman S. Bambara R.A. J. Biol. 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The three subunits of RP-A are likely to play different roles in all these DNA transactions. In particular, mutagenesis studies have begun to reveal different functions of p70, p32, and p14 in DNA replication. All three subunits are required to support DNA replicationin vitro. The p70 subunit contains three functionally distinct domains: an N-terminal domain, a central ssDNA-binding domain, and a C-terminal subunit interaction domain (31Jacobs D.M. Lipton A.S. Isern N.G. Daughdrill G.W. Lowry D.F. Gomes X. Wold M.S. J. Biomol. NMR. 1999; 14: 321-331Crossref PubMed Scopus (83) Google Scholar). The N-terminal domain contains the interaction domain for pol α-primase, which consists of two distinct regions: one (amino acids 1–170) that stimulates pol α synthetic activity and another (amino acids 170–327) that increases pol α processivity. This latter region overlaps with the ssDNA-binding domain (amino acids 168–450); such an activity was shown to be required for pol α processivity stimulation by RP-A (19Braun K.A. Lao Y. He Z. Ingles C.J. Wold M.S. Biochemistry. 1997; 36: 8443-8454Crossref PubMed Scopus (115) Google Scholar). The p32 subunit interacts with XPA and large T-antigen and is phosphorylated in a cell cycle-dependent manner (32Treuner K. Findeisen M. Strausfeld U. Knippers R. J. Biol. Chem. 1999; 274: 15556-15561Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). UV-cross-linking studies with photoreactive primer-templates mapped both p70 and p32 close to the 3′-OH primer end (33Lavrik O.I. Kolpashchikov D.M. Nasheuer H.P. Weisshart K. Favre A. FEBS Lett. 1998; 441: 186-190Crossref PubMed Scopus (30) Google Scholar, 34Lavrik O.I. Nasheuer H.P. Weisshart K. Wold M.S. Prasad R. Beard W.A. Wilson S.H. Favre A. 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Biochemistry. 1999; 38: 3963-3973Crossref PubMed Scopus (79) Google Scholar, 39Lao Y. Lee C.G. Wold M.S. Biochemistry. 1999; 38: 3974-3984Crossref PubMed Scopus (96) Google Scholar, 40Lao Y. Gomes X.V. Ren Y. Taylor J.S. Wold M.S. Biochemistry. 2000; 39: 850-859Crossref PubMed Scopus (82) Google Scholar). These structurally distinct complexes have been proposed to have different subunits rearrangements. Very recently, it has further been demonstrated that RP-A, along with the DNA replication protein Cdc45p, is involved in the recruitment of pol α-primase at the chromosomal DNA replication origins (41Walter J. Newport J. Mol Cell. 2000; 5: 617-627Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar, 42Zou L. Stillman B. Mol. Cell. Biol. 2000; 20: 3086-3096Crossref PubMed Scopus (268) Google Scholar). Thus, both pol α-primase and RP-A are simultaneously present and likely to interact physically during initiation of DNA replication. This makes RP-A a likely candidate for the regulation of the catalytic activity of pol α-primase. In the present work, we have investigated the in vitro role of RP-A on the DNA synthetic activity of pol α using different DNA templates. The results obtained indicated that RP-A could assist pol α in two ways: (i) by increasing the stability of the pol/primer complex and (ii) by reducing the overall misincorporation rate of pol α. [3H]dTTP (40 Ci/mmol) and [γ-32P]ATP (3000 Ci/mmol) were from Amersham Pharmacia Biotech; unlabeled dNTPs, poly(dA), and oligo(dT)12–18, d24-mer, and d66-mer oligodeoxynucleotides were from Roche Molecular Biochemicals. Activated calf thymus DNA was prepared as described (54Weiser T. Gassmann M. Thömmes P. Ferrari E. Hafkemeyer P. Hübscher U. J. Biol. Chem. 1991; 266: 10420-10428Abstract Full Text PDF PubMed Google Scholar). Whatman was the supplier of the GF/C and DE-81 filters. All other reagents were of analytical grade and were purchased from Merck or Fluka. The singly primed d24:d66-mer was prepared by labeling the 5′-end of the d24-mer primer with [γ-32P]ATP and T4 polynucleotide kinase (Ambion) according to the manufacturer's protocol. The d66-mer