DNase I homologous residues in CdtB are critical for cytolethal distending toxin‐mediated cell cycle arrest

Cytolethal distending toxins (CDTs) block cell division by arresting the eukaryotic cell cycle at G2/M. Although previously not recognized in standard blast searches, a position‐specific iterated (psi) blast search of the protein data bank using CDT polypeptides as query sequences indicated that Cdt...

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Bibliographic Details
Published in:Molecular microbiology 2000-08, Vol.37 (4), p.952-963
Main Authors: Elwell, Cherilyn A., Dreyfus, Lawrence A.
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Bacterial Toxins - chemistry
Bacterial Toxins - metabolism
Base Sequence
CdtA protein
CdtB protein
CdtC protein
Cell Cycle
Cytolethal distending toxin
deoxyribonuclease I
Deoxyribonuclease I - metabolism
DNA Primers
HeLa Cells
Humans
Molecular Sequence Data
Sequence Homology, Amino Acid
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Summary:Cytolethal distending toxins (CDTs) block cell division by arresting the eukaryotic cell cycle at G2/M. Although previously not recognized in standard blast searches, a position‐specific iterated (psi) blast search of the protein data bank using CDT polypeptides as query sequences indicated that CdtB bears significant position‐specific homology to type I mammalian DNases. The psiblast sequence alignment reveals that residues of DNase I involved in phosphodiester bond hydrolysis (His134 and His252) are conserved in CdtB as well as their respective hydrogen bond pairs (Glu78 and Asp212). CdtB also contains a pentapeptide motif found in all DNase I enzymes. Further, crude CDT preparations possess detectable DNase activity not associated with identical preparations from control cells. Five CdtB mutations in amino acids corresponding to DNase I active site residues were prepared and expressed together with wild‐type CdtA and CdtC polypeptides. Mutation in four of the five DNase‐specific active site residues resulted in CDT preparations that lacked DNase activity and failed to induce cellular distension or arrest division of HeLa cells. The fifth mutation, Glu86 (Glu78 in DNase I), retained the ability to induce a moderate level of cell cycle arrest and displayed reduced DNase activity relative to wild‐type CDT. Together, these data suggest that the CDT holotoxin has intrinsic DNase activity that is associated with the CdtB polypeptide and that this DNase activity may be responsible for the CDT‐induced cell cycle arrest.
ISSN:0950-382X
1365-2958
DOI:10.1046/j.1365-2958.2000.02070.x