Home LiteratureArticle Details
PMID: 2109323 Published · ppublish English Comparative Study Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Diphtheria toxin and its ADP-ribosyltransferase-defective homologue CRM197 possess deoxyribonuclease activity.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 87 ·No. 8 ·1990-04-00 ·Pages 2995-8

Bruce C, Baldwin RL, Lessnick SL, Wisnieski BJ

Abstract

The cytotoxic mechanism of diphtheria toxin (DTx) is associated with its ability to inhibit protein synthesis by ADP-ribosylation of elongation factor 2. Although DTx intoxication leads to internucleosomal DNA cleavage and cell lysis, these events do not occur when protein synthesis is inhibited by alternative treatments (e.g., cycloheximide). Here we show that endonucleolytic degradation of DNA is an intrinsic activity of DTx and also of the crossreactive mutant protein CRM197. Assays using DNA-impregnated gels as well as linear and supercoiled DNA in solution revealed not only that CRM197 has nuclease activity but also that its specific activity is actually significantly greater than that of the wild-type molecule. Since CRM197 contains a single amino acid substitution that renders it incapable of ADP-ribosylation, we propose that the active sites for ADP-ribosyltransferase and nuclease activities are distinct.

MeSH Terms
Binding Sites DNA, Superhelical Deoxyribonucleases/metabolism Diphtheria Toxin/genetics,metabolism Kinetics NAD/metabolism Plasmids Poly(ADP-ribose) Polymerases/genetics,metabolism Structure-Activity Relationship Substrate Specificity
Chemicals
DNA, Superhelical Diphtheria Toxin NAD Poly(ADP-ribose) Polymerases Deoxyribonucleases
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Bruce C
Department of Microbiology, University of California, Los Angeles 90024.
Baldwin R L
Lessnick S L
Wisnieski B J
References (28)
28 references, click to expand
  1. An endosomal model for acid triggering of diphtheria toxin translocation.
    J Biol Chem. 1988 Oct 25;263(30):15257-9 PMID: 3139667
  2. Localization of the active site of diphtheria toxin.
    Biochemistry. 1988 May 3;27(9):3398-403 PMID: 3390439
  3. Internucleosomal DNA cleavage precedes diphtheria toxin-induced cytolysis. Evidence that cell lysis is not a simple consequence of translation inhibition.
    J Biol Chem. 1989 Sep 15;264(26):15261-7 PMID: 2768263
  4. Second cytotoxic pathway of diphtheria toxin suggested by nuclease activity.
    Science. 1989 Dec 1;246(4934):1165-8 PMID: 2531465
  5. Characterization of the deoxyribonuclease activity of diphtheria toxin.
    J Biol Chem. 1990 Mar 25;265(9):5237-41 PMID: 2318891
  6. How protein toxins enter and kill cells.
    Cancer Treat Res. 1988;37:39-73 PMID: 2908634
  7. Diphtheria toxin and related proteins. I. Isolation and properties of mutant proteins serologically related to diphtheria toxin.
    J Biol Chem. 1973 Jun 10;248(11):3838-44 PMID: 4196584
  8. Diphtheria toxin and related proteins. II. Kinetic studies on intoxication of HeLa cells by diphtheria toxin and related proteins.
    J Biol Chem. 1973 Jun 10;248(11):3845-50 PMID: 4196585
  9. Interaction of fragment A from diphtheria toxin with nicotinamide adenine dinucleotide.
    J Biol Chem. 1974 Apr 10;249(7):2088-97 PMID: 4362061
  10. Occurrence of tryptophan in the enzymically active site of diphtheria toxin fragment A.
    Biochim Biophys Acta. 1977 Mar 28;491(1):286-95 PMID: 849463
  11. Nuclease detection in SDS-polyacrylamide gel electrophoresis.
    Anal Biochem. 1977 May 15;80(1):76-90 PMID: 18945
  12. Ligand interactions of diphtheria toxin. II. Relationships between the NAD site and the P site.
