Zinc is essential for high-affinity DNA binding and recombinase activity of φC31 integrase

McEwan, A.R., Raab, A., Kelly, S.M. , Feldmann, J. and Smith, M.C.M. (2011) Zinc is essential for high-affinity DNA binding and recombinase activity of φC31 integrase. Nucleic Acids Research, 39(14), pp. 6137-6147. (doi:10.1093/nar/gkr220)

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The mechanism through which the large serine recombinases bind DNA is poorly understood. Alignments of phi C31 integrase (Int) and its relatives indicate the presence of a conserved motif containing four cysteines resembling a zinc finger. Inductively coupled plasma-mass spectrometry (ICP-MS) confirmed that an Int monomer contains one atom of zinc. Pre-incubation of Int with ethylenediaminetetraacetic acid (EDTA) was detrimental for both recombination activity and DNA binding affinities but full activity could be restored by adding back Zn(2+). Mutations in the cysteines and other highly conserved residues yielded proteins that were hypersensitive to proteases, suggesting that without zinc the domain is unfolded. Substitutions in the highly charged region between the conserved cysteines led to lowered DNA binding affinities while circular dichroism revealed that these variant Ints were not greatly affected in overall folding. Int was protected from inhibition by EDTA when DNA containing an attachment site was present suggesting that the zinc finger and the DNA are in close proximity. A truncated mutant of Int, hInt V371S(UGA), lacking the putative zinc finger could bind DNA with low affinity. The data are consistent with there being at least two DNA binding motifs in Int one of which is the zinc finger-like motif.

Item Type:Articles
Glasgow Author(s) Enlighten ID:Kelly, Dr Sharon
Authors: McEwan, A.R., Raab, A., Kelly, S.M., Feldmann, J., and Smith, M.C.M.
College/School:College of Medical Veterinary and Life Sciences > Institute of Molecular Cell and Systems Biology
Journal Name:Nucleic Acids Research
Publisher:Oxford University Press
ISSN (Online):1362-4962
Published Online:20 April 2011

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