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Mar 2020
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Real-time Base Excision Repair Assay to Measure the Activity of the 8-oxoguanine DNA Glycosylase 1 in Isolated Mitochondria of Human Skin Fibroblasts
实时碱基切除修复法测定人皮肤纤维细胞的分离线粒体中8-氧鸟嘌呤DNA糖基化酶1的活性   

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Abstract

7,8-dihydro-8-oxoguanine (8-oxoG) is one of the most common and mutagenic oxidative DNA damages induced by reactive oxygen species (ROS). Since ROS is mainly produced in the inner membranes of the mitochondria, these organelles and especially the mitochondrial DNA (mtDNA) contained therein are particularly affected by this damage. Insufficient elimination of 8-oxoG can lead to mutations and thus to severe mitochondrial dysfunctions. To eliminate 8-oxoG, the human body uses the enzyme 8-oxoguanine DNA glycosylase 1 (OGG1), which is the main antagonist to oxidative damage to DNA. However, previous work suggests that the activity of the human OGG1 (hOGG1) decreases with age, leading to an age-related accumulation of 8-oxoG. A better understanding of the exact mechanisms of hOGG1 could lead to the discovery of new targets and thus be of great importance for the development of preventive therapies. Because of this, we developed a real-time base excision repair assay with a specially designed double-stranded reporter oligonucleotides to measure the activity of hOGG1 in lysates of isolated mitochondria. This system presented here differs from the classical assays, in which an endpoint determination is performed via a denaturing acrylamide gel, by the possibility to measure the hOGG1 activity in real-time. In addition, to determine the activity of each enzymatic step (N-glycosylase and AP-lyase activity) of this bifunctional enzyme, a melting curve analysis can also be performed. After isolation of mitochondria from human fibroblasts using various centrifugation steps, they are lysed and then incubated with specially designed reporter oligonucleotides. The subsequent measurement of hOGG1 activity is performed in a conventional real-time PCR system.

Keywords: DNA repair (DNA修复), hOGG1 (人类8-氧代鸟嘌呤DNA糖基化酶1), 7,8-dihydro-8-oxoguanine (7,8-二氢-8-氧鸟嘌呤), Enzyme activity (酶活力), Mitochondria (线粒体)

Background

The human body represents a permanent case of damage. Each of the approximately 1013 body cells suffers tens of thousands to about 100,000 damages to its DNA per day (Janion, 2001; Jackson and Bartek, 2009). 7,8-dihydro-8-oxoguanine (8-oxoG) is one of the most common oxidative DNA damages induced by reactive oxygen species (ROS). By oxidizing the base guanine to 7,8-dihydro-8-oxoguanine, 8-oxoG, in contrast to guanine, is now able to hybridize not only with cytosine but also with adenine. This can lead to a complete base exchange after the second replication and thus trigger serious mutations. The replacement of a guanine/cytosine base pair by thymine/adenine is one of the most frequent mutations in human cancer. This so-called transversion also occurs frequently in the tumor suppressor gene p53 (Hirano, 2008). For this reason, and based on the fact that between 1,000 and 2,000 8-oxoG lesions per day occur in a healthy cell, while in tumor cells the number of 8-oxoG lesions increases up to 1,000,000, 8-oxoG is considered to be one of the major causes of cancer (Ravanat et al., 2000; Nakabeppu, 2014). Furthermore, 8-oxoG is associated with aging processes and degenerative diseases (such as Alzheimer's disease and Parkinson's disease) (Sampath et al., 2012; Nakabeppu, 2014) and thus shows great pathophysiological relevance.


To counteract these processes, the human body uses the enzyme 8-oxoguanine DNA glycosylase 1 (OGG1), which is the main antagonist to oxidative damage to DNA. Before replication, it removes 8-oxoG by means of base excision repair (BER), replaces it with a guanine base and thus prevents the development of mutations (Radicella et al., 1997; Hamann et al., 2009). However, previous studies indicate that the activities of glycosylases and thus also the activity of the human OGG1 (hOGG1) decrease with increasing age (Loft and Poulsen, 1996; Gorbunova et al., 2007). This has the effect on an organism that sooner or later, as a result of the reduced hOGG1 activity and the resulting decrease in the ability to perform BER processes, an age-related accumulation of 8-oxoG occurs. A better understanding of the exact mechanisms of hOGG1 could lead to the discovery of new targets and thus be of great importance for the development of preventive therapies.


For the measurement of the hOGG1 activity we developed an assay that exploits the principle of enzymatic removal of 8-oxoG from DNA as well as the cut into its sugar-phosphate backbone by hOGG1 as a part of the BER. The principle of the enzymatic removal of 8-oxoG from DNA as well as the incision in its sugar-phosphate backbone by hOGG1 during a BER is used. Lysates from isolated mitochondria are incubated with specially designed reporter oligonucleotides for this assay. Each of the two double-stranded constructs consists of 33 nucleotides and identical sequence. In addition, both carry a 6-carboxyfluorescein (6-FAM) molecule at each end of the reporter strand and a black hole quencher (BHQ) at each end of the complementary strand (Figure 1). Thus, in each double-stranded construct, the 6-FAM molecule (fluorescent dye) is opposite of BHQ which suppresses the fluorescence of the dye. The DNA glycosylase present in the lysates from the mitochondria recognizes the damage to the reporter oligonucleotide (CN-8-oxoG) and first cuts out the oxidized base, followed by a cut in the phosphate backbone. The incised DNA strand then dissociates from the counter strand at the incubation temperature of 37 °C and a fluorescence signal can be measured. The strength of the signal correlates with the repair activity or AP lyase activity of the hOGG1 enzyme. The use of an undamaged reporter oligonucleotide (CN-CTRL) serves as a control to exclude unspecific digestion of the oligonucleotides (Figure 2A). To determine the resulting oligonucleotide fragments, a melting curve analysis is performed at a temperature of 95 °C to 20 °C after completion of the real-time activity measurement. For this purpose, the first derivation of the trend of the fluorescence signal as a function of temperature is recorded (-(d/dT)). The resulting minima represent the melting temperatures of the different fragments (Figure 2B).


