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Measurement of kinetic parameters of human platelet DNA polymerase γ
Methods 51 (2010) 374–378
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Measurement of kinetic parameters of human platelet DNA polymerase c Jan-Willem Taanman a,*, Margit Heiske b, Thierry Letellier b a b
Department of Clinical Neurosciences, Institute of Neurology, University College London, Rowland Hill Street, London NW3 2PF, United Kingdom U688 INSERM, Université Victor Segalen Bordeaux 2, 146 Rue Léo Saignat, 33076 Bordeaux-Cedex, France
a r t i c l e
i n f o
Article history: Accepted 9 March 2010 Available online 16 March 2010 Keywords: DNA polymerase c Mitochondria Mitochondrial DNA POLG Polymerase activity Platelets
a b s t r a c t Synthesis of mitochondrial DNA is performed by DNA polymerase c. Mutations in POLG, the gene encoding the catalytic subunit of DNA polymerase c, are a major cause of neurological disease. A large proportion of patients carry rare nucleotide substitutions leading to single amino acid changes. Confirming that these replacements are pathogenic can be problematic without biochemical evidence. Here, we provide a hands-on protocol for an in vitro kinetic assay of DNA polymerase c which allows assessment of the Km and Vmax for the incoming nucleotide of the polymerization reaction. To avoid measurement of contaminating nuclear DNA polymerases, platelet extracts are used since platelets do not contain a nucleus. Moreover, platelets have the advantage of being obtainable relatively non-invasively. Polymerization activity is determined by measurement of the incorporation of radioactive thymidine 50 -triphosphate (dTTP) on the homopolymeric RNA substrate poly(rA)oligo(dT)12–18. To further minimize nuclear DNA polymerase activity, aphidicolin, an inhibitor of most nuclear DNA polymerases, is included in the reaction. In addition, reactions are carried out in the absence and presence of the competitive inhibitor of DNA polymerase c, 20 ,30 -dideoxythymidine 50 -triphosphate (ddTTP), to allow calculation of the ddTTP-sensitive incorporation. With this method, platelets from healthy control subjects extracted with 3% Triton X-100 showed a Km for dTTP of 1.42 lM and a Vmax of 0.83 pmol min1 mg1. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction DNA polymerase c is the only DNA polymerase present in mitochondria. The enzyme is responsible for replication and repair of the mitochondrial DNA (mtDNA). Human DNA polymerase c comprises a 136.9-kDa POLG subunit and a 52.2-kDa POLG2 subunit [1–3]. POLG possesses all three catalytic functions of the enzyme: a DNA polymerase activity required for mtDNA synthesis, a 30 ? 50 exonuclease activity providing proofreading capability, and a 50 -deoxyribose phosphate lyase activity which functions in base excision repair [4,5]. Upon interaction with POLG, POLG2 enhances the affinity of the polymerase for DNA and promotes tighter nucleotide binding, thereby increasing the polymerization rate. This leads to an increase in processivity, i.e. the number of nucleotides incorporated per binding event [6,7]. The POLG gene is highly polymorphic. To date, more than 150 sequence variations have been identified [8]. Pathogenic mutations in POLG lead to mtDNA abnormalities and are an important cause of human disease. The first mutations were reported in families with autosomal dominant progressive external ophtalmoplegia associated with the presence of multiple deletions of mtDNA in muscle biopsies [9]. Since then, POLG mutations have been linked to a spec-
* Corresponding author. Fax: +44 20 7472 6829. E-mail address: [email protected] (J.-W. Taanman). 1046-2023/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ymeth.2010.03.002
trum of clinical phenotypes that can present from neonatal life to late middle age and includes common neurological disorders such as migraine, epilepsy and parkinsonism [10,11]. Although there has been extensive genetic analysis of POLG in patients, functional analysis of mutated POLG has been limited to a number of common or otherwise interesting mutations, studied with reconstituted, recombinant DNA polymerase c or in yeast models (reviewed in: [12]). These studies have provided valuable insights but their methodology is impractical for functional analysis of the large number of POLG sequence variants found in patients. Earlier, we reported assays of DNA polymerase c assembly, activity and processivity, using mitochondrial fractions of cultured skin fibroblasts from pediatric patients harboring POLG mutations [13]. This paper describes an in vitro kinetic assay of DNA polymerase c activity, using platelet extracts. Compared to tissue biopsies, platelets have the advantage of being obtainable relatively noninvasively. Thus, the assay offers a convenient method for clinical laboratories to investigate the functional consequences of POLG sequence variations. 2. Experimental design considerations As mentioned above, DNA polymerase c has several catalytic activities. This paper describes a steady-state kinetic analysis for the incoming deoxynucleoside triphosphate of the polymerase
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reaction. The kinetic parameters are determined at 37 °C in 50 ll reaction mixtures containing: 10 lg of platelet protein extract, 25 mM HEPESKOH (pH 8.0), 100 mM NaCl, 0.5 mM MnCl2, 2.5 mM b-mercaptoethanol, 50 lg/ml of the homopolymeric RNA substrate poly(rA)oligo(dT)12–18, 100 l/ml of acetylated bovine serum albumin (BSA), 0.1 mM aphidicolin, 500 lg/ml of RNasinÒ RNase inhibitor, and [a-32P]thymidine 50 -triphosphate (dTTP; specific activity: 5 Ci/mmol) varied from 0.05 to 10 lM. The composition of this reaction mixture is based on the mixture used by Longley and colleagues [14]. Reactions are carried out in the absence and presence of the inhibitor 20 ,30 -dideoxythymidine 50 -triphosphate (ddTTP) to allow calculation of the ddTTP-sensitive dTTP incorporation. The inhibitory mode of ddTTP has been described as competitive to dTTP [15,16]. To determine the DNA polymerase c kinetic parameters (Vmax, Km, Ki), the DNA polymerase c activity is expressed as a function of the concentrations of dTTP and ddTTP, followed by simultaneous fitting of the experimental data of the saturation curves for dTTP and ddTTP. Clinical samples are inevitably contaminated with nuclear DNA polymerases and RNases. For that reason, assay conditions are chosen that favor measurement of the mitochondrial DNA polymerase activity and prevent RNase activity. Platelets are the enucleate ‘progeny’ of megakaryocytes, formed by cytoplasmic budding of the parent cell. As such, they make an ideal model for the study of DNA polymerase c, since the need to account for nuclear polymerase activity is minimized. Platelets are isolated from blood by differential centrifugation, using a protocol designed to limit platelet activation. Platelets are resuspended in 25 mM HEPESKOH (pH 8.0), 100 mM NaCl, aliquoted and stored at 80 °C. No apparent drop in platelet DNA polymerase c activity has been observed with whole blood storage over a 16-h period at room temperature (not shown). Nevertheless, blood is routinely processed as soon as possible. Also, no apparent drop in activity has been noted after 1 year storage of platelet suspensions at 80 °C (not shown). Others have found that DNA polymerase c activity is labile to freezing and thawing [1], therefore, multiple thaw cycles should be avoided. DNA polymerase c is liberated from the platelets with the nonionic surfactant Triton X-100. Titration experiments have indicated that the detergent is a prerequisite to recover DNA polymerase c activity from platelets, but that concentrations between 1 and 6% (v/v) of Triton X-100 show no significant differences in recovery (not shown). For the kinetic assay, we decided to extract 50 mg/ ml (wet weight) platelet suspensions with 3% (v/v) Triton X-100. It should be noted, however, that the final Triton X-100 concentration in the reaction mixture is 0.3% (v/v) due to a 10-fold dilution of the platelet extract in the reaction mixture. Dose response and time course experiments carried out at a constant substrate concentration of 10 lM dTTP, showed that the ddTTP-sensitive dTTP incorporation was linear for up to 10 min with 12.5 lg of platelet protein per 50 ll reaction mixture, and for longer time periods with lower amounts of protein (Fig. 1). At the 10 min time point, the incorporation rate was proportional to the amount of platelet protein (0.91 ± 0.15, 0.88 ± 0.10 and 0.84 ± 0.24 pmol min1 mg1, respectively, at 12.5, 5 and 3 lg platelet protein per 50 ll reaction mixture). For that reason, 10-min reactions with 10 lg of protein per 50 ll reaction mixture were adopted as the standard condition for the assay. Unlike most nuclear DNA polymerases, DNA polymerase c exhibits a potent RNA-directed DNA polymerase (reverse transcriptase) activity and is particularly active on poly(rA)oligo(dT) primed templates [1]. Therefore, the assay is based on the incorporation of radioactive dTTP on poly(rA)oligo(dT). Further differentiation of mitochondrial DNA polymerase activity from nuclear DNA polymerase activity is achieved by addition of aphidicolin to the reaction mixture. This antibiotic is an inhibitor of most nuclear DNA polymerases, but not of DNA polymerase c [17]. Titrations with aphidic-
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Fig. 1. Reaction progress curves showing the effect of the amount of platelet protein in the reaction mixture. The ddTTP-sensitive dTTP incorporation was measured in 50-ll reaction mixtures containing 10 lM dTTP and 12.5 lg (}), 5 lg (h) or 3 lg (r) of platelet protein as described in the text. Error bars depict standard deviation (n = 3).
