Detection of Bulky Endogenous Oxidative DNA Lesions Derived from 8,5′‐Cyclo‐2′‐deoxyadenosine by 32P‐Postlabeling Assay

Guo‐Dong Zhou1, Bhagavatula Moorthy2

1 Institute of Biosciences and Technology, Texas A&M University Health Science Center, Houston, Texas, 2 Department of Pediatrics, Baylor College of Medicine, Houston, Texas
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 17.17
DOI:  10.1002/0471140856.tx1717s64
Online Posting Date:  May, 2015
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Abstract

8,5′‐Cyclopurine‐2′‐deoxynucleotides represent a class of oxidative DNA lesions that are specifically repaired by nucleotide excision repair but not by base excision repair or direct enzymatic reversion. The 32P‐postlabeling assay is an ultrasensitive method that has been extensively used for the detection of carcinogen‐DNA adducts in laboratory animal and epidemiological studies. This assay under modified chromatographic conditions is also a suitable and sensitive method for the detection of 8,5′‐cyclo‐2′‐deoxyadenosine (cA). After enzymatic digestion of DNA, and enrichment of the oxidative products from the DNA digest, four dinucleotides containing cA, i.e., Ap‐cAp, Cp‐cAp, Gp‐cAp, and Tp‐cAp, are 5′‐labeled with [32P]orthophosphate from [γ‐32P]ATP, mediated by polynucleotide kinase (PNK). The 32P‐labeled cA products are separated by two‐dimensional thin‐layer chromatography (TLC) and quantified by Instant Imager or by a scintillation counter. The assay only requires 1 to 10 μg of DNA sample and is capable of detecting cA lesions at frequencies as low as 1 in 1010 normal nucleotides. © 2015 by John Wiley & Sons, Inc.

Keywords: 8,5′‐cyclopurine‐2′‐deoxynucleotides; 32P‐postlabeling assay; oxidative DNA damage; thin‐layer chromatography

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Enzymatic Digestion of DNA and Enrichment of Dinucleotides Containing Cycloadenosine
  • Basic Protocol 2: 5′‐32P‐Labeling of Dinucleotides Containing Cycloadenosine
  • Basic Protocol 3: Determination of Specific Activity of [γ‐32P]ATP
  • Basic Protocol 4: Preparation of Thin‐Layer Chromatography (TLC) Plates
  • Basic Protocol 5: Thin‐Layer Chromatography (TLC)
  • Basic Protocol 6: Autoradiography and Quantitation of Cycloadenosine
  • Basic Protocol 7: Calculation of Oxidative Dna Lesions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Enzymatic Digestion of DNA and Enrichment of Dinucleotides Containing Cycloadenosine

  Materials
  • DNA of interest (1 to 10 μg)
  • Micrococcal nuclease (MN, 0.4 U/μl, Sigma‐Aldrich, cat. no. M3755)
  • Spleen phosphodiesterase (SPD, 0.02 U/μl, Sigma‐Aldrich, cat. no. P9041)
  • 100 mM calcium chloride (CaCl 2)
  • 300 mM sodium succinate, pH 6.0
  • Nuclease P1 (NP1, 1 U/μl, Sigma‐Aldrich, cat. no. N8630)
  • 1 mM zinc chloride (ZnCl 2)
  • 1 M sodium acetate, pH 5.0
  • SpeedVac evaporator

Basic Protocol 2: 5′‐32P‐Labeling of Dinucleotides Containing Cycloadenosine

  Materials
  • Four dinucleotides containing cycloadenosine (5′‐dAp‐cdAp‐3′, 5′‐dCp‐cdAp‐3′,5′‐dGp‐cdAp‐3′, and 5′‐Tp‐cdAp‐3′; protocol 1, step 10)
  • Cycloadenosine standards (only available from research laboratories, e.g., contact Dr. Yinsheng Wang; )
  • 2‐(Cyclohexylamino)ethanesulfonic acid (CHES, 500 mM, pH 9.5; Sigma‐Aldrich, cat. no. C2885)
  • 150 μCi/μl [γ‐32P]adenosine 5′‐triphosphate ([γ‐32P]ATP, PerkinElmer, cat. no. NEG035C010MC)
  • T4 Polynucleotide kinase (PNK, 30 U/μl, Affymetrix/USB, part. no. 70031X)
  • 10× T4 PNK reaction buffer (500 mM Tris·Cl, pH 7.6, 100 mM MgCl 2, 100 mM 2‐mercaptoethanol; included in T4 PNK package; Affymetrix/USB, part no. 70083)
  • T4 PNK dilution buffer (50 mM Tris·Cl, pH 8.0, included in T4 PNK package; Affymetrix/USB, part no, 71079)
  • Apyrase (40 mU/μl, Sigma‐Aldrich, cat. no. A7646)