Materials and Reagents

  1. 2-Mercaptoethanol (Carl Roth, catalog number: 4227.1 ) 

  2. Benzamidine (Carl Roth, catalog number: CN38.1 )

  3. BHQ (single-stranded) ([BHQ1]GGTATTATTATTATTGCGTTATTATTATTATGG[BHQ1] 100 μM) (Sigma-Aldrich)

  4. Bovine serum albumin (BSA) (New England BioLabs®, catalog number: B9000S )

  5. Cell culture flasks T25, T75, T175 (Sarstedt, catalog numbers: 83.3910.302 , 83.3911.302 , 833912.302 )

  6. CHAPS (Carl Roth, catalog number: 1479.1 )

  7. CN-8-oxoG (single-stranded) ([6FAM]CCATAATAATAATAAC[8-oxo-dG]CAATAATAATAATA

    CC[6FAM] 100 µM) (Sigma-Aldrich)

  8. CN-CTRL (single-stranded) ([6FAM]CCATAATAATAATAACGCAATAATAATAATACC[6FAM] 100 µM) (Sigma-Aldrich)

  9. cOmpleteTM Mini Protease Inhibitor Cocktail (Roche Diagnostics, catalog number: 04693124001 )

  10. DMSO (Carl Roth, catalog number: A994.2 )

  11. Dulbecco’s Modified Eagle’s Medium–high glucose (Sigma-Aldrich, catalog number: D7777-10L )

  12. EGTA (Carl Roth, catalog number: 3054.1 )

  13. Fetal bovine serum (FBS) (Gibco®, catalog number: 26140079 )

  14. Gentamycin (10 mg/ml) (Gibco®, catalog number: 11500506 )

  15. Glycerol (Carl Roth, catalog number: 6967.1 )

  16. Injection cannula (G 20 × 1½”) (B. Braun, catalog number: 4657519 )

  17. KCl (Carl Roth, catalog number: 6781.3 )

  18. KH2PO4 (Carl Roth, catalog number: 3904.2 )

  19. LightCycler® 480 Multiwell Plate 96 (white) (Roche Diagnostics, catalog number: 04729692001 )

  20. LightCycler® 480 Sealing Foil (Roche Diagnostics, catalog number: 04729757001 )

  21. MgCl2 (Carl Roth, catalog number: KK36.1 )

  22. Na2HPO4·12H2O (Carl Roth, catalog number: N350.1 )

  23. NaCl (Carl Roth, catalog number: 3957.3 )

  24. NEBufferTM2 (New England BioLabs®, catalog number: B7002S )

  25. Pipette filter tips: 2.5 µl, 10 µl, 20 µl, 200 µl, 1,000 µl (Sarstedt, catalog numbers: 70.1130.217 , 70.1130.215 , 70.1114.215 , 70.760.216 , 70.762.216 )

  26. QuinoMit®–carrier control (MSE Pharmazeutika)

  27. QuinoMit® Q10-Fluid–ubiquinol (MSE Pharmazeutika)

  28. Reaction Tubes: 0.2 ml, 1.5 ml (Sarstedt, catalog numbers: 72.737.002 , 72.706.201 )

  29. Recombinant Human OGG1 protein (100 µg) (Abcam, catalog number: ab98249 )

  30. Syringe 2 ml (Injekt® 2 ml) (B. Braun, catalog number: 4606027V )

  31. Tris-HCl (Carl Roth, catalog number: 9090.3 )

  32. Tri-sodium citrate dihydrate (≥99%) (Carl Roth, catalog number: 3580.4 )

  33. Trypsin-EDTA (0.5%) (Gibco®, catalog number: 10779413 )

  34. 1× PBS (see Recipes)

  35. CHAPS buffer (see Recipes)

  36. Carrier control (10 mM) (see Recipes)

  37. Ubiquinol formulation (10 mM) (see Recipes)

  38. DMEM for Fibroblasts (see Recipes)

  39. Reaction buffer (see Recipes)

  40. 2 M tri-sodium citrate dihydrate (see Recipes)

Equipment

  1. Centrifuge Eppendorf 5427 R (Eppendorf, catalog number: 5409000210 )

  2. Centrifuge Eppendorf 5804 R (Eppendorf, catalog number: 5805000010 )

  3. Freezer (Liebherr Comfort (Liebherr, catalog number: not available)

  4. Incubator at 37 °C with 5% CO2, 90% humidity (HERA Cell 240, catalog number: 2510-413-01 )

  5. LightCycler® 480 (Roche Diagnostics, catalog number: 05015278001 )

  6. PCR-Cooler (Eppendorf, catalog number: 3881000031 )

  7. pH Meter FiveEasyTM F20 (Mettler Toledo, catalog number: 30266626 )

  8. Pipettes, Eppendorf Research® Plus: 0.1 µl 10 µl, 20 µl, 200 µl, 1,000 µl (Eppendorf, catalog numbers: 3123000012 , 3123000020 , 3123000039 , 3123000055 , 3123000063 )

  9. Sonoplus Ultraschall-Homogenisator HD 3100 (Bandelin Electronic)

  10. Thermocycler MKR13 HLC (DITABIS, catalog number: MKR 13 )

  11. Ultrapure water (ddH2O) preparation unit, Purelab flex 4 (Veolia Water Technologies)

Software

  1. GraphPad Prism, Version 8.2.1 (GraphPad Software®)

  2. LightCycler® 480 Software (instrument software), Version 1.5.0.39 (Roche Diagnostics)

Procedure

  1. Cell culture

    1. First cultivate fibroblasts at 37 °C in the presence of 5% CO2 and 90% humidity in an appropriate cell specific medium with or without test substances.

    2. After an appropriate incubation period, remove the cells with Trypsin-EDTA and transfer 3.0 × 106 cells into a new 1.5 ml reaction tube.

    3. Centrifuge the cells for 5 min at 1,200 × g and 4 °C and resuspend the resulting pellet in 1 ml 1× PBS (washing step).


  2. Isolation and lysis of mitochondria

    1. Take 3.0 × 106 fibroblasts in 1× PBS and centrifuge it for 5 min at 1,200 × g and 4 °C.

    2. Discard the supernatant and resuspend the resulting pellet in 200 µl CHAPS lysis buffer (pH 7.4) supplemented with 3 μl of 2 M tri-sodium citrate dihydrate and incubate on ice for 45 min.

    3. Homogenize the sample with a syringe and a cannula (diameter = 0.9 mm) on ice by pipetting up and down ten times.

    4. To completely separate the mitochondria from the remaining cell structures, centrifuge the samples again for 10 min at 1,200 × g and 4 °C.

    5. Transfer the supernatants with the mitochondria into a new 1.5 ml reaction tube (pre-cooled on ice).

    6. Lyse the isolated mitochondria in a further lysis step on ice with pulsed ultrasound at 20 watts for 1 min (0.5 s pulse on, 1.0 s pulse off).

