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

By Andrew R. McEwan, Andrea Raab, Sharon M. Kelly, Jörg Feldmann and Margaret C. M. Smith

Abstract

The mechanism through which the large serine recombinases bind DNA is poorly understood. Alignments of ϕC31 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 V371SUGA, 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

Topics: Nucleic Acid Enzymes
Publisher: Oxford University Press
OAI identifier: oai:pubmedcentral.nih.gov:3152356
Provided by: PubMed Central

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Citations

  1. (2008). A motif in the C-terminal domain of fC31 integrase controls the directionality of recombination.
  2. (2009). A simple and efficient expression and purification system using two newly constructed vectors. Protein expression and purification,
  3. (1991). Analysis of the integration function of the streptomycete bacteriophage fC31.
  4. (2000). Control of directionality in the site-specific recombination system of the Streptomyces phage fC31.
  5. (2004). DICHROWEB, an online server for protein secondary structure analyses from circular dichroism spectroscopic data.
  6. (2002). Diversity in the serine recombinases.
  7. (2009). DNA binding and synapsis by the large C-terminal domain of fC31 integrase.
  8. (1998). In vitro site-specific integration of bacteriophage DNA catalyzed by a recombinase of the resolvase/invertase family.
  9. (2006). Mechanisms of site-specific recombination.
  10. (2008). Protein secondary structure analyses from circular dichroism spectroscopy: methods and reference databases.
  11. (2007). Sequences in attB that affect the ability of fC31 integrase to synapse and to activate DNA cleavage.
  12. (2010). Site-specific recombination by fC31 integrase and other large serine recombinases.
  13. (2007). Sticky fingers: zinc-fingers as protein-recognition motifs.
  14. (2003). Structural classification of zinc fingers: survey and summary.
  15. (2004). Switching the polarity of a bacteriophage integration system.
  16. (2004). Synapsis and DNA cleavage in fC31 integrase-mediated site-specific recombination.
  17. (2005). Synapsis in phage Bxb1 integration: selection mechanism for the correct pair of recombination sites.
  18. (2008). Tetrameric structure of a serine integrase catalytic domain.
  19. (1990). The crystal structure of the catalytic domain of the site-specific recombination enzyme gamma delta resolvase at 2.7 A resolution.
  20. (2003). The orientation of mycobacteriophage Bxb1 integration is solely dependent on the central dinucleotide of attP and attB.
  21. (2002). The streptomyces genome contains multiple pseudo-attB sites for the fC31-encoded site-specific recombination system.