olin have shown that P0.05 mM aphidicolin results in maximum inhibition of contaminating nuclear DNA polymerases in the in vitro assay (unpublished observations). Therefore, 0.1 mM aphidicolin is present in the reaction mixture. To eradicate nuclear DNA polymerase activity still further, reactions are performed without and with ddTTP, and the ddTTP-sensitive incorporation of dTTP is determined. In contrast to most nuclear DNA polymerases, DNA polymerase c utilizes dideoxynucleotides in vitro as efficiently as natural deoxynucleotides [15,16]. Because these analogs lack the 30 -hydroxyl group, they act as chain terminators once incorporated into DNA. Titrations with ddTTP have indicated that a 1:20 molecular ratio of ddTTP:dTTP results in an almost complete inhibition of DNA polymerase activity of highly purified mitochondria in vitro (unpublished observations). This 1:20 ratio of ddTTP:dTTP is used in the kinetic assay. Unfortunately, the reaction conditions do not abolish all nuclear DNA polymerase activities. DNA polymerase b has reverse transcriptase activity, and DNA polymerases b and h have the same inhibitory properties as DNA polymerase c (i.e. dideoxynucleotide-sensitive and aphidicolin resistant) [17,18]. It is expected, however, that the use of platelets will essentially eliminate these nuclear polymerases. The reaction mixture includes 100 mM NaCl since experiments with reconstituted, recombinant human DNA polymerase c have revealed a salt optimum of around 100 mM NaCl [6]. In addition, the reaction mixture contains 0.5 mM MnCl2 because, with poly(rA)oligo(dT) as template-primer, DNA polymerase c shows optimal activity at 0.5 mM MnCl2 [1]. RNAsinÒ is added to the reaction mixture as a broad spectrum RNase inhibitor. Although RNAsinÒ does not inhibit RNase H activity, the use of MnCl2 eliminates RNase H activity, which requires Mg2+ as cofactor [19].
3. Platelet preparation 3.1. Materials Sodium citrate VacutainersÒ (BD Diagnostics). Prostaglandin I2 sodium salt (Sigma, P6188). Prepare a stock solution of 1 mg/ml in water and store at 80 °C until use. Platelet resuspension buffer: 25 mM HEPESKOH (pH 8.0), 100 mM NaCl (autoclaved). Sterile phosphate-buffered saline (PBS; Gibco or Sigma). Sterile 15-ml tubes, sterile 1.5-ml test tubes, sterile pipettes, microscope slides and coverslips. Specialized equipment: universal centifuge (e.g. Eppendorf centrifuge 5702), phase contrast microscope, ultra low temperature freezer.
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3.2. Protocol 1. Collect blood in sodium citrate VacutainersÒ. Samples should be obtained in accordance with institutional ethics committee guidelines and approval. Observe local health and safety rules for the handling of human blood samples. 2. Pool 10–15 ml of blood in a sterile 15-ml tube. 3. Centrifuge blood for 30 min at 300 g (room temperature). 4. Transfer the top 2/3 of the platelet-rich serum with a sterile pipette to a new 15-ml tube. Pay attention to not disturbing the red cell–serum interface. 5. Add 1 ll of prostaglandin I2 stock solution per ml to the platelet-rich serum to inhibit platelet aggregation. 6. Centrifuge platelet-rich serum for 30 min at 1000 g (room temperature) to pellet platelets. 7. Remove the supernatant using sterile techniques and discard. 8. Resuspend the platelet pellet in PBS at the original volume and add 1 ll/ml of prostaglandin I2 stock solution. 9. Centrifuge resuspended platelets for 15 min at 1000 g (room temperature) to repellet platelets. 10. Remove the supernatant using sterile techniques and discard. 11. Resuspend the platelet pellet in 1 ml of platelet resuspension buffer and add 2 ll of prostaglandin I2 stock solution. 12. Transfer to a sterile, pre-weighted 1.5-ml tube. 13. Centrifuge for 10 min at 1000 g (room temperature) to repellet platelets. 14. Remove all supernatant carefully and determine the wet weight of the platelet pellet. 15. Resuspend platelets in platelet resuspension buffer at a concentration of 100 mg/ml and add 2 ll/ml of prostaglandin I2 stock solution. 16. Assess contamination of the platelet preparations with (nucleated) leukocytes by phase contrast microscopy. Contamination usually consists of only a few nucleated cells per 10 ll of platelet suspension. 17. Prepare 100-ll aliquots of resuspended platelets (10 mg/ 100 ll) in sterile 1.5-ml tubes and store at 80 °C awaiting assays.
4. Polymerase assay 4.1. Materials Water (autoclaved). 50 mM HEPESKOH (pH 8.0), 200 mM NaCl (autoclaved). Extraction buffer: 25 mM HEPESKOH (pH 8.0), 100 mM NaCl, 6% (v/v) Triton X-100. To make 100 ml extraction buffer, add 6.36 g of Triton X-100 to 50 ml of 50 mM HEPESKOH (pH 8.0), 200 mM NaCl and 44 ml of water. Mix until Triton X-100 is fully dissolved. (1 ml of Triton X-100 weights 1.06 g). 1 mM dTTP. To prepare a 1 mM dTTP stock solution, dilute 100 mM dTTP (e.g. Promega, U1235) 100-fold with autoclaved water. Store at 20 °C. 0.1 mM ddTTP. To prepare a 0.1 mM ddTTP stock solution, dilute 100 mM ddTTP (GE Healthcare, 27-2081-01) 1000-fold with autoclaved water. Store at 20°C. 10 mCi/ml [a-32P]dTTP, specific activity: 3000 Ci/mmol (e.g. MP Biomedicals, Thymidine 50 -triphosphate, [a-32P] Isoblue™ stabilized, 0139015X). 50 mM MnCl2 (autoclaved). b-mercaptoethanol (the pure liquid is 14.3 M; e.g. Sigma, M3148). 1 mg/ml of poly(rA)oligo(dT)12–18 (Midland Certified Reagent Company, P-4012B). Dissolve 25 u in 1.25 ml of 25 mM HEPESKOH (pH 8.0), 100 mM NaCl to obtain a concentration
of 20 u/ml. Since 1 u equals 50 lg, 20 u/ml equals 1 mg/ml. Prepare 208-ll aliquots and store at 20 °C. 10 mg/ml acetylated bovine serum albumin (BSA; Promega, R3961). Aphidicolin (Sigma, A0781). To prepare a 20 mM stock solution, dissolve 1 mg of aphidicolin in 148 ll dimethylsulfoxide. Store at 4 °C. This stock solution is stable for >6 weeks. 40 u/ll recombinant RNAsinÒ RNase inhibitor (Promega, N2511 or N2515). DE81 anion exchanger chromatography paper (Whatman, 3658915; Sigma, Z286605). 2 SCC: 300 mM NaCl, 30 mM sodium citrate (pH 7.0). May be made as a 20 SCC stock solution [20] and diluted 10-fold before use. Ethanol (95%). Liquid scintillation cocktail, e.g. Ultima Gold™ liquid scintillation cocktail (Perkin Elmer, 6013329). Protein assay kit (e.g. the BCA™ Protein Assay kit from Pierce, Thermoscientific, 23225). Sterile 96-well PCR plates with lids, 20-ml liquid scintillation vails (e.g. Wheaton; Sigma, V6755), sterile 5 and 1.5-ml test tubes, sterile pipettes. Specialized equipment: thermocycler, vessel sufficiently large to wash a sheet of 500 cm2 DE81 paper in 1 L of 2 SSC on a shaking platform, scintillation counter.
