title: Cross-talk between diverse serine integrases.
abstract: Phage-encoded serine integrases are large serine recombinases that mediate integrative and excisive site-specific recombination of temperate phage genomes. They are well suited for use in heterologous systems and for synthetic genetic circuits as the attP and attB attachment sites are small (<50 bp), there are no host factor or DNA supercoiling requirements, and they are strongly directional, doing only excisive recombination in the presence of a recombination directionality factor. Combining different recombinases that function independently and without cross-talk to construct complex synthetic circuits is desirable, and several different serine integrases are available. However, we show here that these functions are not reliably predictable, and we describe a pair of serine integrases encoded by mycobacteriophages Bxz2 and Peaches with unusual and unpredictable specificities. The integrases share only 59% amino acid sequence identity and the attP sites have fewer than 50% shared bases, but they use the same attB site and there is non-reciprocal cross-talk between the two systems. The DNA binding specificities do not result from differences in specific DNA contacts but from the constraints imposed by the configuration of the component half-sites within each of the attachment site DNAs.
authors.affiliation: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15241, USA.
authors.forename: Shweta
authors.initials: S
authors.lastname: Singh
authors.affiliation: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15241, USA.
authors.forename: Kate
authors.initials: K
authors.lastname: Rockenbach
authors.affiliation: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15241, USA.
authors.forename: Rebekah M
authors.initials: RM
authors.lastname: Dedrick
authors.affiliation: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15241, USA.
authors.forename: Andrew P
authors.initials: AP
authors.lastname: VanDemark
authors.affiliation: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15241, USA. Electronic address: gfh@pitt.edu.
authors.forename: Graham F
authors.initials: GF
authors.lastname: Hatfull
mesh_terms: D001287:Attachment Sites, Microbiological; D001483:Base Sequence; D004269:DNA, Bacterial / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D019426:Integrases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009160:Mycobacteriophages / Q000201:enzymology*; D039002:Prophages / Q000201:enzymology; D011485:Protein Binding; D011995:Recombination, Genetic; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000378:metabolism*; D013379:Substrate Specificity; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D004269:DNA, Bacterial; D004279:DNA, Viral; D014764:Viral Proteins; D012694:Serine; D019426:Integrases
keywords: BSA; C-terminal domain; CC; CTD; N-terminal domain; NTD; RD; ZD; attachment site selection; bovine serum albumin; coiled coil; integrase; phage integration; recombinase domain; serine recombinase; site-specific recombination; zinc-nucleated domain