Single-chain integration host factors as probes for high-precision nucleoprotein complex formation

• Published in: Gene Vol. 343; no. 1; pp. 99 - 106
• Main Authors: Bao, Qiuye, Christ, Nicole, Dröge, Peter
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 08.12.2004
• Subjects: Amino Acid Sequence; Bacteriophage lambda - genetics; DNA architectural protein; DNA bending; DNA Primers; DNA Replication; DNA, Bacterial - genetics; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Escherichia coli; Escherichia coli - genetics; Escherichia coli Proteins - genetics; Integration host factor; Integration Host Factors - chemistry; Integration Host Factors - genetics; Integration Host Factors - metabolism; Models, Molecular; Molecular Sequence Data; Nucleoprotein complex; Phage ^l; Protein Conformation; Protein engineering; Recombination, Genetic; Sequence Deletion; Site-specific recombination; Integration host factor; Protein engineering; attP; DNA replication; snups; IHF; EMSA; attB; Nucleoprotein complex; DNA architectural protein; Site-specific recombination; DNA bending
• ISSN: 0378-1119; 1879-0038
• DOI: 10.1016/j.gene.2004.08.030

Integration host factor (IHF) is a heterodimeric, site-specific DNA-binding and DNA-bending protein from Escherichia coli. It is involved in high-precision DNA transactions where it serves as a key architectural component of specialized nucleoprotein structures (snups). We described recently a novel approach for protein engineering using a single polypeptide chain IHF, termed scIHF2, as a first example. ScIHF2 is made up of the α subunit of IHF which was inserted into the β subunit at peptide bond Q39/G40 via two short linkers. The monomer behaves very similarly to the heterodimeric, parental IHF in biochemical and functional assays. Here, we describe an extension of this approach in which we shortened either one or both linkers by one amino acid, thereby generating three new variants termed scIHF1, 3, and 4. These variants exhibit distinct DNA-binding properties, different phenotypes in site-specific integrative and excisive recombination by phage λ integrase in vitro, as well as in pSC101 replication assays in a ΔIHF E. coli host. We also introduced a K45E substitution within the α domain of scIHF3 and based on electrophoretic mobility shift assays (EMSAs), argue that it significantly changes the DNA trajectory within the protein–DNA complex. Our results indicate that IHF's pleiotropic roles in DNA transactions inside E. coli require different types of high-precision DNA architectural activities. The scIHF variants described here will help to explore further how flexible these requirements are.