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Lookup NU author(s): Dr Steven Evans, Professor Bernard Connolly
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Colicin endonucleases and the H-N-H family of homing enzymes share a common active site structural motif that has similarities to the active sites of a variety of other nucleases such as the non-specific endonuclease from Serratia and the sequence-specific His-Cys box homing enzyme I-PpoI. In contrast to these latter enzymes, however, it remains unclear how H-N-H enzymes cleave nucleic acid substrates. Here, we show that the H-N-H enzyme from colicin E9 (the E9 DNase) shares many of the same basic enzymological characteristics as sequence-specific H-N-H enzymes including a dependence for high concentrations of Mg2+ or Ca2+ with double-stranded substrates, a high pH optimum (pH 8-9) and inhibition by monovalent cations. We also show that this seemingly nonspecific enzyme preferentially nicks double-stranded DNA at thymine bases producing 3′-hydroxy and 5′-phosphate termini, and that the enzyme does not cleave small substrates, such as dinucleotides or nucleotide analogues, which has implications for its mode of inhibition in bacteria by immunity proteins. The E9 DNase will also bind single-stranded DNA above a certain length and in a sequence-independent manner, with transition metals such as Ni2+ optimal for cleavage but Mg2+ a poor cofactor. Ironically, the H-N-H motif of the E9 DNase although resembling the zinc binding site of a metalloenzyme does not support zincmediated hydrolysis of any DNA substrata. Finally, we demonstrate that the E9 DNase also degrades RNA in the absence of metal ions. In the context of current structural information, our data show that the H-N-H motif is an adaptable catalytic centre able to hydrolyse nucleic acid by different mechanisms depending on the substrate and metal ion regime. © 2001 Academic Press.
Author(s): Evans SJ; Connolly BA; Pommer AJ; Moore GR; James R; Kleanthous C; Cal S; Keeble AH; Walker D; Kuhlmann UC; Cooper A; Hemmings AM
Publication type: Article
Publication status: Published
Journal: Journal of Molecular Biology
Year: 2001
Volume: 314
Issue: 4
Pages: 735-749
ISSN (print): 0022-2836
ISSN (electronic): 1089-8638
Publisher: Academic Press
URL: http://dx.doi.org/10.1006/jmbi.2001.5189
DOI: 10.1006/jmbi.2001.5189
PubMed id: 11733993
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