4.2. Protocol This protocol describes the analysis of four platelet samples in parallel. It is not recommended to assay more than four samples together. The serial dilutions of nucleotides (steps 7–9) and the concentrated reaction mixture (steps 10 and 11) can be prepared shortly before and during the platelet extraction (steps 2–6). 1. Draw and mark 195 1.5 1.5 cm squares with a soft pencil on a sheet of DE81 anion exchanger chromatography paper. 2. To prepare the platelet extracts, thaw 100 ll (10 mg wet weight) platelet suspension and add 100 ll ice-cold extraction buffer. 3. Mix thoroughly and leave on ice for 10 min. 4. Microcentrifuge at maximum speed for 10 min at 4 °C to remove particulate matter. 5. Transfer supernatants to a new 1.5-ml tube and discard the pellet. 6. Leave the platelet extracts on ice until required. 7. To prepare the serial dilutions of nucleotides, add to a 1.5-ml tube 336 ll of water, 12 ll of 1 mM dTTP and 6 ll of 10 mCi/ ml [a-32P]dTTP. Mix, spin down briefly and put on ice. Observe local health and safety rules for the use of unsealed radioactive sources. Use Perspex shielding and minimize exposure time, while maximizing the distance from the source. Check for contamination with an appropriate monitor regularly. 8. Transfer 177 ll of the dTTP solution to a new 1.5-ml tube and put on ice. Add 3 ll of water to one tube and 3 ll of 0.1 mM ddTTP to the other tube. Mix, spin down briefly and put on ice. Although the concentration of dTTP in the tubes is 33.3 lM, it is practical to label the tubes ‘‘10 lMddTTP” and ‘‘10 lM + ddTTP” because the solutions will later be diluted 3.33-fold to give reaction mixtures containing 10 lM dTTP. 9. For the serial dilutions, label 1.5-ml tubes: ‘‘5 lM-ddTTP”, ‘‘5 lM + ddTTP”, ‘‘2 lM-ddTTP”, ‘‘2 lM + ddTTP”, ‘‘1 lMddTTP”, ‘‘1 lM + ddTTP”, ‘‘0.5 lM-ddTTP”, ‘‘0.5 lM + ddTTP”, ‘‘0.2 lM-ddTTP”, ‘‘0.2 lM + ddTTP”, ‘‘0.1 lM-ddTTP”, ‘‘0.1 lM + ddTTP”, ‘‘0.05 lM-ddTTP” and ‘‘0.05 lM + ddTTP”.
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Put tubes on ice. Pipette 135 ll of water in the ‘‘2 lM” and ‘‘0.2 lM” tubes, and pipette 90 ll of water in all other tubes. Transfer 90 ll from the ‘‘10 lM-ddTTP” tube to the ‘‘5 lMddTTP” tube and mix thoroughly. Subsequently, transfer 90 ll from the ‘‘5 lM-ddTTP” tube to the ‘‘2 lM-ddTTP” tube and mix thoroughly. Continue making serial dilutions until the ‘‘0.05 lM-ddTTP” tube is reached. Prepare the ‘‘+ddTTP” dilution series in a similar fashion. 10. To prepare 2.5 ml concentrated reaction mixture, add to a 5ml tube on ice: 364 ll of water 1771 ll of 50 mM HEPESKOH (pH 8.0), 200 mM NaCl 42 ll of 50 mM MnCl2 0.73 ll of b-mercaptoethanol 208 ll of 1 mg/ml poly(rA)oligo(dT)12–18 42 ll of 10 mg/ml acetylated BSA 21 ll of 20 mM aphidicolin 52 ll of 40 u/ll RNAsinÒ RNase inhibitor 11. Mix the concentrated reaction mixture thoroughly, spin down briefly and keep on ice. 12. Place a 96-well PCR plate in a thermocycler and maintain the temperature at 4 °C. 13. Pipette 30 ll of concentrated reaction mixture into 8 8 wells. 14. Pipette 16 5 ll of each of the four platelet extracts (step 6) into two rows of 8 wells filled with concentrated reaction mixture. Keep the remainder of the platelet extracts to determine the protein concentration (step 24). 15. For each of the platelet extracts, pipette 15 ll of the serial dTTP dilution minus ddTTP (step 9) into one row of 8 wells filled with concentrated reaction mixture and platelet extract. Mix by pipetting gently up and down. Next, pipette 15 ll of the serial dTTP dilution plus ddTTP (step 9) into the second row of 8 wells and mix by pipetting gently up and down. 16. Close wells of PCR plate and allow the polymerization reactions to proceed for 10 min at 37 °C. 17. Stop the reactions after 10 min by lowering the temperature to 4 °C. 18. Carefully spot 10-ll samples of each reaction onto a 1.5 1.5 cm square of the DE81 anion exchanger chromatography paper in triplicate. Keep the PCR plate for step 22. 19. To remove unincorporated dTTP, wash the sheet of DE81 paper at room temperature three times for 5 min each by gentle rocking in a vessel filled with 1 L of 2 SSC. 20. Wash the sheet of DE81 paper once for 5 min by gentle rocking in 500 ml of 95% ethanol. 21. Carefully remove the sheet of DE81 paper from the vessel and allow to air-dry on filter paper. Air-drying may be speeded up in an oven at 65 °C. 22. To allow calculation of the amount of dTTP incorporation in pmol, spot 1 ll of one of the reaction mixtures containing 1 lM dTTP onto an unused 1.5 1.5 cm square of the DE81 paper in triplicate. Do not wash. 23. Cut out each of the 1.5 1.5 cm squares of DE81 paper and put into a 20-ml scintillation vail with 5 ml of scintillation cocktail. Determine the radioactivity of each square with a scintillation counter. 24. Determine the protein concentration of the platelet extracts (step 14) with a protein assay kit compatible with the presence of 3% (v/v) Triton X-100 in the samples. 25. After counting of the radioactivity (step 23), calculate the means of all triplicates (in cpm). The mean value of the three unwashed squares spotted with 1 ll reaction mixture containing 1 lM dTTP (step 22) represents the radioactivity of 1 pmol dTTP.