template oligonucleotide was then mixed with the complementary labeled d24-mer primer oligonucleotide in a 1:1 molar ratio in 20 mmTris-HCl (pH 8.0) containing 20 mm KCl and 1 mmEDTA, heated at 90 °C for 5 min, and then incubated at 65 °C for 2 h and slowly cooled at room temperature. The sequence of the d66-mer oligonucleotide was: 5′-AGGATGTATGTTTAGTAGGTACATAACTATCTATTGATACAGACCTAAAACAAAAAATTTTCCGAG-3′. The sequence of the d24-mer oligonucleotide was: 5′-CTCGGAAAATTTTTTGTTTTAGGT-3′. Calf thymus pol α was purified as described (54Weiser T. Gassmann M. Thömmes P. Ferrari E. Hafkemeyer P. Hübscher U. J. Biol. Chem. 1991; 266: 10420-10428Abstract Full Text PDF PubMed Google Scholar). The pol α used in this study was 250 units/ml (0.2 mg/ml). 1 unit of pol activity corresponds to the incorporation of 1 nmol of total dTMP into acid-precipitable material for 60 min at 37 °C in a standard assay containing 0.5 μg (nucleotides) of poly(dA)/oligo(dT)10:1 and 20 μm dTTP. Recombinant human RP-A was isolated as described (55Henricksen L.A. Umbricht C.B. Wold M.S. J. Biol. Chem. 1994; 269: 11121-11132Abstract Full Text PDF PubMed Google Scholar). pol α activity on poly(dA)/oligo(dT)10:1 was assayed in a final volume of 25 μl containing 50 mm Tris-HCl (pH 7.6), 0.25 mg/ml bovine serum albumin, 1 mm dithiothreitol, 6 mmMgCl2, and 5 μm [3H]dTTP (5 Ci/mmol), unless otherwise indicated in the figure legends. All reactions were incubated for 15 min at 37 °C unless otherwise stated, and the DNA was precipitated with 10% trichloroacetic acid. Insoluble radioactive material was determined as described (56Hübscher U. Kornberg A. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 6284-6288Crossref PubMed Scopus (53) Google Scholar). When the singly primed d24:d66-mer oligodeoxynucleotide was used as template, a final volume of 25 μl contained 50 mmTris-HCl (pH 7.6), 0.25 mg/ml bovine serum albumin, 1 mmdithiothreitol, 6 mm MgCl2, and 10 μm each [3H]dATP (5 Ci/mmol), dGTP, dCTP, and [3H]dTTP (5 Ci/mmol). Enzymes and proteins were added as indicated in the figure legends. All reactions were incubated for 15 min at 37 °C unless otherwise stated and stopped by the addition of 0.1 m EDTA and 1 μg of calf thymus DNA as carrier. 20 μl of the reaction mixture were then spotted onto DE-81 cellulose filters. Filters were washed to remove unincorporated dNTPs as described (57Focher F. Verri A. Maga G. Spadari S. Hubscher U. FEBS Lett. 1990; 259: 349-352Crossref PubMed Scopus (5) Google Scholar), and incorporated radioactivity was monitored by scintillation counting. For incorporation studies with the singly primed d24:d66-mer oligodeoxynucleotide as template, a final volume of 10 μl contained 50 mm Tris-HCl (pH 7.6), 0.25 mg/ml bovine serum albumin, 1 mm dithiothreitol, 6 mm MgCl2, and 20 nm (3′-OH ends) of the 5′ 32P-labeled d24:d66-mer DNA template. Enzymes, proteins, and unlabeled dNTPs were added as indicated in the figure legends. All reactions were incubated for 15 min at 37 °C, samples were mixed with denaturing gel loading buffer (95% v/v formamide, 10 mm EDTA, 0.25 mg/ml bromphenol blue, 0.25 mg/ml xylene cyanol), heated at 95 °C for 5 min, and then subjected to electrophoresis on a 7 m urea, 20% polyacrylamide gel. Quantification of the reaction products on the gel was performed using a Molecular Dynamics PhosphorImager and ImageQuant software. Km, Vmax, and [RP-A]50 values were calculated according to the Michaelis-Menten equation in the form, ν=kcatE01+Km[S]Equation 1 where kcat E0 =Vmax. The Ki values for incorrect dNTPs were determined from inhibition assays with increasing concentrations of the selected dNTP in the presence of different fixed amounts of RP-A and were calculated according to a simple competitive mechanism of inhibition as described by the equation,ν=kcatE01+Km[S]1+[I]KiEquation 2 Computer fitting of the experimental data to the equations was performed with the program MacCurveFitTM 1.5 using the least squares curve-fitting quasi-Newton method, based on the Davidon-Fletcher-Powell algorithm (59Fletcher R. Practical Methods of Optimization. 