Tris‐dependent oxidative DNA strand scission during electrophoresis

Abstract

The DNA of two Streptomyces species contains site‐specific labile modifications. During gel electrophoresis the DNA can undergo Tris‐dependent strand scission at the positions of these modifications. Our investigations into the nucleolytic activity which reacts with the modifications implicate a peracid derivative of Tris formed at the anode; the kinetics of production and decay of this activity were followed using both a DNA cleavage assay and a reduced methyl viologen assay to measure oxidant. Anode activation could be chemically mimicked by addition of peracetic acid to Tris buffers. We tested the DNA cleavage activity of several other compounds after anode or chemical activation; we used an analogue of Tris lacking a primary amine group and also several reagents known to promote DNA strand cleavage by amine‐catalysis at abasic sites. Anode generation of oxidant could be detected for compounds containing either hydroxyl or carboxyl groups. However, DNA cleavage activity correlated with oxidant formation only for those compounds also containing primary amine groups. These results support a mechanism of DNA strand scission at modification sites via concerted peracid‐mediated oxidative and amine‐catalysed reactions. The novel finding of Tris‐dependent formation of a long‐lived reactive oxidant at the anode suggests that this compound is unsuited as an electrophoresis buffer for certain biological macromolecules.