26. To calculate the ddTTP-sensitive dTTP incorporation for each reaction in pmol min1 mg1, first subtract the mean incorporation plus ddTTP from the mean incorporation minus ddTTP to obtain the ddTTP-sensitive incorporation in cpm 10 min1 10 ll1 reaction mixture. Divide this value by the value that represents 1 pmol dTTP (step 25) to obtain the ddTTP-sensitive incorporation in pmol 10 min1 10 ll1 reaction mixture. Divide this value by 10 to obtain the ddTTP-sensitive incorporation in pmol min1 10 ll1 reaction mixture. Divide this value by the protein concentration in lg ll1 (step 24) to obtain the ddTTP-sensitive incorporation in pmol min1 lg1 (because each 10-ll spot contains: 10 ll [5 ll platelet extract/50 ll total reaction mixture] = 1 ll platelet extract). Multiply this value by 1000 to obtain the ddTTP-sensitive incorporation in pmol min1 mg1. 4.3. Calculation of the kinetic constants The reactions catalyzed by DNA polymerase c can be written as:
dT n þ dTTP ! dT nþ1
ð1Þ
dT n þ ddTTP ! dT n ddT
ð2Þ
where (1) describes the elongation of the chain by adding a dTTP and (2) describes the termination of the chain by adding a ddTTP. We use the irreversible Michaelis–Menten kinetics to describe the first process (1). Assuming a constant concentration of DNA 30 -hydroxyl groups ([dTn]), the velocity can be expressed as:
v¼
V max ½dTTP K mðdTTPÞ þ ½dTTP
ð3Þ
where Km (dTTP) is the Michaelis–Menten constant for dTTP and Vmax the maximal velocity, which can be expressed as the total amount of enzyme multiplied by its specific activity: Vmax = [DNA polymerase c] kcat. Because the inhibitory mode of ddTTP has been described as competitive to dTTP [15,16], we express the DNA polymerase c activity (reactions 1 and 2) as a function of the concentrations of dTTP and ddTTP:
v¼
V max ½dTTP K mðdTTPÞ ð1 þ K½ddTTP Þ þ ½dTTP
ð4Þ
iðddTTPÞ
where Ki (ddTTP) is the inhibition constant for ddTTP. In order to obtain the DNA polymerase c kinetic parameters (Vmax, Km, Ki), we simultaneously fit the experimental data of the saturation curves for [dTTP] and [ddTTP] using equation (4), i.e. we fit the experimental curves of the effect of substrate (dTTP) and inhibitor (ddTTP) concentrations on the ddTTP-sensitive dTTP incorporation (DNA polymerase c activity). The kinetic data are fitted via nonlinear regression using Scilab software (INRIA) [21]. The raw data of the saturation curves are put into a Microsoft Excel file in three columns: dTTP concentration, ddTTP concentration and ddTTP-sensitive dTTP incorporation, respectively. We wrote a Scilab routine to fit the data imported from this Excel file. For the nonlinear regression, we used the Scilab built-in function leastsq (for least square), which minimizes the sum of the squares of the difference between experimental and theoretical values. This procedure allows a good determination of the different kinetics parameters of DNA polymerase c and includes a direct determination of the Ki of ddTTP. The Scilab routine is given in the supplementary file.
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netic values of similar magnitudes but we do not think that these represent the Km (dTTP) and Vmax (dTTP) of platelet DNA polymerase c. Earlier, we reported impaired DNA polymerase c activity in mitochondrial preparations of cultured fibroblasts from patients carrying POLG mutations, assayed under similar conditions as described here [13]. Although this previous study demonstrated that cultured fibroblasts are a useful source to study DNA polymerase c function, we were keen to explore a more convenient source of patient DNA polymerase c that is relatively free of contaminating nuclear DNA polymerases. In addition, we wanted to use a more thorough kinetic analysis. With this in mind, we developed the protocol described here. We hope that this protocol will prove useful to investigate the catalytic consequences of POLG sequence variations found in patients. Fig. 2. Reaction saturation curves showing the effect of ddTTP. The dTTP incorporation was measured in 50-ll reaction mixtures containing 10 lg of platelet protein and 0.5–10 lM dTTP, in the absence or presence of the competitive inhibitor ddTTP. The incorporation in the presence of ddTTP was subtracted from the incorporation in the absence of ddTTP to calculate the ddTTP-sensitive dTTP incorporation (j). The dTTP incorporation in the absence of ddTTP is also shown (h). Error bars depict standard deviation (n = 3).
5. Discussion Using the protocol and calculations described above, we found a mean Km (±SD) of 1.42 (±0.37) lM and a mean Vmax of 0.83 (±0.19) pmol min1 mg1 for the incoming dTTP when we determined the steady-state kinetic values of platelet extracts from six adult control subjects. Since we consider the inhibitory mode of ddTTP as competitive, we were also able to calculate its inhibition constant, Ki (ddTTP). We found a mean of 9.57 (±0.03) lM. This value is a somewhat higher than previously reported (0.05 lM) [15]; however, this discrepancy may be explained by experimental differences and the use of extensively purified bovine testes DNA polymerase c. On the other hand, our apparent Km (dTTP) corresponds well to published Km (dTTP) values of 0.8–2.0 lM for isolated bovine enzyme preparations [15,22] and to Km (dTTP) values of 0.4–5.7 lM for reconstituted, recombinant human DNA polymerase c [6, 23–25]. Our Km (dTTP) value is also consistent with the Km (dTTP) of 1.43 lM found by Naviaux and colleagues, who analyzed mitochondrial fractions from human skeletal muscle biopsies [26]. Unlike purified enzyme preparations, mitochondria are possibly contaminated with nuclear DNA polymerases. Although Naviaux and colleagues used quite similar assay conditions as described in this communication, they did not determine the ddTTP-sensitive dTTP incorporation to calculate the enzyme’s activity and reaction constants. In the presence of ddTTP, their mitochondrial preparations showed a residual polymerase activity of 1.9% on poly(rA)oligo(dT)12–18, suggesting the virtual absence of contaminating nuclear DNA polymerase activity [26]. When we assayed human dermal fibroblast mitochondria, we found a residual polymerase activity of 20% in the presence of ddTTP and, therefore, we determined the ddTTP-sensitive dTTP incorporation to calculate the DNA polymerase c activity [13]. Also with platelet extracts, we found that determination of the ddTTPsensitive dTTP incorporation is essential, because the dTTP incorporation in the absence of ddTTP was several fold higher than the ddTTP-sensitive dTTP incorporation (Fig. 2). Kapsa and colleagues [27], who measured DNA polymerase c activity in human platelet extracts but did not use ddTTP, found a Km (dTTP) of 150 lM and a Vmax 1 mg1. In the absence of ddTTP, we found ki(dTTP) of 11.8 pmol min
Acknowledgments The authors thank Rita Smith for the blood sample collection. Thanks are extended to the volunteers who kindly donated their blood and also to Dr. Matthew E. Gegg for critical reading of the manuscript. This work was supported by the Association Française contre les Myopathy Grant 14343 to J.-W.T and T.L. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ymeth.2010.03.002. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13]
[14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27]
H. Gray, T.W. Wong, J. Biol. Chem. 267 (1992) 5835–5841. P.A. Ropp, W.C. Copeland, Genomics 36 (1996) 449–458. J.A. Carrodeguas, D.F. Bogenhagen, Nucl. Acids Res. 28 (2000) 1237–1244. L.S. Kaguni, Annu. Rev. Biochem. 73 (2004) 293–320. M.A. Graziewicz, M.J. Longley, W.C. Copeland, Chem. Rev. 106 (2006) 383–405. S.E. Lim, M.J. Longley, W.C. Copeland, J. Biol. Chem. 274 (1999) 38197–38203. A.A. Johnson, Y. Tsai, S.W. Graves, K.A. Johnson, Biochemistry 39 (2000) 1702– 1708. Available from:
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Methods journal homepage: www.elsevier.com/locate/ymeth
Measurement of kinetic parameters of human platelet DNA polymerase c Jan-Willem Taanman a,*, Margit Heiske b, Thierry Letellier b a b
Department of Clinical Neurosciences, Institute of Neurology, University College London, Rowland Hill Street, London NW3 2PF, United Kingdom U688 INSERM, Université Victor Segalen Bordeaux 2, 146 Rue Léo Saignat, 33076 Bordeaux-Cedex, France
a r t i c l e
i n f o
Article history: Accepted 9 March 2010 Available online 16 March 2010 Keywords: DNA polymerase c Mitochondria Mitochondrial DNA POLG Polymerase activity Platelets
