Activation of site-specific DNA integration in human cells by a single chain integration host factor Journal Article
| Authors: | Corona, T.; Bao, Q.; Christ, N.; Schwartz, T.; Li, J.; Dröge, P. |
| Article Title: | Activation of site-specific DNA integration in human cells by a single chain integration host factor |
| Abstract: | 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 poly-peptide chain IHF, termed scIHF2. In a novel approach for protein engineering, we inserted almost the entire α-subunit of IHF into the β-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 λ integrase and for pSC101 replication in a ΔIHF 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. |
| Keywords: | immunohistochemistry; controlled study; protein expression; dna binding protein; human cell; dna-binding proteins; dna replication; protein conformation; protein function; protein analysis; dna recombination; mammalia; protein binding; in vitro study; hela cell; hela cells; dna; amino acid sequence; molecular sequence data; recombination, genetic; escherichia coli; cellular distribution; crystallography, x-ray; binding sites; cell nucleus; nucleic acid conformation; protein subunits; alpha chain; dna transcription; mammal cell; integrase; integrases; polypeptide; curved dna; beta chain; dna integration; bacteriophage; protein engineering; bacteriophage lambda; humans; human; priority journal; article; integration host factor; integration host factors |
| Journal Title: | Nucleic Acids Research |
| Volume: | 31 |
| Issue: | 17 |
| ISSN: | 0305-1048 |
| Publisher: | Oxford University Press |
| Date Published: | 2003-09-01 |
| Start Page: | 5140 |
| End Page: | 5148 |
| Language: | English |
| DOI: | 10.1093/nar/gkg711 |
| PUBMED: | 12930965 |
| PROVIDER: | scopus |
| PMCID: | PMC212810 |
| DOI/URL: | |
| Notes: | Export Date: 12 September 2014 -- Source: Scopus |
