Characterization of a cloned temperature-sensitive construct of the diphtheria toxin A domain.

Our goal was to determine how the DTM mutant construct of the A domain of diphtheria toxin (DTx) causes temperature-sensitive effects in Drosophila and yeast [Bellen, H. J., D'Evelyn, D., Harvey, M., Elledge, S. J. (1992) Development 114, 787-796]. Because DTM fortuitously bears the same point mutation as found in the A chain of CRM197, an ADP-ribosyltransferase (ADPrT)-deficient form of DTx, we hypothesized that the dramatic low-temperature-sensitive effects did not stem from ADP-ribosylation of elongation factor 2 (EF-2). To rule out acquisition of ADPrT activity at low temperatures, we assayed mutant forms of the A domain of DTx produced by in vitro transcription/translation and found that DTM has no ability to ADP-ribosylate EF-2 at 18 or 30 degrees C. Because the DTM gene results in a protein with a 23-amino acid missense carboxy-terminal extension, we also constructed a form without this extension. Assays for nuclease activity revealed that nuclease activity comigrated with the two distinguishable E. coli-cloned mutant proteins DTM and DTM-23, regardless of whether electrophoresis was conducted under denaturing or nondenaturing conditions in gels embedded with DNA. Studies with CRM197 showed that Ca(2+) and Mg(2+) promote single-strand DNA nicks, whereas Mn(2+) promotes double-strand DNA breaks. Evidence that the cation-dependent nuclease and NAD-dependent ADPrT enzymic sites are distinct is that NAD protected only the A domain of DTx from proteolytic cleavage, whereas DNA protected the A domains of both DTx and CRM197. We conclude that the nuclease activity of DTM is responsible for the temperature-sensitive effects associated with its expression in both yeast and Drosophila.