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

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.

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

Procedures of ³²P-postlabeling assay. cA, cycloadenosine; N, normal nucleotides; p, phosphate; *P, [³²P]phosphate; *pNpcAp, ³²P-labeled dinucleotide containing cycloadenosine.

Figure 3

Template of D1 chromatogram (TLC plate).

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

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

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

Typical patterns of ³²P-postlabeled cycloadenosins. AcA, CcA, GcA, and TcA represent different dinucleotides containing 8,5'-cycloadenosine-2'-deoxynucleotides.

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.