    7. After a further centrifugation step for 45 min at 21,000 × g and 4 °C, the supernatants can be stored on ice until further use within a few hours. Keep supernatants at -20 °C for overnight or longer storage.


  3. Preparation of the reporter oligonucleotides

    1. Mix 5 µl of each of the single-stranded reporter oligonucleotides CN-8-oxoG ([6FAM]CCATAATAATAATAAC[8-oxo-dG]CAATAATAATAATACC[6FAM]) (100 µM) and CN-CTRL ([6FAM]CCATAATAATAATAACGCAATAATAATAATACC[6FAM]) (100 µM) each in a separate 0.2 ml reaction tube with 10 μl of the BHQ-1-tagged complementary strands ([BHQ1]GGTATTATTATTATTGCGTTATTATTA TTATGG[BHQ1]) (100 μM) (all Sigma Aldrich GmbH) and 35 μl NEBufferTM2. A schematic representation of the reporter oligonucleotides can be seen in Figure 1.

    2. Incubate the oligo mixtures for 15 min at 95 °C in the thermocycler, followed by cooling down of the finished constructs in the freezer at -20 °C (annealing during cooling phase–annealing temperature = 50 °C). The subsequent storage of the finished constructs take place at -20 °C.



      Figure 1. Basic principle of the assay. A double-stranded nucleotide strand carrying an 8-oxoG base (shown in red) is used to detect the activity of hOGG1. The strand with the damage carries at both ends a 6-FAM molecule as a fluorescence reporter and on the opposing quencher are added at both ends. In the uncut reporter oligonucleotide both strands are hybridized with each other whereby the fluorescence signal of the 6-FAM molecules is intercepted by the quenchers. If the strand with the damage is cut, the two nucleotide strands dissociate from each other and the fluorescence signal can be measured.


  4. Base excision repair assay

    1. Pipette the samples and controls into a white 96-well plate as shown in Table 1, homogenizing the samples by pipetting up and down (two wells are required per approach – one for CN-8-oxoG and one for CN-CTRL).


      Table 1. Components of the different reaction approaches

      Positive control Negative control Sample
      Reaction buffer 94.3 µl 94.3 µl 75.3 µl
      Tri-sodium citrate dihydrate [2M] 3.7 µl 3.7 µl 3.7 µl

      Reporter constructs [CN-8-oxoG 0.1 µM/
      CN-CTRL 0.2 µM]

      1 µl (CN-8-oxoG)/
      (CN-CTRL)
      1 µl (CN-8-oxoG)/
      (CN-CTRL)
      1 µl (CN-8-oxoG)/
      (CN-CTRL)
      Recomb. OGG1 1 µl -- --
      ddH2O -- 1 µl --
      Sample -- -- 20 µl


    2. Seal the plate with a sealing foil and centrifuge the plate for 5 min at 720 × g and 4 °C.

    3. For a real-time detection of the base excision activity, perform the measurement of the fluorescence signals in a Light Cycler 480 (Roche Diagnostics) with a total of 80 cycles of 1 min each at 37 °C. Excitation of the 6-FAM molecule occurs at 465 nm and detection of the fluorescence signals in the wavelength range of 483-533 nm (Figure 2A).

    4. To determine the resulting oligonucleotide fragments, perform a melting curve analysis at a temperature of 95 °C to 20 °C after completion of the real-time activity measurement (Figure 2B).



      Figure 2. Schematic representation of a determination of the hOGG1 activity by fluorescence and melting curve analysis. A. Fluorescence curves of the reporter oligonucleotides CN-8-oxoG (with damage) and CN-CTRL (without damage) after digestion with 1.6 units of commercially purchased hOGG1 as well as hOGG1 obtained from cell lysates over a period of 80 cycles of 1 min each at 37 °C. B. Melting curves of the reporter oligonucleotides CN-8-oxoG and CN-CTRL after digestion with 1.6 units of commercially purchased hOGG1 and hOGG1 obtained from cell lysates.

Data analysis

  1. The base excision repair activity of hOGG1 can be determined by means of the end point (maximum fluorescence) of the fluorescence curve of the defective reporter oligonucleotide (CN-8-oxoG) minus the nonspecific digestion (CN-CTRL) (Figure 2A).

  2. A fragment analysis can be carried out after completion of the real-time activity measurement using the function of the recorded melting curves. For this, the first negative derivative of the trend of the fluorescence signal is recorded at a temperature change from 95 °C to 20 °C (-(d/dT)). The resulting minima represent the melting temperatures of the different fragments. The peak at position 1 represents a complete cut substrate of hOGG1 activity. In this substrate, both the modified base (8-oxoG) was excised from the DNA and the sugar-phosphate backbone of the DNA was cut. The peak labeled at position 2 represents substrate with an AP site in which the N-glycosylase function of the enzyme was already active but the AP lyase was not. At position 3 there is uncut substrate, in which neither the glycosylase nor the ligase function became active. Depending on where the larger peak is located, statements can be made about the activity of the respective enzymatic steps of hOGG1 (Figure 2B).

  3. Data can be analyzed using GraphPad Prism, Version 8.2.1 (GraphPad Software®). Values are presented as mean ± SEM, or individual values. Three replicates per approach are recommended.


Representative data

Measurement of base excision repair activity of hOGG1 in lysates of isolated mitochondria (Figure 3) according to the proceeding steps. In this case however, to test the effect of CoQ10 on the enzymatic activity of hOGG1, an ubiquinol formulation (QuinoMit® Q10-Fluid–ubiquinol, MSE Pharmazeutika) and a carrier control (CC) (QuinoMit®–carrier control, MSE Pharmazeutika) were additionally added to the different approaches directly before the measurement. This was done because it is suspected that hOGG1, like other redox-dependent transcription factors are actively regulated by the redox-active properties of antioxidants. The carrier control was administrated exactly as described for QuinoMit® Q10-Fluid. QuinoMit® Q10-Fluid and carrier control are emulsions of phospholipid nanoparticles (30-90 nm). Both were always freshly weighed and diluted with ultrapure water. The exact composition of these test compounds can be found in Table 2 below.



Figure 3. Influence of ubiquinol on hOGG1 enzymatic activity isolated from mitochondria. Addition of different concentrations (10-1,000 μM) of ubiquinol and carrier control (CC) to reporter oligonucleotides and hOGG1 from mitochondrial lysates, isolated from 3.0 × 106 human fibroblasts. As a negative control (NC) one approach with test solution and reporter oligonucleotides but without hOGG1 were included. The fluorescence signals were recorded with a LightCycler® 480 in a wavelength range between 483 and 533 nm. The fluorescence signals of the digested reporter oligonucleotides based on the hOGG1 activity are shown (n = 3, mean ± SEM, one-way ANOVA with Bonferroni’s multiple comparison test, *P < 0.05). Values normalized to the positive control (PC = all assay components without test solution). PC correspond to 1.0. Data obtained from Schniertshauer et al. (2020).


