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. 2015 May 4:64:17.17.1-17.17.14.
doi: 10.1002/0471140856.tx1717s64.

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

Guo-Dong Zhou  1 , Bhagavatula Moorthy  2
Affiliations

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

Guo-Dong Zhou et al. Curr Protoc Toxicol. .

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

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Figures

Figure 1
Figure 1
Structure and formation of 8,5’-cycloadenosine-2’-deoxynucleotide (cA), that is induced by hydroxyl radical attack on 2’-deoxyadenosine (dA).
Figure 2
Figure 2
Procedures of 32P-postlabeling assay. cA, cycloadenosine; N, normal nucleotides; p, phosphate; *P, [32P]phosphate; *pNpcAp, 32P-labeled dinucleotide containing cycloadenosine.
Figure 3
Figure 3
Template of D1 chromatogram (TLC plate).
Figure 4
Figure 4
Template of 2-D chromatograms (TLC plates). L, lower cutout (donor) from D 1 TLC plate; C, central cutout (donor) from D 1 TLC plate.
Figure 5
Figure 5
Template of chromatogram for [γ-32P]ATP specific activity and locations of major labeled products after development in 0.28 M NH4(SO4)2 + 50 mM NaH2PO4, pH 6.7.
Figure 6
Figure 6
Profiles of DNA samples (1–4) after D1 chromatography and chromatographic mobilities of the four possible dinucleotides containing A, C, G, or T, 5’ to cA. L, lower cutout; C, central cutout. These two cutouts are used to contact-transfer to 2D TLC plates.
Figure 7
Figure 7
Typical patterns of 32P-postlabeled cycloadenosins. AcA, CcA, GcA, and TcA represent different dinucleotides containing 8,5'-cycloadenosine-2'-deoxynucleotides.
Figure 8
Figure 8
A new chromatographic method combining L and C cutouts in a single 2D plate. Contact-transfer of L and C is displayed in panel A. Typical patterns of 32P-postlabeled AcA, CcA, GcA, and TcA are shown in panel B.
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