32P-labeling test for DNA damage.

Abstract

Covalent adducts formed by the reaction of DNA with chemical carcinogens and mutagens may be detected by a 32P-labeling test. DNA preparations exposed to chemicals known to bind covalently to DNA [N-methyl-N-nitrosourea, dimethyl sulfate, formaldehyde, .beta.-propiolactone, propylene oxide, streptozotocin, nitrogen mustard and 1,3-bis(2-chloroethyl)-1-nitrosourea] were digested to a mixture of deoxynucleoside 3''-monophosphates by incubation with micrococcal endonuclease (EC 3.1.31.1) and spleen exonuclease (EC 3.1.16.1). The digests were treated with [.gamma.-32P]ATP and T4 polynucleotide kinase (ATP:5''-dephosphopolynucleotide 5''-phosphotransferase, EC 2.7.1.78) to convert the monophosphates to 5''.cntdot.32P-labeled deoxynucleoside 3'', 5''-bisphosphates. These compounds were separated on polyethyleneimine-cellulose thin layers in ammonium formate and ammonium sulfate solutions. Autoradiograms of the chromatograms obtained by this high-resolution procedure showed the presence of nucleotides derived from chemically altered as well as normal DNA constituents. Maps from DNA exposed to any of the chemicals used exhibited a spot pattern typical for the particular chemical. This method detected a single adduct in 105 DNA nucleotides without requiring that the compound under investigation be radioactive and thus provides a useful test to screen chemicals for their capacity to damage DNA by covalent binding.