Table 2. Exact composition of QuinoMit® Q10-Fluid and carrier control

CoQ10 formulations and carrier control
QuinoMit® Q10-Fluid
ubiquinol (red.)
carrier control
Glycerin + +
Ubiquinol + -
Deionized water + +
Soy lecithin + +
Niacinamide + +

Notes

  1. Measurement of base excision repair activity in lysates of isolated mitochondria we described here was highly reproducible.

  2. Tri-sodium citrate is required to prevent unspecific digestion of the reporter oligonucleotides by DNases contained in the cell lysates. The optimal concentration of tri-sodium citrate dihydrate for inhibition of DNase was established for fibroblasts and could be adapted to the use of other cell lines.

  3. The assay presented here addresses the activity of the repair enzyme hOGG1. An adaptation to other repair enzymes of BER should be possible and depends on the reporter oligonucleotides used.

  4. Reporter oligonucleotides used here are not commercially available, but were designed by Sigma Aldrich and generated on behalf of a customer.

  5. This method was developed on primary fibroblasts but according to initial findings it also works for lysates of PBMCs. Further cell lines must first be established.

  6. The measurement as described here was performed in a light cycler, but can be adapted to any other RT-PCR system.

  7. Measurement of fluorescence signals in the wavelength range between 483 and 533 is based on the fluorescent markers used (6-FAM; Ex 465 nm/Em 510 nm).

  8. Preparation of the approaches in the 96-well plate should be carried out on cold packs (PCR-Cooler) to avoid premature enzyme activity as far as possible.

  9. The increase in fluorescence in the real-time image is caused–as described above–by the incised fragments. These fragments have a melting temperature of about 36 °C for the reporter oligonucleotides, while the uncut oligonucleotide has a melting temperature of about 52 °C.

  10. Human fibroblasts were isolated from skin biopsies which were received from the Kreiskrankenhaus Sigmaringen, general surgery unit, Germany; from Aesthetic Perfection Lake Constance, plastic surgery unit, Germany; or from the Chirurgische Gemeinschaftspraxis Dr. Fuhrer, H. Nonnenmacher, Dr. Astfalk und Dr. Fauser, Reutlingen, Germany. All experiments were conducted in accordance with the Declaration of Helsinki and approved by the Ethics Commission of the State Medical Association of Baden-Württemberg, Germany (187-03). Patients were informed in advance and gave their written consent to the use of their samples. Cells were isolated as described by Burger et al. (2010).

Recipes

  1. 1× PBS

    8 g NaCl

    0.20 g KCl

    2.88 g Na2HPO4·12H2O

    1.24 g KH2PO4

    Adjust to 1 L ddH2O, pH 7.4

  2. 2 M tri-sodium citrate dihydrate

    5.882 g tri-sodium citrate dihydrate

    Adjust to 10 ml ddH2O

  3. Carrier control (10 mM)

    0.904 g QuinoMit®–carrier control

    Adjust to 5 ml ddH2O, pH 7.4

  4. CHAPS buffer

    1.25 ml Glycerol (80%)

    1 ml CHAPS (5%)

    100 µl Tris-HCl (1 M)

    100 µl EGTA (0.1 M)

    10 µl MgCl2 (1 M)

    5 µl Benzamidine (0.2 M)

    3.52 µl 2-Mercaptoethanol (14.19 M)

    Adjust to 10 ml ddH2O, pH 7.4

    Add one tablet cOmpleteTM Mini Protease Inhibitor Cocktail

  5. DMEM for Fibroblasts

    500 ml DMEM

    50 ml FBS

    2.5ml Gentamycin (50 µg/ml)

  6. Reaction buffer

    320 µl DMSO

    160 µl NEBufferTM2

    16 µl BSA (20 µg/µl)

    752 µl ddH2O

  7. Ubiquinol formulation (10 mM)

    0.904 g QuinoMit® Q10-Fluid

    Adjust to 5 ml ddH2O, pH 7.4

Acknowledgments

The authors thank Dr. Franz Enzmann and Dr. Alexander Bürkle for scientific advice. This study was supported by the BMBFFHprofUnt2012 “MitoFunk” (03FH022PX2) and by the Baden-Württemberg Ministry of Science, Research and Art.

Competing interests

Jörg Bergemann has consulting contracts with MSE Pharmazeutika GmbH, Bad Homburg, Germany, and Beiersdorf AG, Hamburg, Germany.

References

  1. Burger, K., Matt, K., Kieser, N., Gebhard, D. and Bergemann, J. (2010). A modified fluorimetric host cell reactivation assay to determine the repair capacity of primary keratinocytes, melanocytes and fibroblasts. BMC Biotechnol 10: 46.
  2. Gorbunova, V., Seluanov, A., Mao, Z. and Hine, C. (2007). Changes in DNA repair during aging. Nucleic Acids Res 35(22): 7466-7474.
  3. Hamann, I., Schwerdtle, T. and Hartwig, A. (2009). Establishment of a non-radioactive cleavage assay to assess the DNA repair capacity towards oxidatively damaged DNA in subcellular and cellular systems and the impact of copper. Mutat Res 669(1-2): 122-130.
  4. Hirano, T. (2008). Repair system of 7, 8-dihydro-8-oxoguanine as a defense line against carcinogenesis.J Radiat Res 49(4): 329-340.
  5. Jackson, S. P. and Bartek, J. (2009). The DNA-damage response in human biology and disease. Nature 461(7267): 1071-1078.
  6. Janion, C. (2001). Some provocative thoughts on damage and repair of DNA. J Biomed Biotechnol 1(2): 50-51.
  7. Loft, S. and Poulsen, H. E. (1996). Cancer risk and oxidative DNA damage in man. J Mol Med (Berl) 74(6): 297-312.
  8. Nakabeppu, Y. (2014). Cellular levels of 8-oxoguanine in either DNA or the nucleotide pool play pivotal roles in carcinogenesis and survival of cancer cells. Int J Mol Sci 15(7): 12543-12557.
  9. Radicella, J. P., Dherin, C., Desmaze, C., Fox, M. S. and Boiteux, S. (1997). Cloning and characterization of hOGG1, a human homolog of the OGG1 gene of Saccharomyces cerevisiae.Proc Natl Acad Sci U S A 94(15): 8010-8015.
  10. Ravanat, J. L., Di Mascio, P., Martinez, G. R., Medeiros, M. H. and Cadet, J. (2000). Singlet oxygen induces oxidation of cellular DNA. J Biol Chem 275(51): 40601-40604.
  11. Sampath, H., Vartanian, V., Rollins, M. R., Sakumi, K., Nakabeppu, Y. and Lloyd, R. S. (2012). 8-Oxoguanine DNA glycosylase (OGG1) deficiency increases susceptibility to obesity and metabolic dysfunction.PLoS One 7(12): e51697.
  12. Schniertshauer, D., Gebhard, D., van Beek, H., Noth, V., Schon, J. and Bergemann, J. (2020). The activity of the DNA repair enzyme hOGG1 can be directly modulated by ubiquinol.DNA Repair (Amst) 87: 102784.

