Mechanistic aspects of the deoxyribonuclease activity of diphtheria toxin.
Here we examined the intrinsic nuclease activity of diphtheria toxin (DTx) to determine the mechanism by which it catalyzes DNA degradation. Results show that DTx degrades double-stranded DNA (dsDNA) by non-processive, endonucleolytic attack, without apparent specificity for nucleotide sequence. Moreover, divalent cation composition determines whether supercoiled dsDNA is cleaved by the introduction of single-strand nicks or double-strand breaks. Circular single-stranded DNA (ssDNA) is also a substrate for endonucleolytic attack. Pre-incubation of DTx with a 2000-fold excess of NAD, the natural substrate for the toxin's ADP-ribosyltransferase (ADPrT) activity, inhibited the transfer of radiolabeled ADP-ribose to elongation factor 2 but had no effect on the degradation of radiolabeled DNA. Based on this result and the fact that compounds known to inhibit the ADPrT activity of DTx had no effect on its nuclease activity and pre-incubation of DTx with DNA had no effect on ADPrT activity, we conclude that the ADPrT and nuclease active sites of DTx are functionally and spatially distinct. Moreover, studies with an ADPrT-inactivated form of DTx indicate that nuclease activity alone can lead to target cell lysis.
Lee
Jason W
JW
Department of Microbiology, Immunology and Molecular Genetics, The Molecular Biology Institute, and The Jonsson Comprehensive Cancer Center, University of California, Los Angeles, CA 90095, USA.
Nakamura
Lawrence T
LT
Chang
Michael P
MP
Wisnieski
Bernadine J
BJ
D036002:ADP Ribose Transferases / Q000037:antagonists & inhibitors / Q000494:pharmacology / Q000502:physiology; D000241:Adenosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D001386:Azides / Q000494:pharmacology; D001665:Binding Sites / Q000502:physiology; D002384:Catalysis; D002413:Cations, Divalent / Q000378:metabolism / Q000494:pharmacology; D002460:Cell Line; D003513:Cycloheximide / Q000494:pharmacology; D004247:DNA / Q000187:drug effects / Q000378:metabolism*; D004167:Diphtheria Toxin / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000494:pharmacology; D004706:Endodeoxyribonucleases / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D009243:NAD / Q000494:pharmacology; D009536:Niacinamide / Q000494:pharmacology; D009843:Oligoribonucleotides / Q000494:pharmacology; D020652:Peptide Elongation Factor 2 / Q000502:physiology; D014176:Protein Biosynthesis / Q000187:drug effects; D013379:Substrate Specificity
D001386:Azides; D002413:Cations, Divalent; D004167:Diphtheria Toxin; D009843:Oligoribonucleotides; D020652:Peptide Elongation Factor 2; D009243:NAD; C043846:adenylyl-(3'-5')-uridine 3'-monophosphate; D000246:Adenosine Diphosphate Ribose; D009536:Niacinamide; C012760:8-azidoadenosine; D004247:DNA; D003513:Cycloheximide; D036002:ADP Ribose Transferases; D004706:Endodeoxyribonucleases; D000241:Adenosine