Chen Jin-zhong, Ji Chao-neng, Xu Guan-lan, Pang Rong-yan, Yao Ji-hua, Zhu Huan-zhang, Xue Jing-lun, Jia William
State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, China.
Nucleic Acids Res. 2006;34(21):6298-304. doi: 10.1093/nar/gkl890. Epub 2006 Nov 10.
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.
噬菌体PhiC31整合酶有潜力作为将治疗性基因插入人类基因组特定位点的一种手段。然而，PhiC31整合酶与细胞蛋白之间可能的相互作用从未被研究过。我们以pLexA-PhiC31整合酶作为诱饵，筛选了一个pB42AD-人类胎儿脑cDNA文库，以寻找潜在的相互作用细胞蛋白。从10⁶个独立克隆中分离出的61个阳性克隆中，有51个包含DAXX的C末端片段。DAXX与PhiC31之间的强相互作用通过共免疫沉淀进一步得到证实。缺失分析表明，DAXX的fas结合结构域也是与PhiC31结合的区域。PhiC31整合酶肽阵列与HEK293细胞提取物之间的杂交显示，PhiC31 C末端的一个四聚体451RFGK454负责与DAXX的相互作用。这个四聚体对于PhiC31整合酶活性也是必需的，因为去除这个四聚体导致整合酶活性完全丧失。在HEK293衍生的PhiC31整合酶活性报告细胞系中，DAXX与PhiC31整合酶共表达显著降低了PhiC31介导的重组率。用DAXX特异性双链RNA敲低DAXX导致重组效率提高。因此，内源性DAXX可能与PhiC31相互作用，对整合效率产生轻微抑制。这是首次证明PhiC31与一种重要的细胞蛋白相互作用，这种相互作用的潜在影响值得进一步研究。
