Immunological Detection of Carcinogen-modified DNA Fragments after in Vivo Modification of Cellular and Viral Chromatin

Abstract

Antibodies specific for DNA modified by (±)-trans-7,8-dihydrobenzo(a)pyrene-7,8-diol-9,10-epoxide have been used to quantitate the relative modification level in fragments derived from pBR322 DNA from cellular DNA and in the coding and noncoding strands of simian virus 40 DNA. DNA fragments with a covalent molar modification level ranging from less than 1 to over 200 are resolved by agarose gel electrophoresis and transferred to diazobenzyloxymethyl cellulose paper. The paper is incubated with antibodies specific to carcinogen-modified DNA, and the location of the antibody is visualized by autoradiography after incubation with ¹²⁵I-protein A. The binding of antibodies is directly proportional to the level of DNA modification. Using this technique, we find that linker DNA is about 2.5- to 3-fold more accessible to (±)-trans-7,8-dehydrobenzo(a)pyrene-7,8-diol-9,10-epoxide than nucleosomal core DNA and that under in vivo conditions the coding and noncoding strands of the simian virus 40 chromosome are equally accessible to trans-7,8-dihydrobenzo[a]pyrene-7,8-diol-9,10-epoxide. The approach described allows assessment of the relative level of modification in any DNA sequence which can be subjected to gel electrophoresis.