1. Wiley, New York1980Google Scholar). When data points were derived from densitometric analysis of the intensities of the products bands, the values of integrated gel band intensities in dependence of the nucleotide substrate concentrations were fitted to the equation (58Creighton S. Bloom L.B. Goodman M.F. Methods Enzymol. 1995; 262: 232-256Crossref PubMed Scopus (226) Google Scholar),I*T/IT−1=Vmax[dNTP]/(Km+[dNTP])Equation 3 where, T = target site, the template position of interest; and I*T = the sum of the integrated intensities at positions T,T+1 … T+n. Before being inserted in the above equation, the intensities of the single bands of interest were first normalized by dividing for the total intensity of the lane. This was done to reduce the variability because of manual gel loading. An empty portion of the gel was scanned, and the resulting value was subtracted as background. The goodness of the interpolated curve was assessed by computer-aided calculation of the sum of squares of errors and the correlation coefficient R 2. Standard errors were provided by the computer program MacCurveFitTM 1.5. The standard errors are calculated from the variance-covariance matrix, and the values displayed are the square roots of the diagonal elements. The variance-covariance matrix is calculated from the Jacobian matrix (59Fletcher R. Practical Methods of Optimization. 1. Wiley, New York1980Google Scholar). Different concentrations of RP-A were tested for their effect on nucleotide incorporation catalyzed by pol α on different DNA templates (Fig. 1). RP-A was able to stimulate pol α activity on either homo- or heteropolymeric deoxyoligonucleotides, within a range of concentrations close to the 3′-OH primer concentration used in the assay (Fig.1 A). Variation of the 3′-OH primer concentration resulted in a shift of the RP-A concentration giving the maximal stimulation, which was observed at equimolar amounts of RP-A to 3′-OH primer (Fig.1 B, arrows). To investigate the effects of RP-A on the nucleotide incorporation reaction catalyzed by pol α, increasing concentrations of 3′-OH primer were tested in the presence of different fixed amounts of RP-A. As shown in Fig. 2 A, theKm value of pol α for the 3′-OH primer was decreased by increasing concentrations of RP-A. TheVmax/Km ratio, which is an estimate of the association rate for the pol α·3′-OH primer complex formation, was also increased by RP-A (Fig. 2 B). A comparison of the variation of the reaction velocity in dependence of the 3′-OH primer concentration (Fig. 2 C) with the observed decrease of Km values in dependence of RP-A concentration (Fig. 2 D) showed that [RP-A]50, the RP-A concentration giving half of the maximal decrease, was 41 nm, a value very close to the Km of pol α for the 3′-OH primer, which is 39 nm. Next, the effect of RP-A on the ability of pol α to incorporate a wrong nucleotide was tested. It is known that the misincorporation efficiency of a pol is influenced by the nature of the mismatch resulting from the misalignment of the template-encoded base and the incoming nucleotide (43Mendelman L.V. Boosalis M.S. Petruska J. Goodman M.F. J. Biol. Chem. 1989; 264: 14415-14423Abstract Full Text PDF PubMed Google Scholar). Thus, to directly compare the effects of RP-A on the ability of pol α to incorporate a wrong nucleotide, the homopolymeric substrate poly(dA)/oligo(dT) was used, which contains only adenines as template. As shown in Fig. 3 A, the incorporation of radioactively labeled dTTP catalyzed by pol α on such a template can be inhibited by the addition of unlabeled dNTPs as competitors. Each individual dNTP inhibited the reaction with different potencies, as indicated by the different Ki values (Table I). The calculatedKi values and theKi/Km ratios, which are an estimate of the accuracy of nucleotide incorporation by pol α, are listed in Table I. pol α discriminates 330-fold against the C-A