DAXX interacts with phage PhiC31 integrase and inhibits recombination.
Phage PhiC31 integrase has potential as a means of inserting therapeutic genes into specific sites in the human genome. However, the possible interactions between PhiC31 integrase and cellular proteins have never been investigated. Using pLexA-PhiC31 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 PhiC31 was further confirmed by co-immunoprecipitation. Deletion analysis revealed that the fas-binding domain of DAXX is also the region for PhiC31 binding. Hybridization between a PhiC31 integrase peptide array and an HEK293 cell extract revealed that a tetramer, 451RFGK454, in the C-terminus of PhiC31 is responsible for the interaction with DAXX. This tetramer is also necessary for PhiC31 integrase activity as removal of this tetramer resulted in a complete loss of integrase activity. Co-expression of DAXX with PhiC31 integrase in a HEK293-derived PhiC31 integrase activity reporter cell line significantly reduced the PhiC31-mediated recombination rate. Knocking down DAXX with a DAXX-specific duplex RNA resulted in increased recombination efficiency. Therefore, endogenous DAXX may interact with PhiC31 causing a mild inhibition in the integration efficiency. This is the first time that PhiC31 was shown to interact with an important cellular protein and the potential effect of this interaction should be further studied.
Chen
Jin-zhong
JZ
State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, China.
Ji
Chao-neng
CN
Xu
Guan-lan
GL
Pang
Rong-yan
RY
Yao
Ji-hua
JH
Zhu
Huan-zhang
HZ
Xue
Jing-lun
JL
Jia
William
W
D048868:Adaptor Proteins, Signal Transducing / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D001435:Bacteriophages / Q000201:enzymology*; D001665:Binding Sites; D002460:Cell Line; D056970:Co-Repressor Proteins; D006801:Humans; D047468:Immunoprecipitation; D019429:Integrase Inhibitors / Q000378:metabolism; D019426:Integrases / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D018832:Molecular Chaperones; D009687:Nuclear Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D011995:Recombination, Genetic; D020798:Two-Hybrid System Techniques
D048868:Adaptor Proteins, Signal Transducing; D056970:Co-Repressor Proteins; C106985:DAXX protein, human; D019429:Integrase Inhibitors; D018832:Molecular Chaperones; D009687:Nuclear Proteins; D019426:Integrases