简介

[摘要] 7,8-二氢-8-氧鸟嘌呤(8-oxoG)是由活性氧(ROS)引起的最常见且诱变的氧化DN A损伤之一。由于ROS主要在线粒体的内膜中产生,因此这些细胞器,特别是其中所含的线粒体DNA(mtDNA)受到这种损害的特别影响。消除8-oxoG可能会导致突变,从而导致严重的线粒体功能障碍。为了消除8-oxoG,人体使用了8-氧代鸟嘌呤DNA糖基化酶1(OGG1),它是DNA氧化损伤的主要拮抗剂。但是,先前的研究表明,人类OGG1的活性(h OGG1)随着年龄的增长而减少,导致与年龄相关的8-oxoG积累。更好地了解hOGG1的确切机制可能会导致发现新的靶标,因此对于开发预防性疗法具有重要意义。因此,我们开发了一种实时碱基切除修复测定法,该测定法采用了专门设计的双链报告寡核苷酸来测量分离的线粒体裂解物中hOGG1的活性。这里介绍的该系统与经典测定法不同,在经典测定法中,可以通过实时测量hOGG1活性通过变性丙烯酰胺凝胶进行终点测定。另外,为了确定该双功能酶的每个酶促步骤的活性(N-糖基化酶和AP-裂解酶活性),还可以进行解链曲线分析。使用各种离心步骤从人成纤维细胞中分离线粒体后,将其裂解,然后与专门设计的报告寡核苷酸一起孵育。hOGG1活性的后续测量是在常规实时PCR系统中进行的。

[背景]人体是永久的损害案例。每天约10 13个人体细胞中的每个细胞每天都对其DNA遭受数万至约100,000的损害(Janion,2001 ;Jackson和Bartek,2009)。7,8-二氢-8-氧鸟嘌呤(8-oxoG)是活性氧(ROS)引起的最常见的DNA氧化损伤之一。通过将基础鸟嘌呤氧化为7,8-二氢-8-氧代鸟嘌呤,与鸟嘌呤相比,8-oxoG现在不仅可以与胞嘧啶杂交,还可以与腺嘌呤杂交。这可能导致第二次复制后发生完全的碱基交换,从而引发严重的突变。用胸腺嘧啶/腺嘌呤替代鸟嘌呤/胞嘧啶碱基对是人类癌症中最常见的突变之一。这种所谓的颠换也经常在肿瘤抑制基因p53中发生(Hirano,2008)。因此,基于在健康细胞中每天发生1,000至2,000个8-oxoG损伤的事实,而在肿瘤细胞中,8-oxoG损伤的数量增加至1,000,000,因此8-oxoG被认为是其中之一。导致癌症的主要原因(Ravanat et al。,2000; Nakabeppu,2014)。此外,8-oxoG与衰老过程和退行性疾病(例如阿尔茨海默氏病和帕金森氏病)相关(Sampath等,2012; Nakabeppu,2014),因此显示出极大的病理生理意义。

为了抵消这些过程,人体使用了8-氧代鸟嘌呤DNA糖基化酶1(OGG1),它是DNA氧化损伤的主要拮抗剂。在复制之前,其通过碱基切除修复(BER)去除8-​​oxoG,用鸟嘌呤碱基替代它,从而防止了突变的发展(Radicella等,1997; Hamann等,2009)。但是,先前的研究表明,糖基化酶的活性以及人OGG1(hOGG1)的活性随着年龄的增长而降低(Loft和Poulsen,1996 ;Gorbunova等,2007)。由于降低了hOGG1的活性并降低了进行BER的能力,这对有机体产生了迟早的影响,出现了年龄相关的8-o xoG积累。更好地了解hOGG1的确切机制可能会导致发现新的靶标,因此对于开发预防性疗法具有重要意义。

为了测量hOGG1的活性,我们开发了一种测定方法,该方法利用了酶从DNA上去除8-oxoG的原理以及hOGG1作为BER的一部分将其切成糖糖主链的原理。使用从DNA中酶促去除8-oxoG的原理,以及在BER期间hOGG1在其糖-磷酸主链上切开的原理。将来自分离的线粒体的裂解物与专门设计的报道分子寡核苷酸进行温育以进行此测定。每两个双链构建体包括小号的33个核苷酸和相同序列。另外,无论是在记者链和在互补链(图中的每个端部的黑洞猝灭剂(BHQ)的每个端部携带一个6-羧基(6-FAM)分子URE 1)。因此,在每个双链构建体中,6-FAM分子(荧光染料)是相反的BHQ这抑制了染料的荧光。线粒体裂解液中存在的DNA糖基化酶识别对报告寡核苷酸(CN-8-oxoG)的破坏,首先切出氧化的碱基,然后切开磷酸酯骨架。然后,在37°C的孵育温度下,切割的DNA链与对链解离,可以测量荧光信号。信号强度与hOGG1酶的修复活性或AP裂解酶活性相关。使用未损坏的报告寡核苷酸(CN-CTRL)作为对照,以排除寡核苷酸的非特异性消化(图2A)。为了确定所得的寡核苷酸片段,在完成实时活性测量之后,在95℃至20℃的温度下进行解链曲线分析。为此,记录荧光信号随温度变化的趋势的一阶导数(-(d / dT))。产生的最小值代表不同片段的熔化温度(图2B)。

关键字:DNA修复, 人类8-氧代鸟嘌呤DNA糖基化酶1, 7,8-二氢-8-氧鸟嘌呤, 酶活力, 线粒体

材料和试剂

1. 2-巯基乙醇(卡尔·罗斯,目录号:4227.1)

2.苄am(Carl Roth,目录号:CN38.1)     

