Shiga toxin 1: damage to DNA in vitro

Shiga toxins share with plant ribosome-inactivating proteins the same enzymatic mechanism of action: the removal of a specific adenine from 28S RNA when 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 activity, on both ribosomes and DNA, Shiga toxins require activation by trypsin, urea and dithiothreitol which release the enzymatically active A(1) fragment. Activation by the classical procedure leaves large amounts of urea and DTT which interfere in the DNA depurination assay and completely abolish depurination at physiological pH. A consistent release of [3H]adenine from DNA at 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 proteolytic cleavage and reduction. Damage to single-stranded DNA by Shiga toxin 1 (Stx1) primarily involves depurination. A spontaneous DNA breakdown appears in fact only after extensive base removal, a behavior similar to that observed with uracil-DNA glycosylase, a simple glycosylase devoid of lyase activity. NaCl inhibits the activity of Stx1, probably by minimizing the sliding distance traveled by the enzyme along DNA in search of its target sites and promoting dissociation of the substrate-enzyme complex.