Corona Teresa, Bao Qiuye, Christ Nicole, Schwartz Thomas, Li Jinming, Dröge Peter
Institute of Genetics, University of Cologne, Weyertal 121, D-50931 Cologne, Germany.
Nucleic Acids Res. 2003 Sep 1;31(17):5140-8. doi: 10.1093/nar/gkg711.
The heterodimeric integration host factor (IHF) is a site-specific DNA-binding and DNA-bending protein from Escherichia coli. It plays essential roles in a variety of DNA transactions including recombination, transcription and DNA replication. IHF's ability for concerted binding and bending of DNA is key to its biological function. Here we report the design, characterization and application of a single polypeptide chain IHF, termed scIHF2. In a novel approach for protein engineering, we inserted almost the entire alpha-subunit of IHF into the beta-subunit. DNA binding and DNA bending assays revealed that purified wild-type IHF and scIHF2 behave very similarly. Further, scIHF2 is required for site-specific integrative recombination by phage lambda integrase and for pSC101 replication in a DeltaIHF E.coli host. It also triggers site-specific integrative and excisive recombination in vitro to the same extent as the wild-type protein. We also demonstrate that scIHF2 is stably expressed in HeLa cells, that it is localized primarily in the cell nucleus and that it triggers integrative recombination in mammalian cells by wild-type integrase. Hence, scIHF2 may be used as a novel regulatory cofactor for recombination or other DNA transactions in mammalian cells that require or benefit from sequence-specific high precision DNA bending.
异二聚体整合宿主因子（IHF）是一种来自大肠杆菌的位点特异性DNA结合和DNA弯曲蛋白。它在包括重组、转录和DNA复制在内的多种DNA事务中发挥着重要作用。IHF协同结合和弯曲DNA的能力是其生物学功能的关键。在此，我们报告了一种单多肽链IHF（称为scIHF2）的设计、表征及应用。在一种新颖的蛋白质工程方法中，我们将几乎整个IHF的α亚基插入到β亚基中。DNA结合和DNA弯曲分析表明，纯化的野生型IHF和scIHF2表现非常相似。此外，scIHF2是噬菌体λ整合酶进行位点特异性整合重组以及在缺失IHF的大肠杆菌宿主中pSC101复制所必需的。它在体外引发位点特异性整合和切除重组的程度与野生型蛋白相同。我们还证明，scIHF2在HeLa细胞中稳定表达，主要定位于细胞核，并且它通过野生型整合酶在哺乳动物细胞中引发整合重组。因此，scIHF2可作为一种新型调节辅因子，用于哺乳动物细胞中需要或受益于序列特异性高精度DNA弯曲的重组或其他DNA事务。
