Protein–protein interaction between monomers of coliphage HK022 excisionase

• Published in: FEBS letters 2004-11, Vol.577 (1), p.17-20
• Main Authors: Gottfried, Pnina, Kolot, Mikhail, Silberstein, Nava, Yagil, Ezra
• Format: Article
• Language: English
• Subjects: Bacteriophage HK022; bp, base pairs; Chromatography, Gel; Coliphages - enzymology; DNA Nucleotidyltransferases - chemistry; DNA Nucleotidyltransferases - metabolism; DSG, disuccinimidyl glutarate; DSS, disuccinimidyl suberate; Excisionase; FITC, fluorescein-5-isothiocyanate; Fluorescence Resonance Energy Transfer; FRET, fluorescence resonance energy transfer; IHF, integration host factor; Int, integrase; IPTG, isopropyl β-d-thiogalactopyranoside; PBS, phosphate buffer saline; Protein Binding; Protein–protein interaction; RDF, recombination directionality factor; SDS–PAGE, polyacrylamide gel electorphoresis in the presence of sodium dodecyl sulphate; Site-specific recombination; TRITC, tetramethylrhodamine-5- (and 6) isothiocyanate; Viral Proteins - chemistry; Viral Proteins - metabolism; Xis, excisionase; β-ME, β-mercaptoethanol
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/j.febslet.2004.09.045

Excisionase (Xis) is an accessory protein that is required for the site-specific excision reaction of the coliphages HK022 and λ. Xis binds in a strong cooperative manner to two tandem binding sites (X1 and X2) located on the P arm of the attachment ( att) sites on the phage genome. As a result of crosslinking experiments in vivo and in vitro of Xis-overexpressing cells, by gel filtration of purified Xis and by FRET analyses we show that Xis monomers of HK022 interact and form dimers that are not dependent on the single Cys residue of the protein and on the presence of DNA. The formation of the dimers may explain the strong binding cooperativity of Xis to its sites on DNA.