    J Biol Chem. 1980 Dec 25;255(24):12016-9 PMID: 7440582
  13. Intracellular stability of diphtheria toxin fragment A in the presence and absence of anti-fragment A antibody.
    Proc Natl Acad Sci U S A. 1982 Jan;79(2):461-5 PMID: 6952197
  14. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity.
    Anal Biochem. 1983 Jul 1;132(1):6-13 PMID: 6312838
  15. The amino-acid sequence of two non-toxic mutants of diphtheria toxin: CRM45 and CRM197.
    Nucleic Acids Res. 1984 May 25;12(10):4063-9 PMID: 6427753
  16. NAD binding site of diphtheria toxin: identification of a residue within the nicotinamide subsite by photochemical modification with NAD.
    Proc Natl Acad Sci U S A. 1984 Jun;81(11):3307-11 PMID: 6145155
  17. Mechanism of insertion of diphtheria toxin: peptide entry and pore size determinations.
    Proc Natl Acad Sci U S A. 1984 Jun;81(11):3341-5 PMID: 6328510
  18. "A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity". Addendum.
    Anal Biochem. 1984 Feb;137(1):266-7 PMID: 6329026
  19. Interaction of diphtheria toxin with adenylyl-(3',5')-uridine 3'-monophosphate. II. The NAD-binding site and determinants of dinucleotide affinity.
    J Biol Chem. 1984 Dec 25;259(24):15159-62 PMID: 6511789
  20. Circular dichroism of diphtheria toxin, Pseudomonas aeruginosa exotoxin A, and various derivatives.
    Biochim Biophys Acta. 1985 Apr 5;828(2):138-43 PMID: 3919768
  21. Binding properties of diphtheria toxin to cells are altered by mutation in the fragment A domain.
    J Biol Chem. 1985 Oct 5;260(22):12148-53 PMID: 4044590
  22. Transmembrane transport of diphtheria toxin, related toxins, and colicins.
    Annu Rev Biochem. 1986;55:195-224 PMID: 3527042
  23. Diphtheria toxin and its mutant crm 197 differ in their interaction with lipids.
    FEBS Lett. 1987 May 4;215(1):73-8 PMID: 3569541
  24. Conformational changes in diphtheria toxoids. Analysis with monoclonal antibodies.
    FEBS Lett. 1987 Jun 29;218(2):271-6 PMID: 2439381
  25. Single mutation in the A domain of diphtheria toxin results in a protein with altered membrane insertion behavior.
    Biochim Biophys Acta. 1987 Aug 7;902(1):24-30 PMID: 3607056
  26. Highly frequent single amino acid substitution in mammalian elongation factor 2 (EF-2) results in expression of resistance to EF-2-ADP-ribosylating toxins.
    J Biol Chem. 1987 Sep 5;262(25):12298-305 PMID: 2887567
  27. Exotoxin A of Pseudomonas aeruginosa: substitution of glutamic acid 553 with aspartic acid drastically reduces toxicity and enzymatic activity.
    J Bacteriol. 1987 Nov;169(11):4967-71 PMID: 2889718
  28. Histidine 21 is at the NAD+ binding site of diphtheria toxin.
    J Biol Chem. 1989 Jul 25;264(21):12385-8 PMID: 2526125
Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
0027-8424
DOI
10.1073/pnas.87.8.2995
Published
1990-04-00
Pages
2995-8
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC53820
Subset
IM
Grants
NHLBI NIH HHS · 2 T32 HL07386 · United States
NIGMS NIH HHS · GM22240 · United States
External Links
PubMed source DOI: 10.1073/pnas.87.8.2995 PMC Full Text View journal details
Analysis Services
Analysis Services

Contact

No. 2 Wenbo Road, Zhangqiu District, Jinan, Shandong

Qilu Normal University · Genelibs Bioinformatics Lab

750 Shunhua Rd, Jinan

2F, Bldg F, University Science Park

Tel: 0531-88819269

WeChat Official Account

Follow our WeChat subscription account for real-time updates and the latest in medical and biological research.


Business Email

E-mail: [email protected]

Copyright © 2026.Genelibs Jinan Honghui Biotechnology Co., Ltd. 鲁ICP备20007159号-2