mismatch, but only 60-fold against the A-A and 30-fold against the G-A misincorporations, respectively. Thus, pol α showed different misincorporation efficiencies for the resulting mismatches, in the order G-A > (i.e. more efficiently generated) A-A > C-A. When similar experiments were performed in the presence of RP-A, the misincorporation efficiencies for the G-A and A-A mismatches were reduced to about the level observed for the C-A misincorporation (see below and Table I).Table IEffect of RP-A on the inhibition by different dNTPs of dTTP incorporation catalyzed by pol α on poly(dA)/oligo(dT)SubstrateCompetitor−RP-A+RP-AReduction of misincorporation 1-aCalculated as the ratio (Ki/Km)+RP-A/(Ki/Km)−RP-A.KmKiKi/KmKmKiKi/Kmμmμm-folddTTP10.411.5dCTP3440 1-bAll of the values for the kinetic constants were calculated by computer simulation as described under "Materials and Methods." Standard deviations (±S.D.) were in all cases ≤±10%. Values reported are the mean of three independent experiments. The significance of the differences between the mean values obtained from reactions with and without RP-A were tested by a Student's t test under the null hypothesis that the true mean values were equal in all cases. The probability value that the reference hypothesis was true was p ≤± 0.05, and thus the observed differences were considered statistically significant.330.7n.d. 1-cn.d., not determined.n.d.dATP61058.63303287.25.4dGTP32030.71900165.25.91-a Calculated as the ratio (Ki/Km)+RP-A/(Ki/Km)−RP-A.1-b All of the values for the kinetic constants were calculated by computer simulation as described under "Materials and Methods." Standard deviations (±S.D.) were in all cases ≤±10%. Values reported are the mean of three independent experiments. The significance of the differences between the mean values obtained from reactions with and without RP-A were tested by a Student's t test under the null hypothesis that the true mean values were equal in all cases. The probability value that the reference hypothesis was true was p ≤± 0.05, and thus the observed differences were considered statistically significant.1-c n.d., not determined. Open table in a new tab Fig. 3 Bshows the effect of different amounts of RP-A on the inhibition by dGTP of dTTP incorporation catalyzed by pol α on poly(dA)/oligo(dT). The plot of the increase in the Ki value of dGTPversus RP-A concentration showed a typical saturation kinetics (Fig. 3 C), with a half-maximal stimulatory RP-A concentration (or [RP-A]50) of 47 nm, very close to the value observed for the 3′-OH primer binding stimulation (Fig. 2 D). Maximal increase of the Kivalues for dGTP inhibition was observed at RP-A concentrations close to the concentration of 3′-OH. Similar results were obtained when dATP inhibition was tested (data not shown). Because the template sequence was known, it was possible to force pol α to make specific mismatches by adding the appropriate combinations of nucleotides in a reaction mixture. Fig. 4shows the products of a reaction containing the heteropolymeric deoxyoligonucleotide resolved by denaturing polyacrylamide gel electrophoresis. The addition of different combinations of nucleotides generated strong pausing sites at the positions immediately preceding the mismatch, suggesting that incorporation of a wrong nucleotide was rate-limiting with respect to the elongation of a mismatched primer. For example, in the presence of the first encoded nucleotide, dCTP, a strong signal was detected at position +1 (lane 2), whereas the addition of the first two nucleotides, dCTP and dTTP, resulted in the generation of a strong band at position +2 (lane 3). Significant misincorporation products were detected with all of the combinations used (lanes 2-4), confirming the relatively low fidelity of pol α in DNA synthesis. The same reactions were performed in the presence of RP-A (Fig. 4, lanes 5-8). The first observation was that RP-A stimulated the correct incorporation of nucleotides by pol α as judged by the increasing