Journal Article

Mutagenic scan of the H‐N‐H motif of colicin E9: implications for the mechanistic enzymology of colicins, homing enzymes and apoptotic endonucleases

David C. Walker, Theonie Georgiou, Ansgar J. Pommer, Daniel Walker, Geoffrey R. Moore, Colin Kleanthous and Richard James

in Nucleic Acids Research

Volume 30, issue 14, pages 3225-3234
Published in print July 2002 | ISSN: 0305-1048
Published online July 2002 | e-ISSN: 1362-4962 | DOI: http://dx.doi.org/10.1093/nar/gkf420
Mutagenic scan of the H‐N‐H motif of colicin E9: implications for the mechanistic enzymology of colicins, homing enzymes and apoptotic endonucleases

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Colicin E9 is a microbial toxin that kills bacteria through random degradation of chromosomal DNA. Within the active site of the cytotoxic endonuclease domain of colicin E9 (the E9 DNase) is a 32 amino acid motif found in the H‐N‐H group of homing endonucleases. Crystal structures of the E9 DNase have implicated several conserved residues of the H‐N‐H motif in the mechanism of DNA hydrolysis. We have used mutagenesis to test the involvement of these key residues in colicin toxicity, metal ion binding and catalysis. Our data show, for the first time, that the H‐N‐H motif is the site of DNA binding and that Mg2+‐dependent cleavage of double‐stranded DNA is responsible for bacterial cell death. We demonstrate that more active site residues are required for catalysis in the presence of Mg2+ ions than transition metals, consistent with the recent hypothesis that the E9 DNase hydrolyses DNA by two distinct, cation‐dependent catalytic mechanisms. The roles of individual amino acids within the H‐N‐H motif are discussed in the context of the available structural information on this and related DNases and we address the possible mechanistic similarities between caspase‐activated DNases, responsible for the degradation of chromatin in eukaryotic apoptosis, and H‐N‐H DNases.

Journal Article.  7368 words.  Illustrated.

Subjects: Chemistry ; Biochemistry ; Bioinformatics and Computational Biology ; Genetics and Genomics ; Molecular and Cell Biology

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