a b s t r a c t Synthesis of mitochondrial DNA is performed by DNA polymerase c. Mutations in POLG, the gene encoding the catalytic subunit of DNA polymerase c, are a major cause of neurological disease. A large proportion of patients carry rare nucleotide substitutions leading to single amino acid changes. Confirming that these replacements are pathogenic can be problematic without biochemical evidence. Here, we provide a hands-on protocol for an in vitro kinetic assay of DNA polymerase c which allows assessment of the Km and Vmax for the incoming nucleotide of the polymerization reaction. To avoid measurement of contaminating nuclear DNA polymerases, platelet extracts are used since platelets do not contain a nucleus. Moreover, platelets have the advantage of being obtainable relatively non-invasively. Polymerization activity is determined by measurement of the incorporation of radioactive thymidine 50 -triphosphate (dTTP) on the homopolymeric RNA substrate poly(rA)oligo(dT)12–18. To further minimize nuclear DNA polymerase activity, aphidicolin, an inhibitor of most nuclear DNA polymerases, is included in the reaction. In addition, reactions are carried out in the absence and presence of the competitive inhibitor of DNA polymerase c, 20 ,30 -dideoxythymidine 50 -triphosphate (ddTTP), to allow calculation of the ddTTP-sensitive incorporation. With this method, platelets from healthy control subjects extracted with 3% Triton X-100 showed a Km for dTTP of 1.42 lM and a Vmax of 0.83 pmol min1 mg1. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction DNA polymerase c is the only DNA polymerase present in mitochondria. The enzyme is responsible for replication and repair of the mitochondrial DNA (mtDNA). Human DNA polymerase c comprises a 136.9-kDa POLG subunit and a 52.2-kDa POLG2 subunit [1–3]. POLG possesses all three catalytic functions of the enzyme: a DNA polymerase activity required for mtDNA synthesis, a 30 ? 50 exonuclease activity providing proofreading capability, and a 50 -deoxyribose phosphate lyase activity which functions in base excision repair [4,5]. Upon interaction with POLG, POLG2 enhances the affinity of the polymerase for DNA and promotes tighter nucleotide binding, thereby increasing the polymerization rate. This leads to an increase in processivity, i.e. the number of nucleotides incorporated per binding event [6,7]. The POLG gene is highly polymorphic. To date, more than 150 sequence variations have been identified [8]. Pathogenic mutations in POLG lead to mtDNA abnormalities and are an important cause of human disease. The first mutations were reported in families with autosomal dominant progressive external ophtalmoplegia associated with the presence of multiple deletions of mtDNA in muscle biopsies [9]. Since then, POLG mutations have been linked to a spec-
* Corresponding author. Fax: +44 20 7472 6829. E-mail address: [email protected] (J.-W. Taanman). 1046-2023/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ymeth.2010.03.002
trum of clinical phenotypes that can present from neonatal life to late middle age and includes common neurological disorders such as migraine, epilepsy and parkinsonism [10,11]. Although there has been extensive genetic analysis of POLG in patients, functional analysis of mutated POLG has been limited to a number of common or otherwise interesting mutations, studied with reconstituted, recombinant DNA polymerase c or in yeast models (reviewed in: [12]). These studies have provided valuable insights but their methodology is impractical for functional analysis of the large number of POLG sequence variants found in patients. Earlier, we reported assays of DNA polymerase c assembly, activity and processivity, using mitochondrial fractions of cultured skin fibroblasts from pediatric patients harboring POLG mutations [13]. This paper describes an in vitro kinetic assay of DNA polymerase c activity, using platelet extracts. Compared to tissue biopsies, platelets have the advantage of being obtainable relatively noninvasively. Thus, the assay offers a convenient method for clinical laboratories to investigate the functional consequences of POLG sequence variations. 2. Experimental design considerations As mentioned above, DNA polymerase c has several catalytic activities. This paper describes a steady-state kinetic analysis for the incoming deoxynucleoside triphosphate of the polymerase
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reaction. The kinetic parameters are determined at 37 °C in 50 ll reaction mixtures containing: 10 lg of platelet protein extract, 25 mM HEPESKOH (pH 8.0), 100 mM NaCl, 0.5 mM MnCl2, 2.5 mM b-mercaptoethanol, 50 lg/ml of the homopolymeric RNA substrate poly(rA)oligo(dT)12–18, 100 l/ml of acetylated bovine serum albumin (BSA), 0.1 mM aphidicolin, 500 lg/ml of RNasinÒ RNase inhibitor, and [a-32P]thymidine 50 -triphosphate (dTTP; specific activity: 5 Ci/mmol) varied from 0.05 to 10 lM. The composition of this reaction mixture is based on the mixture used by Longley and colleagues [14]. Reactions are carried out in the absence and presence of the inhibitor 20 ,30 -dideoxythymidine 50 -triphosphate (ddTTP) to allow calculation of the ddTTP-sensitive dTTP incorporation. The inhibitory mode of ddTTP has been described as competitive to dTTP [15,16]. To determine the DNA polymerase c kinetic parameters (Vmax, Km, Ki), the DNA polymerase c activity is expressed as a function of the concentrations of dTTP and ddTTP, followed by simultaneous fitting of the experimental data of the saturation curves for dTTP and ddTTP. Clinical samples are inevitably contaminated with nuclear DNA polymerases and RNases. For that reason, assay conditions are chosen that favor measurement of the mitochondrial DNA polymerase activity and prevent RNase activity. Platelets are the enucleate ‘progeny’ of megakaryocytes, formed by cytoplasmic budding of the parent cell. As such, they make an ideal model for the study of DNA polymerase c, since the need to account for nuclear polymerase activity is minimized. Platelets are isolated from blood by differential centrifugation, using a protocol designed to limit platelet activation. Platelets are resuspended in 25 mM HEPESKOH (pH 8.0), 100 mM NaCl, aliquoted and stored at 80 °C. No apparent drop in platelet DNA polymerase c activity has been observed with whole blood storage over a 16-h period at room temperature (not shown). Nevertheless, blood is routinely processed as soon as possible. Also, no apparent drop in activity has been noted after 1 year storage of platelet suspensions at 80 °C (not shown). Others have found that DNA polymerase c activity is labile to freezing and thawing [1], therefore, multiple thaw cycles should be avoided. DNA polymerase c is liberated from the platelets with the nonionic surfactant Triton X-100. Titration experiments have indicated that the detergent is a prerequisite to recover DNA polymerase c activity from platelets, but that concentrations between 1 and 6% (v/v) of Triton X-100 show no significant differences in recovery (not shown). For the kinetic assay, we decided to extract 50 mg/ ml (wet weight) platelet suspensions with 3% (v/v) Triton X-100. It should be noted, however, that the final Triton X-100 concentration in the reaction mixture is 0.3% (v/v) due to a 10-fold dilution of the platelet extract in the reaction mixture. Dose response and time course experiments carried out at a constant substrate concentration of 10 lM dTTP, showed that the ddTTP-sensitive dTTP incorporation was linear for up to 10 min with 12.5 lg of platelet protein per 50 ll reaction mixture, and for longer time periods with lower amounts of protein (Fig. 1). At the 10 min time point, the incorporation rate was proportional to the amount of platelet protein (0.91 ± 0.15, 0.88 ± 0.10 and 0.84 ± 0.24 pmol min1 mg1, respectively, at 12.5, 5 and 3 lg platelet protein per 50 ll reaction mixture). For that reason, 10-min reactions with 10 lg of protein per 50 ll reaction mixture were adopted as the standard condition for the assay. Unlike most nuclear DNA polymerases, DNA polymerase c exhibits a potent RNA-directed DNA polymerase (reverse transcriptase) activity and is particularly active on poly(rA)oligo(dT) primed templates [1]. Therefore, the assay is based on the incorporation of radioactive dTTP on poly(rA)oligo(dT). Further differentiation of mitochondrial DNA polymerase activity from nuclear DNA polymerase activity is achieved by addition of aphidicolin to the reaction mixture. This antibiotic is an inhibitor of most nuclear DNA polymerases, but not of DNA polymerase c [17]. Titrations with aphidic-
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Fig. 1. Reaction progress curves showing the effect of the amount of platelet protein in the reaction mixture. The ddTTP-sensitive dTTP incorporation was measured in 50-ll reaction mixtures containing 10 lM dTTP and 12.5 lg (}), 5 lg (h) or 3 lg (r) of platelet protein as described in the text. Error bars depict standard deviation (n = 3).
