Shiga toxin 1: damage to DNA in vitro

Shiga toxins share with plant ribosome-inactivating proteins the same enzym atic mechanism of action: the removal of a specific adenine from 28S RNA wh en acting on ribosomes and the removal of multiple adenines when acting on DNA in vitro. The activity on DNA, only recently reported, is particularly evident, and has been studied mostly at acidic pH. For the in vitro activit y, on both ribosomes and DNA, Shiga toxins require activation by trypsin, u rea and dithiothreitol which release the enzymatically active Al fragment. Activation by the classical procedure leaves large amounts of urea and DTT which interfere in the DNA depurination assay and completely abolish depuri nation at physiological pH. A consistent release of [H-3]adenine from DNA a t neutral pH is instead observed when the toxin is activated in vitro by an improved method which removes most of the drastic reagents required for pr oteolytic cleavage and reduction. Damage to single-stranded DNA by Shiga to xin 1 (Stx1) primarily involves depurination. A spontaneous DNA breakdown a ppears in fact only after extensive base removal, a behavior similar to tha t observed with uracil-DNA glycosylase, a simple glycosylase devoid of lyas e activity. NaCl inhibits the activity of Stx1, probably by minimizing the sliding distance traveled by the enzyme along DNA in search of its target s ites and promoting dissociation of the substrate-enzyme complex. (C) 2000 E lsevier Science Ltd. All rights reserved.