TRIS-DEPENDENT OXIDATIVE DNA STRAND SCISSION DURING ELECTROPHORESIS

The DNA of two Streptomyces species contains site-specific labile modi fications. During gel electrophoresis the DNA can undergo Tris-depende nt strand scission at the positions of these modifications. Our invest igations into the nucleolytic activity which reacts with the modificat ions implicate a peracid derivative of Tris formed at the anode; the k inetics of production and decay of this activity were followed using b oth a DNA cleavage assay and a reduced methyl viologen assay to measur e oxidant. Anode activation could be chemically mimicked by addition o f 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 acid also several r eagents known to promote DNA strand cleavage by amine-catalysis at aba sic 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 al so containing primary amine groups. These results support a mechanism of DNA strand scission at modification sites via concerted peracid-med iated oxidative and amine-catalysed reactions. The novel finding of Tr is-dependent formation of a long-lived reactive oxidant at the anode s uggests that this compound is unsuited as an electrophoresis buffer fo r certain biological macromolecules.