Zinc is essential for high-affinity DNA binding and recombinase activity of phi C31 integrase

Andrew R. McEwan, Andrea Raab, Sharon M. Kelly, Joerg Feldmann, Margaret C. M. Smith

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

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 Zn2+. 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.

Original languageEnglish
Pages (from-to)6137-6147
Number of pages11
JournalNucleic Acids Research
Volume39
Issue number14
Early online date20 Apr 2011
DOIs
Publication statusPublished - Aug 2011

Fingerprint

Integrases
Recombinases
Zinc
Zinc Fingers
DNA
Cysteine
Edetic Acid
Nucleotide Motifs
Circular Dichroism
Serine
Genetic Recombination
Mass Spectrometry
Peptide Hydrolases
Mutation
Proteins

Keywords

  • site-specific recombination
  • secondary structure analyses
  • C-terminal domain
  • serine recombinases
  • catalytic domain
  • BXB1 integration
  • system
  • recognition
  • synapsis
  • ATTB

Cite this

Zinc is essential for high-affinity DNA binding and recombinase activity of phi C31 integrase. / McEwan, Andrew R.; Raab, Andrea; Kelly, Sharon M.; Feldmann, Joerg; Smith, Margaret C. M.

In: Nucleic Acids Research, Vol. 39, No. 14, 08.2011, p. 6137-6147.

Research output: Contribution to journalArticle

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