olin have shown that P0.05 mM aphidicolin results in maximum inhibition of contaminating nuclear DNA polymerases in the in vitro assay (unpublished observations). Therefore, 0.1 mM aphidicolin is present in the reaction mixture. To eradicate nuclear DNA polymerase activity still further, reactions are performed without and with ddTTP, and the ddTTP-sensitive incorporation of dTTP is determined. In contrast to most nuclear DNA polymerases, DNA polymerase c utilizes dideoxynucleotides in vitro as efficiently as natural deoxynucleotides [15,16]. Because these analogs lack the 30 -hydroxyl group, they act as chain terminators once incorporated into DNA. Titrations with ddTTP have indicated that a 1:20 molecular ratio of ddTTP:dTTP results in an almost complete inhibition of DNA polymerase activity of highly purified mitochondria in vitro (unpublished observations). This 1:20 ratio of ddTTP:dTTP is used in the kinetic assay. Unfortunately, the reaction conditions do not abolish all nuclear DNA polymerase activities. DNA polymerase b has reverse transcriptase activity, and DNA polymerases b and h have the same inhibitory properties as DNA polymerase c (i.e. dideoxynucleotide-sensitive and aphidicolin resistant) [17,18]. It is expected, however, that the use of platelets will essentially eliminate these nuclear polymerases. The reaction mixture includes 100 mM NaCl since experiments with reconstituted, recombinant human DNA polymerase c have revealed a salt optimum of around 100 mM NaCl [6]. In addition, the reaction mixture contains 0.5 mM MnCl2 because, with poly(rA)oligo(dT) as template-primer, DNA polymerase c shows optimal activity at 0.5 mM MnCl2 [1]. RNAsinÒ is added to the reaction mixture as a broad spectrum RNase inhibitor. Although RNAsinÒ does not inhibit RNase H activity, the use of MnCl2 eliminates RNase H activity, which requires Mg2+ as cofactor [19].
3. Platelet preparation 3.1. Materials Sodium citrate VacutainersÒ (BD Diagnostics). Prostaglandin I2 sodium salt (Sigma, P6188). Prepare a stock solution of 1 mg/ml in water and store at 80 °C until use. Platelet resuspension buffer: 25 mM HEPESKOH (pH 8.0), 100 mM NaCl (autoclaved). Sterile phosphate-buffered saline (PBS; Gibco or Sigma). Sterile 15-ml tubes, sterile 1.5-ml test tubes, sterile pipettes, microscope slides and coverslips. Specialized equipment: universal centifuge (e.g. Eppendorf centrifuge 5702), phase contrast microscope, ultra low temperature freezer.
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3.2. Protocol 1. Collect blood in sodium citrate VacutainersÒ. Samples should be obtained in accordance with institutional ethics committee guidelines and approval. Observe local health and safety rules for the handling of human blood samples. 2. Pool 10–15 ml of blood in a sterile 15-ml tube. 3. Centrifuge blood for 30 min at 300 g (room temperature). 4. Transfer the top 2/3 of the platelet-rich serum with a sterile pipette to a new 15-ml tube. Pay attention to not disturbing the red cell–serum interface. 5. Add 1 ll of prostaglandin I2 stock solution per ml to the platelet-rich serum to inhibit platelet aggregation. 6. Centrifuge platelet-rich serum for 30 min at 1000 g (room temperature) to pellet platelets. 7. Remove the supernatant using sterile techniques and discard. 8. Resuspend the platelet pellet in PBS at the original volume and add 1 ll/ml of prostaglandin I2 stock solution. 9. Centrifuge resuspended platelets for 15 min at 1000 g (room temperature) to repellet platelets. 10. Remove the supernatant using sterile techniques and discard. 11. Resuspend the platelet pellet in 1 ml of platelet resuspension buffer and add 2 ll of prostaglandin I2 stock solution. 12. Transfer to a sterile, pre-weighted 1.5-ml tube. 13. Centrifuge for 10 min at 1000 g (room temperature) to repellet platelets. 14. Remove all supernatant carefully and determine the wet weight of the platelet pellet. 15. Resuspend platelets in platelet resuspension buffer at a concentration of 100 mg/ml and add 2 ll/ml of prostaglandin I2 stock solution. 16. Assess contamination of the platelet preparations with (nucleated) leukocytes by phase contrast microscopy. Contamination usually consists of only a few nucleated cells per 10 ll of platelet suspension. 17. Prepare 100-ll aliquots of resuspended platelets (10 mg/ 100 ll) in sterile 1.5-ml tubes and store at 80 °C awaiting assays.
4. Polymerase assay 4.1. Materials Water (autoclaved). 50 mM HEPESKOH (pH 8.0), 200 mM NaCl (autoclaved). Extraction buffer: 25 mM HEPESKOH (pH 8.0), 100 mM NaCl, 6% (v/v) Triton X-100. To make 100 ml extraction buffer, add 6.36 g of Triton X-100 to 50 ml of 50 mM HEPESKOH (pH 8.0), 200 mM NaCl and 44 ml of water. Mix until Triton X-100 is fully dissolved. (1 ml of Triton X-100 weights 1.06 g). 1 mM dTTP. To prepare a 1 mM dTTP stock solution, dilute 100 mM dTTP (e.g. Promega, U1235) 100-fold with autoclaved water. Store at 20 °C. 0.1 mM ddTTP. To prepare a 0.1 mM ddTTP stock solution, dilute 100 mM ddTTP (GE Healthcare, 27-2081-01) 1000-fold with autoclaved water. Store at 20°C. 10 mCi/ml [a-32P]dTTP, specific activity: 3000 Ci/mmol (e.g. MP Biomedicals, Thymidine 50 -triphosphate, [a-32P] Isoblue™ stabilized, 0139015X). 50 mM MnCl2 (autoclaved). b-mercaptoethanol (the pure liquid is 14.3 M; e.g. Sigma, M3148). 1 mg/ml of poly(rA)oligo(dT)12–18 (Midland Certified Reagent Company, P-4012B). Dissolve 25 u in 1.25 ml of 25 mM HEPESKOH (pH 8.0), 100 mM NaCl to obtain a concentration
of 20 u/ml. Since 1 u equals 50 lg, 20 u/ml equals 1 mg/ml. Prepare 208-ll aliquots and store at 20 °C. 10 mg/ml acetylated bovine serum albumin (BSA; Promega, R3961). Aphidicolin (Sigma, A0781). To prepare a 20 mM stock solution, dissolve 1 mg of aphidicolin in 148 ll dimethylsulfoxide. Store at 4 °C. This stock solution is stable for >6 weeks. 40 u/ll recombinant RNAsinÒ RNase inhibitor (Promega, N2511 or N2515). DE81 anion exchanger chromatography paper (Whatman, 3658915; Sigma, Z286605). 2 SCC: 300 mM NaCl, 30 mM sodium citrate (pH 7.0). May be made as a 20 SCC stock solution [20] and diluted 10-fold before use. Ethanol (95%). Liquid scintillation cocktail, e.g. Ultima Gold™ liquid scintillation cocktail (Perkin Elmer, 6013329). Protein assay kit (e.g. the BCA™ Protein Assay kit from Pierce, Thermoscientific, 23225). Sterile 96-well PCR plates with lids, 20-ml liquid scintillation vails (e.g. Wheaton; Sigma, V6755), sterile 5 and 1.5-ml test tubes, sterile pipettes. Specialized equipment: thermocycler, vessel sufficiently large to wash a sheet of 500 cm2 DE81 paper in 1 L of 2 SSC on a shaking platform, scintillation counter.