3. BHQ (单链)([BHQ1] GGTATTATTATTATTGCGTTATTATTATTATGG [BHQ1] 100 μM)(Sigma-Aldrich公司)       

4.牛血清白蛋白(BSA)(New England Biolabs公司® ,目录号:B9000S)     

5.细胞培养瓶T25,T75,T175(Sarstedt,目录号:83.3910.302、83.3911.302、833912.302 )     

6. CHAPS(卡尔·罗斯,目录号:1479.1)     

7. CN-8-oxoG(单链)([6FAM] CCATAATAATAATAAC [8-oxo-dG] CAATAATAATAATA     

CC [6FAM] 100 µM)(Sigma-Aldrich)


8. CN-CTRL(单链)([6 FAM] CCATAATAATAATAACGCAATAATAATAATACC [6FAM] 100 µM)(Sigma-Aldrich)     

9. cOmplete TM迷你蛋白酶抑制剂鸡尾酒(Roche Diagnostics,目录号:04693124001)     

10. DMSO(卡尔·罗斯,目录号:A994.2) 

11. Dulbecco的改良版Eagle媒介    –高葡萄糖(Sigma-Aldrich,目录号:D7777-10L)


12. EGTA(卡尔·罗斯,目录号:3054.1) 

13.胎牛血清(FBS)(Gibco公司® ,目录号:26140079) 

14.庆大霉素(10毫克/毫升)(Gibco公司® ,目录号:11500506) 

15.甘油(Carl Roth,目录号:6967.1) 

16.注射套管(G 20 × 1½”)(B。Braun,目录号:4657519) 

17. KCl(卡尔·罗斯,目录号:6781.3) 

18. KH 2 PO 4 (卡尔·罗斯,目录号:3904.2) 

19.的LightCycler ® 480多孔板96(白色)(Roche Diagnostics公司,目录号:04729692001) 

20.的LightCycler ® 480密封膜(Roche Diagnostics公司,目录号:04729757001) 

21. MgCl 2 (卡尔·罗斯,目录号:KK36.1) 

22. Na 2 HPO 4 · 12H 2 O(Carl Roth,目录号:N350.1) 

23.氯化钠(卡尔·罗斯,目录号:3957.3) 

24. NEBuffer TM 2(新英格兰生物实验室® ,目录号:B 7002S) 

25.移液器过滤嘴:2.5 µl,10 µl,20 µl,200 µl,1和000 µl(Sarstedt,目录号:70.1130.217、70.1130.215、70.1114.215、70.760.216、70.762.216) 

26. QuinoMit ®    –航母控制(MSE Pharmazeutika)


27. QuinoMit ® Q10-液-泛醇(MSE Pharmazeutika) 

28.反应管:0.2 ml,1.5 ml(Sarstedt,目录号:72.737.002、72.706.201) 

29.重组人OGG1蛋白(100微克)(Abcam,目录号:ab98249) 

30.注射器2毫升(Injekt ® 2ml)中的(贝朗,目录号:4606027V) 

31. Tris-HCl(Carl Roth,目录号:9090.3) 

32.柠檬酸三钠二水合物(≥99%)(卡尔·罗斯,目录号:3580.4) 

33.胰蛋白酶-EDTA(0 。5%)(Gibco公司® ,目录号:10779413) 

34. 1 × PBS(请参阅食谱) 

35. CHAPS缓冲区(请参阅食谱) 

36.载波控制(10 mM)(请参阅食谱) 

37.泛醇配方(10毫米)(请参阅食谱) 

38.用于成纤维细胞的DMEM(请参阅食谱) 

39.反应缓冲液(请参见食谱) 

40. 2 M柠檬酸三钠二水合物(请参阅食谱) 



设备

离心机Eppendorf 5427 R(Eppendorf,目录号:5409000210)
离心机Eppendorf 5804 R(Eppendorf,目录号:5805000010)
冰柜(利勃海尔Comfort(利勃海尔,目录号:不提供)
培养箱中在37℃,5%CO 2,90%湿度(HERA细胞240,目录号:2510-413-01)
的LightCycler ® 480(罗氏诊断,目录号:05015278001)
PCR冷却器(Eppendorf,目录号:3881000031)
pH计FiveEasy TM F20 (梅特勒-托利多(Mettler Toledo),目录号:30266626)
移液器,微量离心研究®加:0 。1 µl 10 µl,20 µl,200 µl,1和000 µl(Eppendorf,目录号:3123000012、3123000020、3123000039、3123000055、3123000063)
Sonoplus Ultraschall-Homogenisator HD 3100(班德林电子)
热循环仪MKR13 HLC(DITABIS,目录号:MKR 13)
超纯水(ddH 2 O)制备装置,Purelab flex 4(Veolia Water Technologies)

软件

的GraphPad Prism,8.2版本0.1(格拉夫派得软件® )
的LightCycler ® 480软件(仪器软件),版本1.5.0.39 (罗氏诊断)

程序

细胞培养
在适当的细胞特异性培养基中,在有或没有测试物质的情况下,首先在37°C下以5%CO 2和90%湿度的形式培养成纤维细胞。
在适当的温育期后,用胰蛋白酶-EDTA去除细胞,并将3.0 × 10 6个细胞转移到新的1.5 ml反应管中。   
在1200 × g和4°C下将细胞离心5分钟,然后将得到的沉淀重悬于1 ml 1 × PBS中(洗涤步骤)。

线粒体的分离与裂解
取3.0 × 10 6成纤维细胞于1 × PBS中,在1,200 × g和4°C下离心5分钟。
弃去上清液,将得到的沉淀重悬于200 µl CHAPS裂解缓冲液(pH 7.4)中,该缓冲液中补充有3 µl 2 M柠檬酸三钠二水合物,并在冰上孵育45分钟。
用注射器和套管(直径= 0.9 mm)在冰上通过上下吹打10次使样品均质化。
为了将线粒体与其余细胞结构完全分离,将样品在1200 × g和4°C下再次离心10分钟。
将带有线粒体的上清液转移到新的1.5 ml反应管中(在冰上预冷)。
在冰上进一步裂解步骤中,用20瓦脉冲超声将分离的线粒体裂解1分钟(打开0.5 s脉冲,关闭1.0 s脉冲)。
在21,000 × g和4°C下进一步离心45分钟后,可以将上清液保存在冰上,直到数小时内可以再次使用。将上清液在-20°C下保存过夜或更长时间。

