title: Zinc is essential for high-affinity DNA binding and recombinase activity of ΦC31 integrase.
abstract: The mechanism through which the large serine recombinases bind DNA is poorly understood. Alignments of 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.
authors.affiliation: Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK.
authors.forename: Andrew R
authors.initials: AR
authors.lastname: McEwan
authors.forename: Andrea
authors.initials: A
authors.lastname: Raab
authors.forename: Sharon M
authors.initials: SM
authors.lastname: Kelly
authors.forename: Jörg
authors.initials: J
authors.lastname: Feldmann
authors.forename: Margaret C M
authors.initials: MC
authors.lastname: Smith
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001287:Attachment Sites, Microbiological; D002614:Chelating Agents / Q000494:pharmacology; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D019426:Integrases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D011485:Protein Binding; D055550:Protein Stability; D017433:Protein Structure, Secondary; D011995:Recombination, Genetic; D016415:Sequence Alignment; D017384:Sequence Deletion; D017906:Siphoviridae / Q000201:enzymology*; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015032:Zinc / Q000032:analysis / Q000378:metabolism / Q000502:physiology*; D016335:Zinc Fingers
chemical_list: D002614:Chelating Agents; D004268:DNA-Binding Proteins; D014764:Viral Proteins; D004492:Edetic Acid; D019426:Integrases; D015032:Zinc; D003545:Cysteine