4.2. Protocol This protocol describes the analysis of four platelet samples in parallel. It is not recommended to assay more than four samples together. The serial dilutions of nucleotides (steps 7–9) and the concentrated reaction mixture (steps 10 and 11) can be prepared shortly before and during the platelet extraction (steps 2–6). 1. Draw and mark 195 1.5 1.5 cm squares with a soft pencil on a sheet of DE81 anion exchanger chromatography paper. 2. To prepare the platelet extracts, thaw 100 ll (10 mg wet weight) platelet suspension and add 100 ll ice-cold extraction buffer. 3. Mix thoroughly and leave on ice for 10 min. 4. Microcentrifuge at maximum speed for 10 min at 4 °C to remove particulate matter. 5. Transfer supernatants to a new 1.5-ml tube and discard the pellet. 6. Leave the platelet extracts on ice until required. 7. To prepare the serial dilutions of nucleotides, add to a 1.5-ml tube 336 ll of water, 12 ll of 1 mM dTTP and 6 ll of 10 mCi/ ml [a-32P]dTTP. Mix, spin down briefly and put on ice. Observe local health and safety rules for the use of unsealed radioactive sources. Use Perspex shielding and minimize exposure time, while maximizing the distance from the source. Check for contamination with an appropriate monitor regularly. 8. Transfer 177 ll of the dTTP solution to a new 1.5-ml tube and put on ice. Add 3 ll of water to one tube and 3 ll of 0.1 mM ddTTP to the other tube. Mix, spin down briefly and put on ice. Although the concentration of dTTP in the tubes is 33.3 lM, it is practical to label the tubes ‘‘10 lMddTTP” and ‘‘10 lM + ddTTP” because the solutions will later be diluted 3.33-fold to give reaction mixtures containing 10 lM dTTP. 9. For the serial dilutions, label 1.5-ml tubes: ‘‘5 lM-ddTTP”, ‘‘5 lM + ddTTP”, ‘‘2 lM-ddTTP”, ‘‘2 lM + ddTTP”, ‘‘1 lMddTTP”, ‘‘1 lM + ddTTP”, ‘‘0.5 lM-ddTTP”, ‘‘0.5 lM + ddTTP”, ‘‘0.2 lM-ddTTP”, ‘‘0.2 lM + ddTTP”, ‘‘0.1 lM-ddTTP”, ‘‘0.1 lM + ddTTP”, ‘‘0.05 lM-ddTTP” and ‘‘0.05 lM + ddTTP”.
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Put tubes on ice. Pipette 135 ll of water in the ‘‘2 lM” and ‘‘0.2 lM” tubes, and pipette 90 ll of water in all other tubes. Transfer 90 ll from the ‘‘10 lM-ddTTP” tube to the ‘‘5 lMddTTP” tube and mix thoroughly. Subsequently, transfer 90 ll from the ‘‘5 lM-ddTTP” tube to the ‘‘2 lM-ddTTP” tube and mix thoroughly. Continue making serial dilutions until the ‘‘0.05 lM-ddTTP” tube is reached. Prepare the ‘‘+ddTTP” dilution series in a similar fashion. 10. To prepare 2.5 ml concentrated reaction mixture, add to a 5ml tube on ice: 364 ll of water 1771 ll of 50 mM HEPESKOH (pH 8.0), 200 mM NaCl 42 ll of 50 mM MnCl2 0.73 ll of b-mercaptoethanol 208 ll of 1 mg/ml poly(rA)oligo(dT)12–18 42 ll of 10 mg/ml acetylated BSA 21 ll of 20 mM aphidicolin 52 ll of 40 u/ll RNAsinÒ RNase inhibitor 11. Mix the concentrated reaction mixture thoroughly, spin down briefly and keep on ice. 12. Place a 96-well PCR plate in a thermocycler and maintain the temperature at 4 °C. 13. Pipette 30 ll of concentrated reaction mixture into 8 8 wells. 14. Pipette 16 5 ll of each of the four platelet extracts (step 6) into two rows of 8 wells filled with concentrated reaction mixture. Keep the remainder of the platelet extracts to determine the protein concentration (step 24). 15. For each of the platelet extracts, pipette 15 ll of the serial dTTP dilution minus ddTTP (step 9) into one row of 8 wells filled with concentrated reaction mixture and platelet extract. Mix by pipetting gently up and down. Next, pipette 15 ll of the serial dTTP dilution plus ddTTP (step 9) into the second row of 8 wells and mix by pipetting gently up and down. 16. Close wells of PCR plate and allow the polymerization reactions to proceed for 10 min at 37 °C. 17. Stop the reactions after 10 min by lowering the temperature to 4 °C. 18. Carefully spot 10-ll samples of each reaction onto a 1.5 1.5 cm square of the DE81 anion exchanger chromatography paper in triplicate. Keep the PCR plate for step 22. 19. To remove unincorporated dTTP, wash the sheet of DE81 paper at room temperature three times for 5 min each by gentle rocking in a vessel filled with 1 L of 2 SSC. 20. Wash the sheet of DE81 paper once for 5 min by gentle rocking in 500 ml of 95% ethanol. 21. Carefully remove the sheet of DE81 paper from the vessel and allow to air-dry on filter paper. Air-drying may be speeded up in an oven at 65 °C. 22. To allow calculation of the amount of dTTP incorporation in pmol, spot 1 ll of one of the reaction mixtures containing 1 lM dTTP onto an unused 1.5 1.5 cm square of the DE81 paper in triplicate. Do not wash. 23. Cut out each of the 1.5 1.5 cm squares of DE81 paper and put into a 20-ml scintillation vail with 5 ml of scintillation cocktail. Determine the radioactivity of each square with a scintillation counter. 24. Determine the protein concentration of the platelet extracts (step 14) with a protein assay kit compatible with the presence of 3% (v/v) Triton X-100 in the samples. 25. After counting of the radioactivity (step 23), calculate the means of all triplicates (in cpm). The mean value of the three unwashed squares spotted with 1 ll reaction mixture containing 1 lM dTTP (step 22) represents the radioactivity of 1 pmol dTTP.