报告寡核苷酸的制备
混合5μl每个单链报告子寡核苷酸CN-8-oxoG([6FAM] CCATAATAATAATAAC [8-oxo-dG] CAATAATAATAATACC [6F AM])(100 µM)和CN-CTRL([6FAM] CCATAATAATAATAACGCAATAATAATACC [6FAM] ])(100μM)分别与10微升BHQ1标记的互补链的([BHQ1] GGTATTATTATTATTGCGTTATTATTA TTATGG [BHQ1])(100μM)(所有西格玛奥德里奇GmbH)和35微升的单独0.2毫升反应管NE缓冲液TM 2. A中的报道寡核苷酸中可以看到的示意图˚F igure 1。
将寡核苷酸混合物在热循环仪中于95°C孵育15分钟,然后在-20 °C的冰箱中冷却完成的构建物(冷却阶段退火–退火温度= 50°C)。Ť他最终构建体的随后的存储发生在-20℃。





图1.测定的基本原理。带有8-oxoG碱基的双链核苷酸链(以红色显示)用于检测hOGG1的活性。带有损伤的链的两端带有6-FAM分子作为荧光报告分子,两端带有相对的淬灭剂。在未切割的报告寡核苷酸中,两条链彼此杂交,从而6-FAM分子的荧光信号被淬灭剂截获。如果具有损伤的链被切断,则两个核苷酸链彼此解离并且可以测量荧光信号。

碱基切除修复测定
移液管的样品和对照如表1所示,通过上下移液均质化样品放入白色96孔板(两个孔是必需的每Appro公司ACH - ø NE为CN -8-氧鸟嘌呤和一个用于CN-CTRL) 。

表1.不同反应方法的组成

积极控制


负控制


样本


反应缓冲液


94.3微升


94.3微升


75.3微升


柠檬酸三钠二水合物[2M]


3.7微升


3.7微升


3.7微升


记者构建的[CN-8-oxoG 0.1 µM /


CN-CTRL 0.2 µM]


1微升(CN-8-oxoG)/


(CN-CTRL)


1微升(CN-8-oxoG)/


(CN-CTRL)


1微升(CN-8-oxoG)/


(CN-CTRL)


矩形。OGG1


1微升


--


--


ddH 2 O


--


1微升


--


样本


--


--


20微升

用密封箔将板密封,并在720 × g和4°C下将板离心5分钟。
为了实时检测基本切除活性,请在Light Cycler 480(Roche Diagnostics)中执行荧光信号的测量,共37个温度,每个周期80分钟,每次1分钟。的6-FAM分子的激发发生在波长范围内的483的荧光信号的465nm处和检测- 533ñ米(图2A) 。
为了确定所得的寡核苷酸片段,在完成实时活性测量(图2B)后,在95°C至20°C的温度下进行熔解曲线分析。





图2.通过荧光和解链曲线分析确定hOGG1活性的示意图。一。用1.6单位商业购买的hOGG1以及从细胞裂解物中获得的hOGG1消化80分钟后每次1分钟的报告寡核苷酸CN-8-oxoG(有损伤)和CN-CTRL(无损伤)的荧光曲线在37°C下。B.中号报道的elting曲线与从细胞裂解物获得1.6单位商业购买和hOGG1基因hOGG1基因的消化后的寡核苷酸CN-8-氧鸟嘌呤和CN-CTRL。

数据分析

hOGG1的碱基切除修复活性可以通过缺陷报告寡核苷酸(CN-8-oxoG)减去非特异性消化(CN-CTRL)的荧光曲线的终点(最大荧光)来确定(图2A) 。
实时活性测量完成后,可以使用记录的熔解曲线进行片段分析。为此,在从95°C到20°C (-(d / dT))的温度变化下记录荧光信号趋势的一阶负导数。所得的最小值代表不同片段的熔化温度。位置1处的峰表示hOGG1活性的完整切割底物。在该底物中,从DNA上切下两个修饰的碱基(8-oxoG),并切割DNA的糖-磷酸主链。在位置2处标记的峰代表具有AP位点的底物,其中该酶的N-糖基化酶功能已经具有活性,而AP裂解酶则没有。在位置3处有未切割的底物,其中糖基化酶和连接酶功能均未激活。根据较大峰的位置,可以对hOGG1各个酶促步骤的活性做出说明(图2B)。
数据可以用分析的GraphPad Prism,8.2.1版(格拉夫派得软件® )。值以平均值±SEM或单个值表示。建议每种方法重复三次。

代表数据


根据进行的步骤测量分离的线粒体裂解物中hOGG1的碱基切除修复活性(图3)。然而在这种情况,为了测试在hOGG1基因,一个泛醇制剂的酶活性辅酶Q10的效果(QuinoMit ® Q10-流体-泛醇,MSE Pharmazeutika)和载体控制(CC)(QuinoMit ® -载波控制,MSE Pharmazeutika)被附加地添加到不同的测量之前直接接近。这样做是因为怀疑与其他依赖氧化还原的转录因子一样,hOGG1受到抗氧化剂的氧化还原活性特性的积极调控。完全按照QuinoMit®Q10-Fluid所述的方法进行载体对照的给药。QuinoMit®Q10-流体和载波控制是磷脂纳米颗粒(30乳液- 90纳米)。两者始终都是新鲜称量的,并用超纯水稀释。这些测试化合物的确切组成可以在下表2中找到。





图3。泛醇对从线粒体分离的hOGG1酶活性的影响。不同浓度的加成(10 - 1 ,ubiquino的000μM)升和载波控制(CC),以从线粒体裂解物报道寡核苷酸和hOGG1基因,从3.0分离× 10 6人成纤维细胞。作为阴性对照(NC),包括一种使用测试溶液和报告寡核苷酸但不使用hOGG1的方法。用LightCycler ?480在483和533 nm之间的波长范围内记录荧光信号。显示了基于hOGG1活性的消化的报告寡核苷酸的荧光信号(n = 3,平均值 ± SEM,采用Bonferroni多重比较测试的单向方差分析,* P <0.05)。标准化为阳性对照的值(PC =没有测试溶液的所有测定成分)。PC对应于1.0。从Schniertshauer等获得的数据。(2020年)。

表2 。的确切组成QuinoMit ® Q10-流体和载体控制

辅酶Q10配方和载体控制

QuinoMit ® Q10-液


泛醇(红色)


