DAXX interacts with phage ΦC31 integrase and inhibits recombination

Phage Phi C31 integrase has potential as a means of inserting therapeutic genes into specific sites in the human genome. However, the possible interactions between Phi C31 integrase and cellular proteins have never been investigated. Using pLexA-Phi C31 integrase as bait, we screened a pB42AD-human fetal brain cDNA library for potential interacting cellular proteins. Among 61 positives isolated from 10(6) independent clones, 51 contained DAXX C-terminal fragments. The strong interaction between DAXX and Phi C31 was further confirmed by co-immunoprecipitation. Deletion analysis revealed that the fas-binding domain of DAXX is also the region for Phi C31 binding. Hybridization between a Phi C31 integrase peptide array and an HEK293 cell extract revealed that a tetramer, 451RFGK454, in the C-terminus of Phi C31 is responsible for the interaction with DAXX. This tetramer is also necessary for Phi C31 integrase activity as removal of this tetramer resulted in a complete loss of integrase activity. Co-expression of DAXX with Phi C31 integrase in a HEK293-derived Phi C31 integrase activity reporter cell line significantly reduced the Phi C31-mediated recombination rate. Knocking down DAXX with a DAXX-specific duplex RNA resulted in increased recombination efficiency. Therefore, endogenous DAXX may interact with Phi C31 causing a mild inhibition in the integration efficiency. This is the first time that Phi C31 was shown to interact with an important cellular protein and the potential effect of this interaction should be further studied.