intensities of the bands corresponding to complementary nucleotide incorporation (Fig. 4, compare positions 0, +1, and +2 in lanes 2–4 with the same positions inlanes 5–8). In particular, the band at position 0 decreased significantly; this was expected, because RP-A imposed a block to misincorporation, which forced pol α to elongate mainly the d24-mer primer to a d25-mer product but did not allow further elongation because of the lack of the complementary correct nucleotides required. The intensities of the bands were quantified by densitometric analysis, and the relative amounts of synthesized products at each position along the template were calculated as described in the figure legend, both in the absence and in the presence of RP-A. As shown in Fig.5, with the different combinations of nucleotides tested, RP-A significantly decreased the amount of products generated by elongation of a mismatched primer by pol α, thereby increasing the accumulation of products at the position immediately preceding the misincorporation site (compare panel A withB in Fig. 5). To investigate more closely the mechanism by which RP-A decreases the misincorporation efficiency of pol α, a detailed kinetic analysis of correct versus incorrect single nucleotide incorporation was performed. Reactions were carried out as shown in Fig. 4, lanes 2 and 6, respectively, but in the presence of different concentrations of dCTP. Quantification of the products at position +1 (C-G base pair) and +2 (C-A mismatch) at each nucleotide concentration allowed the calculation of theKm and Vmax values for the correct and incorrect incorporation reactions, as well as the specificity constant Vmax/Km. The computed values are listed in TableII. RP-A specifically decreased both the affinity and the reaction velocity for the misincorporation reactions, whereas it only slightly stimulated the correct nucleotide incorporation (∼2-fold increase in theVmax/Km value). A comparison of the ratio between the Vmax and theKm values for correct and incorrect nucleotide incorporation in the absence and the presence of RP-A showed that pol α alone discriminated ∼540-fold against the C-A mismatch, a value comparable with the one derived with poly(dA)/oligo(dT) (Table I). This value was increased to more than 3300-fold by the addition of RP-A, thus reducing the misincorporation more than 6-fold (Table II).Table IIEffect of RP-A on the kinetic parameters for correct (C-G) versus incorrect (C-A) single nucleotide incorporation by pol α−RP-A+RP-ACorrect (C-G)Incorrect (C-A)Correct (C-G)Incorrect (C-A)Km(μm)9.3 (±2)2-aAll of the values for the kinetic constants were calculated by computer simulation as described under "Materials and Methods." Standard deviations (±S.D.) are indicated. Values reported are the mean of three independent experiments. Significance of the differences between the mean values obtained from reactions with and without RP-A were tested by a Student's t test under the null hypothesis that the true mean values were equal in all cases. The probability value that the reference hypothesis was true was p ≤ 0.05, and thus the observed differences were considered statistically significant.1230 (±5)7.0 (±3)2392 (±7)Vmax (pmol × min−1 × mg−1)12 (±2)3 (±0.5)15 (±3)1.5 (±0.1)Vmax/Km1.30.00242.10.00063(Vmax/Kmcorr.)/(Vmax/Kmincorr.)54033332-a All of the values for the kinetic constants were calculated by computer simulation as described under "Materials and Methods." Standard deviations (±S.D.) are indicated. Values reported are the mean of three independent experiments. Significance of the differences between the mean values obtained from reactions with and without RP-A were tested by a Student's t test under the null hypothesis that the true mean values were equal in all cases. The probability value that the reference hypothesis was true was p ≤ 0.05, and thus the observed differences were considered statistically significant. Open table in a new tab
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