Probing Cu(II)/H2O2 damage in DNA with a damage-specific DNA binding protein

Abstract

A human damage-specific DNA binding protein has been employed as a sensitive probe of damage introduction by the combination of Cu(II) and H2O2. Optimal conditions for the introduction of protein-recognizable lesions into DNA in the Cu(II)/H2O2 system were obtained with 10(-5)-M CuCl2 and 0.10-mM H2O2. The absolute requirement for the presence of a metal ion suggests the involvement of a metal catalyzed Fenton reaction. However, damage introduction in the presence of KI and dimethylsulfoxide indicate that hydroxyl radical, while responsible for the introduction of strand breaks, is not the primary species responsible for lesion introduction. Protein-recognizable damage was introduced into DNA and poly d(G-C), but not into poly d(A-T). Loss of label from the five position of cytosine was also observed at high peroxide levels.