Basic Protocol 3: Determination of Specific Activity of [γ‐32P]ATP

  Materials
  • 2′‐Deoxyadenosine 3′‐monophosphate (dAp, 2.0 μM, Sigma‐Aldrich, cat. no. D3139)
  • 2‐(Cyclohexylamino)ethanesulfonic acid (CHES; (50 mM, pH 9.5; Sigma‐Aldrich, cat. no. C2885)
  • Additional reagents and equipment for preparing labeling mix ( protocol 2, step 4)

Basic Protocol 4: Preparation of Thin‐Layer Chromatography (TLC) Plates

  Materials
  • Polyethyleneimine (PEI)‐impregnated cellulose TLC plates (thickness, 0.5 mm; 20 × 20 cm; Macherey‐Nagel, cat. no. 801053)
  • Whatman filter paper (46 × 57 cm; Fisher Scientific, cat. no. 1001917)
  • Extra‐soft pencil
  • Stapler
  • Paper trimmer

Basic Protocol 5: Thin‐Layer Chromatography (TLC)

  Materials
  • Labeled digests of dinucleotides containing cycloadenosine, and of cycloadenosine standards ( protocol 2)
  • D1 solvent: 2.8 M NaH 2PO 4, pH 5.2; Fisher Scientific, cat. no. S369)
  • Ink with 99Tc: add 56 μCi of 99Tc (PerkinElmer, cat. no. NEZ085) in a 100‐μl volume to 800 μl India ink and add 400 μl H 2O
  • 2‐D solvent, first‐dimension: 2.12 M lithium formate/ 3.75 M urea, pH 3.35)
  • 2‐D solvent, second‐dimension for L cutout (0.4 M NaH 2PO 4, 4.25 M urea, 0.25 M Tris·Cl, pH 8.2)
  • 2‐D solvent, second‐dimension for C cutout (1.0 M NaH 2PO 4, pH 6.0)
  • d*pAp solution for specific activity determination (see protocol 3, step 5)
  • Solvent for specific activity [0.28 M (NH 4) 2SO 4 + 50 mM NaH 2PO 4, pH 6.7]
  • TLC plates:
    • PEI‐impregnated cellulose TLC plate for D1 ( protocol 4, “a” steps)
    • PEI‐impregnated cellulose TLC 2‐D (acceptor) plate ( protocol 4, “b” steps)
    • PEI‐impregnated cellulose TLC plate for specific activity ( protocol 4, “c” steps)
  • Rectangular glass TLC developing tank with lid (Fisher Scientific, cat. no. K416180)
  • Kodak X‐Omat LS Film (Fisher Scientific, cat. no. 864 6770)
  • Wolf Cassettes (8 in. × 10 in.; Medical Solution, cat. no. 11118)
  • BioMax MS Intensifying Screen (8 in. × 10 in., Fisher Scientific, cat. no. 856 8706)
  • Saran wrap
  • Hair dryer
  • Extra‐soft pencil
  • Scissors
  • Whatman filter paper (46 × 57 cm; Fisher Scientific, cat. no. 1001917)
  • Stapler
  • Magnet button (General Hardware Mfg. Co., Inc., cat. no. 372A)

Basic Protocol 6: Autoradiography and Quantitation of Cycloadenosine

  Materials
  • TLC plates from labeled digests of dinucleotides containing cycloadenosine and cycloadenosine standards ( protocol 5)
  • Ink with 14C: add 20 μCi of 14C (MP Biomedicals, cat. no. 11743691) in a 20‐μl volume to 1300 μl India ink, and add 700 μl H 2O)
  • Kodak XAR Film (Fisher Scientific, cat. no. 165 1454)
  • Wolf Cassettes (8 in. × 10 in.; Medical Solution, #11118)
  • BioMax MS Intensifying Screen (8 in. × 10 in., Fisher Scientific, cat. no. 856 8706)
  • Imager: Instant Imager (Packard Instruments), Phosphorimager (GE Healthcare), or scintillation analyzer (PerkinElmer; Randerath et al., )
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Figures

Videos

Literature Cited

Literature Cited
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