ABSTRACT
Streptomyces phage ØC31 encodes a serine integrase which recombines the host (attB) and phage (attP) attachment sites to form new attL and attR sites. For attL and attR to be recombined by the integrase, the presence of its cognate recombination directionality factor (RDF) is required. It is hypothesized that the RDF binds the coiled-coil domain of the integrase to accomplish this switch in recombination directionality. Yet, nothing is known about the site of interaction between the RDF and integrase. Here, we tried to identify the region on the coiled-coil domain of ØC31 integrase to which its cognate RDF (gp3) might bind. Mutant integrases fused to their cognate RDF (gp3) were created and selected for inactivity in attL x attR recombination. It was not possible to characterize the mutants and identify the Int-RDF binding region, due to unforeseen errors that occurred during the construction of the mutant library, but we demonstrated that simple experimental approaches could be used to identify this region. Despite this, an integrase mutant (P398L D595N) fused to gp3 was characterized. This mutant was catalyzing a more unidirectional attL x attR recombination reaction with reduced attP x attB recombination compared to the wild-type integrase. This mutant was selected since it could be used within the field of molecular biology to construct inversion switches which are a key element by which cells can be computerized.
By the time this work was uploaded on bioRxiv, there is now a publication that investigated the Int-RDF interaction interface and several residues at the base of the coiled-coil that affected both the interaction and recombinase activity were identified (Paul C M Fogg, Ellen Younger, Booshini D Fernando, Thanafez Khaleel, W Marshall Stark, Margaret C M Smith, Recombination directionality factor gp3 binds PhiC31 integrase via the zinc domain, potentially affecting the trajectory of the coiled-coil motif, Nucleic Acids Research, Volume 46, Issue 3, 16 February 2018, Pages 1308-1320, https://doi.org/10.1093/nar/gkx1233).
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
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The text in the abstract was updated to include a reference to this paper: Paul C M Fogg, Ellen Younger, Booshini D Fernando, Thanafez Khaleel, W Marshall Stark, Margaret C M Smith, Recombination directionality factor gp3 binds ϕC31 integrase via the zinc domain, potentially affecting the trajectory of the coiled-coil motif, Nucleic Acids Research, Volume 46, Issue 3, 16 February 2018, Pages 1308-1320, https://doi.org/10.1093/nar/gkx1233
Abbreviations used
- Int
- Integrase
- Int-gp3
- Integrase-gp3
- RDF
- Recombination directionality factor
- CC
- Coiled-coil
- GFP
- Green Fluorescent Protein
- RFP
- Red Fluorescent Protein