运营商控制


甘油


+


+


泛醇


+


--


去离子水


+


+


大豆卵磷脂


+


+


烟酰胺


+


+



笔记

我们在此描述的分离的线粒体裂解物中的碱基切除修复活性的测量具有很高的重现性。
需要柠檬酸三钠来防止细胞裂解物中所含的DNase对报告寡核苷酸的非特异性消化。确定了抑制DNase的柠檬酸三钠二水合物的最佳浓度,适用于成纤维细胞,并且可以适应于其他细胞系的使用。
此处介绍的测定法解决了修复酶hOGG1的活性。对BER的其他修复酶的适应性应该是可能的,并取决于所使用的报道寡核苷酸。
此处使用的报告基因寡核苷酸不是可商购的,而是由Sigma Aldrich设计并代表客户生成的。
此方法是在原发性纤维上开发的,但根据初步发现,它也适用于PBMC的裂解物。首先必须建立更多的细胞系。
如此处所述的测量是在光循环仪中进行的,但可以适用于任何其他RT-PCR系统。
基于所使用的荧光标记(6-FAM; Ex 465 nm / Em 510 nm),在483至533之间的波长范围内测量荧光信号。
应在冷袋(PCR-Cooler)上进行96孔板中方法的准备,以尽可能避免酶的过早活性。
实时图像中的荧光的增加引起-如上所述-通过切开的片段。对于报告寡核苷酸,这些片段的解链温度为约36℃,而未切割的寡核苷酸的解链温度为约52℃。
从皮肤活检组织中分离出人成纤维细胞,所述皮肤活检组织是从德国普通外科部门Kreiskrankenhaus Sigmaringen获得的。来自德国整形外科部门Constance Aesthetic Perfection Lake Constance;或来自德国罗伊特林根的Fuhrer博士,H。Nonnenmacher博士,Astfalk博士和Fauser博士。所有实验均根据赫尔辛基宣言进行,并经德国巴登-符腾堡州医学协会国家伦理委员会批准(187-03)。事先通知患者,并书面同意使用他们的样品。如Burger等人所述分离细胞。(2010年)。

菜谱

1 × PBS
8克氯化钠


0.20克氯化钾


2.88克Na 2 HPO 4· 12H 2 O


1.24克KH 2 PO 4


甲djust到1升的DDH 2 O,pH 7.4的


2 M柠檬酸三钠二水合物
5.882 g柠檬酸三钠二水合物


甲djust到10毫升的DDH 2 ö


载波控制(10 mM)
0 。904克QuinoMit ® -载体控制


甲djust至5 ml的的DDH 2 O,pH 7.4的


CHAPS缓冲区
1.25毫升甘油(80%)


1毫升CHAPS(5%)


100 µl Tris-HCl(1 M)


100 µl EGTA(0.1 M)


10 µl MgCl 2 (1 M)


5 µl苄am (0.2 M)


3.52 µl 2-巯基乙醇(14.19 M)


甲djust到10毫升的DDH 2 O,pH 7.4的


甲DD一个片剂完整TM迷你蛋白酶抑制剂混合物


用于成纤维细胞的DMEM
500毫升DMEM


50毫升FBS


2.5毫升庆大霉素(50微克/毫升)


反应缓冲液
320微升DMSO


160 µl NEBuffer TM 2


16 µl BSA(20 µg / µl)


752微升ddH 2 O


泛醇配方(10毫米)
0 。904克QuinoMit ® Q10-液


甲djust至5 ml的的DDH 2 O,pH 7.4的

致谢

作者感谢Franz Enzmann博士和AlexanderBürkle博士的科学建议。这项研究得到了BMBFFHprofUnt2012 “ MitoFunk ” (03FH022PX2)和巴登-符腾堡州科学,研究与艺术部的支持。

利益争夺

JörgBergemann与德国巴特洪堡的MSE Pharmazeutika GmbH和德国汉堡的Beiersdorf AG签订了咨询合同。



参考

Burger,K.,Matt,K.,Kieser,N.,Gebhard,D.和Bergemann,J.(2010)。一种改进的荧光宿主细胞活化测定法,用于确定原代角质形成细胞,黑素细胞和成纤维细胞的修复能力。BMC生物技术10 :46。
Gorbunova,V.,Seluanov,A.,Mao,Z.和Hine,C.(2007)。老化过程中DNA修复的变化。Nucleic Acids Res 35(22):7466-7474。
Hamann,I.,Schwerdtle,T.和Hartwig,A.(2009)。建立非放射性裂解测定法以评估DNA对亚细胞和细胞系统中氧化损伤的DNA的修复能力以及铜的影响。Mutat Res 669(1-2):122-130。
Hirano,T.(2008年)。7、8-二氢-8-氧鸟嘌呤的修复系统,可抵抗癌变。辐射研究杂志49(4):329-340。
SP.Jackson和J.Bartek,J。(2009年)。人类生物学和疾病中的DNA损伤反应。自然461(7267):1071-1078。
Janion,C.(2001年)。关于DNA损伤和修复的一些挑衅性思想。生物医学技术杂志1(2):50-51。
Loft,S。和Poulsen,HE(1996)。人体患癌症的风险和氧化DNA的损害。分子医学杂志(Berl)74(6):297-312。
Nakabeppu,Y.(2014年)。DNA或核苷酸库中细胞中的8-氧鸟嘌呤水平在癌细胞的癌变和存活中起关键作用。国际分子科学杂志15(7):12543-12557。
Radicella,JP,Dherin,C.,Desmaze,C.,Fox,MS和Boiteux,S。(1997)。hOGG1的克隆和鉴定,hOGG1是酿酒酵母OGG1基因的人类同源物。美国国家科学院院刊94(15):8010-8015。
Ravanat,JL,Di Mascio,P.,Martinez,GR,Medeiros,MH和Cadet,J.(2000)。单线态氧诱导细胞DNA氧化。生物化学杂志275(51):40601-40604。
Sampath,H.,Vartanian,V.,Rollins,MR,Sakumi,K.,Nakabeppu,Y.和Lloyd,RS(2012)。8-氧鸟嘌呤DNA糖基化酶(OGG1)缺乏症会增加对肥胖症和代谢功能障碍的敏感性。PLoS One 7(12):e51697。
Schniertshauer,D.,Gebhard,D.,van Beek,H.,Noth,V.,Schon,J.和Bergemann,J.(2020年)。DNA修复酶hOGG1的活性可以被泛醇直接调节。DNA Repair(Amst)87:102784。
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引用:Schniertshauer, D., Gebhard, D. and Bergemann, J. (2021). Real-time Base Excision Repair Assay to Measure the Activity of the 8-oxoguanine DNA Glycosylase 1 in Isolated Mitochondria of Human Skin Fibroblasts. Bio-protocol 11(6): e3954. DOI: 10.21769/BioProtoc.3954.
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