HUMAN-CELLS CONTAIN PROTEIN SPECIFICALLY BINDING TO A SINGLE 1,N6-ETHENOADENINE IN A DNA FRAGMENT

A human DNA binding protein has been characterized from cell-free extracts of liver, placenta, and cultured cells. This protein, apparent molecular mass almost-equal-to 35 kDa, to our knowledge, does not resemble other proteins reported to bind to carcinogen-modified DNA. The probe used for characterization was a 25-base oligonucleotide containing a single site-specifically placed 1,N6-ethenoadenine (epsilon-A), a product of vinyl chloride metabolism. When annealed to form an epsilon-A.T or epsilon-A.C pair, a strong affinity to the protein was observed, with a binding constant of almost-equal-to 1 x 10(9) M-1. In contrast, very little binding was found with an epsilon-A.A pair and none was found with an epsilon-A.G pair. This suggests protein recognition of a specific structural alteration. Other defined probes with alkyl adducts did not bind. In addition, the human cell extracts and a rat liver extract were found to nick specifically at the 5' side of the epsilon-A adduct, which could indicate a possible associated repair activity.