Activation of site-specific DNA integration in human cells by a single chain integration host factor.
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.
Corona
Teresa
T
Institute of Genetics, University of Cologne, Weyertal 121, D-50931 Cologne, Germany.
Bao
Qiuye
Q
Christ
Nicole
N
Schwartz
Thomas
T
Li
Jinming
J
Dröge
Peter
P
D000595:Amino Acid Sequence; D010582:Bacteriophage lambda / Q000201:enzymology; D001665:Binding Sites / Q000235:genetics; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D019426:Integrases / Q000378:metabolism; D034982:Integration Host Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic
D004268:DNA-Binding Proteins; D034982:Integration Host Factors; D021122:Protein Subunits; D004247:DNA; D019426:Integrases