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.
Lee
Jason W
JW
Department of Microbiology, Immunology, and Molecular Genetics, Molecular Biology Institute, and Jonsson Comprehensive Cancer Center, University of California, Los Angeles, California 90095, USA.
Cho
Eun
E
Aghaian
Elda
E
Aghaian
Elsa
E
Der
Joshua
J
Wisnieski
Bernadine J
BJ
D036002:ADP Ribose Transferases / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002413:Cations, Divalent / Q000737:chemistry; D003001:Cloning, Molecular / Q000379:methods; D004247:DNA / Q000737:chemistry; D043224:Deoxyribonuclease (Pyrimidine Dimer) / Q000235:genetics / Q000378:metabolism; D004167:Diphtheria Toxin / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000737:chemistry; D010446:Peptide Fragments / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D014176:Protein Biosynthesis; D011489:Protein Denaturation / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D011817:Rabbits; D013696:Temperature; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D002413:Cations, Divalent; D004167:Diphtheria Toxin; D010446:Peptide Fragments; C029795:diphtheria toxin fragment A; C028581:CRM197 (non-toxic variant of diphtheria toxin); D009243:NAD; D004247:DNA; D036002:ADP Ribose Transferases; D043224:Deoxyribonuclease (Pyrimidine Dimer)