Critical amino acid residues within the φC31 integrase DNA-binding domain affect recombination activities in mammalian cells.
The bacteriophage-derived ϕC31 integrase system represents an attractive tool for site-directed recombination in mammalian cells. Its integration reaction is based on recombination between the attachment site attB within an episomal substrate plasmid and either the bacteriophage-derived wild-type attachment site attP or pseudo-attP attachment sites (attP') present in the mammalian genome. In the present study we aimed at increasing the safety and efficiency of ϕC31 integrase-mediated recombination by mutating the DNA-binding domain located at the C terminus. Using an alanine mutagenesis approach, we generated 22 ϕC31 point mutants that were screened for activities in mammalian cells. Intramolecular excision assays based on recombination between attB and attP revealed five mutants with 2-fold enhanced excision activity. Importantly, we also identified mutants showing enhanced recombination activities between attB and three previously described attP' sites detected in the mammalian genome, indicating that there may be enhanced specificity for these hot spots. Several mutants showed, in mammalian cells, integration activities that increased in a cell line-dependent manner. The combination of beneficial mutations in addition to optimization of the integrase plasmid dose enhanced integration efficiencies up to 5.5-fold. We also identified three ϕC31 integrase mutants that were recombination defective in all applied assays, suggesting that these amino acid residues are essential for the functionality of ϕC31 integrase in mammalian cells. In summary, we identified critical amino acid residues within the ϕC31 DNA-binding domain. With respect to site-directed recombination and genome engineering these findings have important implications for improved ϕC31 protein design.
Liesner
Raphael
R
Department of Virology, Max von Pettenkofer Institute, Ludwig Maximilians University Munich, Germany.
Zhang
Wenli
W
Noske
Nadja
N
Ehrhardt
Anja
A
D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D001287:Attachment Sites, Microbiological / Q000235:genetics; D001435:Bacteriophages / Q000201:enzymology*; D055162:Biocatalysis; D001681:Biological Assay; D045744:Cell Line, Tumor; D005164:Factor IX; D006801:Humans; D019426:Integrases / Q000737:chemistry* / Q000378:metabolism*; D008099:Liver / Q000378:metabolism; D008322:Mammals / Q000378:metabolism*; D051379:Mice; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D017434:Protein Structure, Tertiary; D011995:Recombination, Genetic; D013329:Structure-Activity Relationship; D016662:Virus Integration / Q000502:physiology
D000596:Amino Acids; D050505:Mutant Proteins; D005164:Factor IX; D019426:Integrases