26. To calculate the ddTTP-sensitive dTTP incorporation for each reaction in pmol min1 mg1, first subtract the mean incorporation plus ddTTP from the mean incorporation minus ddTTP to obtain the ddTTP-sensitive incorporation in cpm 10 min1 10 ll1 reaction mixture. Divide this value by the value that represents 1 pmol dTTP (step 25) to obtain the ddTTP-sensitive incorporation in pmol 10 min1 10 ll1 reaction mixture. Divide this value by 10 to obtain the ddTTP-sensitive incorporation in pmol min1 10 ll1 reaction mixture. Divide this value by the protein concentration in lg ll1 (step 24) to obtain the ddTTP-sensitive incorporation in pmol min1 lg1 (because each 10-ll spot contains: 10 ll [5 ll platelet extract/50 ll total reaction mixture] = 1 ll platelet extract). Multiply this value by 1000 to obtain the ddTTP-sensitive incorporation in pmol min1 mg1. 4.3. Calculation of the kinetic constants The reactions catalyzed by DNA polymerase c can be written as:
dT n þ dTTP ! dT nþ1
ð1Þ
dT n þ ddTTP ! dT n ddT
ð2Þ
where (1) describes the elongation of the chain by adding a dTTP and (2) describes the termination of the chain by adding a ddTTP. We use the irreversible Michaelis–Menten kinetics to describe the first process (1). Assuming a constant concentration of DNA 30 -hydroxyl groups ([dTn]), the velocity can be expressed as:
v¼
V max ½dTTP K mðdTTPÞ þ ½dTTP
ð3Þ
where Km (dTTP) is the Michaelis–Menten constant for dTTP and Vmax the maximal velocity, which can be expressed as the total amount of enzyme multiplied by its specific activity: Vmax = [DNA polymerase c] kcat. Because the inhibitory mode of ddTTP has been described as competitive to dTTP [15,16], we express the DNA polymerase c activity (reactions 1 and 2) as a function of the concentrations of dTTP and ddTTP:
v¼
V max ½dTTP K mðdTTPÞ ð1 þ K½ddTTP Þ þ ½dTTP
ð4Þ
iðddTTPÞ
where Ki (ddTTP) is the inhibition constant for ddTTP. In order to obtain the DNA polymerase c kinetic parameters (Vmax, Km, Ki), we simultaneously fit the experimental data of the saturation curves for [dTTP] and [ddTTP] using equation (4), i.e. we fit the experimental curves of the effect of substrate (dTTP) and inhibitor (ddTTP) concentrations on the ddTTP-sensitive dTTP incorporation (DNA polymerase c activity). The kinetic data are fitted via nonlinear regression using Scilab software (INRIA) [21]. The raw data of the saturation curves are put into a Microsoft Excel file in three columns: dTTP concentration, ddTTP concentration and ddTTP-sensitive dTTP incorporation, respectively. We wrote a Scilab routine to fit the data imported from this Excel file. For the nonlinear regression, we used the Scilab built-in function leastsq (for least square), which minimizes the sum of the squares of the difference between experimental and theoretical values. This procedure allows a good determination of the different kinetics parameters of DNA polymerase c and includes a direct determination of the Ki of ddTTP. The Scilab routine is given in the supplementary file.
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netic values of similar magnitudes but we do not think that these represent the Km (dTTP) and Vmax (dTTP) of platelet DNA polymerase c. Earlier, we reported impaired DNA polymerase c activity in mitochondrial preparations of cultured fibroblasts from patients carrying POLG mutations, assayed under similar conditions as described here [13]. Although this previous study demonstrated that cultured fibroblasts are a useful source to study DNA polymerase c function, we were keen to explore a more convenient source of patient DNA polymerase c that is relatively free of contaminating nuclear DNA polymerases. In addition, we wanted to use a more thorough kinetic analysis. With this in mind, we developed the protocol described here. We hope that this protocol will prove useful to investigate the catalytic consequences of POLG sequence variations found in patients. Fig. 2. Reaction saturation curves showing the effect of ddTTP. The dTTP incorporation was measured in 50-ll reaction mixtures containing 10 lg of platelet protein and 0.5–10 lM dTTP, in the absence or presence of the competitive inhibitor ddTTP. The incorporation in the presence of ddTTP was subtracted from the incorporation in the absence of ddTTP to calculate the ddTTP-sensitive dTTP incorporation (j). The dTTP incorporation in the absence of ddTTP is also shown (h). Error bars depict standard deviation (n = 3).
5. Discussion Using the protocol and calculations described above, we found a mean Km (±SD) of 1.42 (±0.37) lM and a mean Vmax of 0.83 (±0.19) pmol min1 mg1 for the incoming dTTP when we determined the steady-state kinetic values of platelet extracts from six adult control subjects. Since we consider the inhibitory mode of ddTTP as competitive, we were also able to calculate its inhibition constant, Ki (ddTTP). We found a mean of 9.57 (±0.03) lM. This value is a somewhat higher than previously reported (0.05 lM) [15]; however, this discrepancy may be explained by experimental differences and the use of extensively purified bovine testes DNA polymerase c. On the other hand, our apparent Km (dTTP) corresponds well to published Km (dTTP) values of 0.8–2.0 lM for isolated bovine enzyme preparations [15,22] and to Km (dTTP) values of 0.4–5.7 lM for reconstituted, recombinant human DNA polymerase c [6, 23–25]. Our Km (dTTP) value is also consistent with the Km (dTTP) of 1.43 lM found by Naviaux and colleagues, who analyzed mitochondrial fractions from human skeletal muscle biopsies [26]. Unlike purified enzyme preparations, mitochondria are possibly contaminated with nuclear DNA polymerases. Although Naviaux and colleagues used quite similar assay conditions as described in this communication, they did not determine the ddTTP-sensitive dTTP incorporation to calculate the enzyme’s activity and reaction constants. In the presence of ddTTP, their mitochondrial preparations showed a residual polymerase activity of 1.9% on poly(rA)oligo(dT)12–18, suggesting the virtual absence of contaminating nuclear DNA polymerase activity [26]. When we assayed human dermal fibroblast mitochondria, we found a residual polymerase activity of 20% in the presence of ddTTP and, therefore, we determined the ddTTP-sensitive dTTP incorporation to calculate the DNA polymerase c activity [13]. Also with platelet extracts, we found that determination of the ddTTPsensitive dTTP incorporation is essential, because the dTTP incorporation in the absence of ddTTP was several fold higher than the ddTTP-sensitive dTTP incorporation (Fig. 2). Kapsa and colleagues [27], who measured DNA polymerase c activity in human platelet extracts but did not use ddTTP, found a Km (dTTP) of 150 lM and a Vmax 1 mg1. In the absence of ddTTP, we found ki(dTTP) of 11.8 pmol min
Acknowledgments The authors thank Rita Smith for the blood sample collection. Thanks are extended to the volunteers who kindly donated their blood and also to Dr. Matthew E. Gegg for critical reading of the manuscript. This work was supported by the Association Française contre les Myopathy Grant 14343 to J.-W.T and T.L. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ymeth.2010.03.002. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13]
[14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27]
H. Gray, T.W. Wong, J. Biol. Chem. 267 (1992) 5835–5841. P.A. Ropp, W.C. Copeland, Genomics 36 (1996) 449–458. J.A. Carrodeguas, D.F. Bogenhagen, Nucl. Acids Res. 28 (2000) 1237–1244. L.S. Kaguni, Annu. Rev. Biochem. 73 (2004) 293–320. M.A. Graziewicz, M.J. Longley, W.C. Copeland, Chem. Rev. 106 (2006) 383–405. S.E. Lim, M.J. Longley, W.C. Copeland, J. Biol. Chem. 274 (1999) 38197–38203. A.A. Johnson, Y. Tsai, S.W. Graves, K.A. Johnson, Biochemistry 39 (2000) 1702– 1708. Available from: