title: Zinc-finger recombinase activities in vitro.
abstract: Zinc-finger recombinases (ZFRs) are chimaeric proteins comprising a serine recombinase catalytic domain linked to a zinc-finger DNA binding domain. ZFRs can be tailored to promote site-specific recombination at diverse 'Z-sites', which each comprise a central core sequence flanked by zinc-finger domain-binding motifs. Here, we show that purified ZFRs catalyse efficient high-specificity reciprocal recombination between pairs of Z-sites in vitro. No off-site activity was detected. Under different reaction conditions, ZFRs can catalyse Z-site-specific double-strand DNA cleavage. ZFR recombination activity in Escherichia coli and in vitro is highly dependent on the length of the Z-site core sequence. We show that this length effect is manifested at reaction steps prior to formation of recombinants (binding, synapsis and DNA cleavage). The design of the ZFR protein itself is also a crucial variable affecting activity. A ZFR with a very short (2 amino acids) peptide linkage between the catalytic and zinc-finger domains has high activity in vitro, whereas a ZFR with a very long linker was less recombination-proficient and less sensitive to variations in Z-site length. We discuss the causes of these phenomena, and their implications for practical applications of ZFRs.
authors.affiliation: Institute of Infection, Immunity and Inflammation, University of Glasgow, GBRC, Glasgow G12 8QQ, Scotland, UK.
authors.forename: Marko M
authors.initials: MM
authors.lastname: Prorocic
authors.forename: Dong
authors.initials: D
authors.lastname: Wenlong
authors.forename: Femi J
authors.initials: FJ
authors.lastname: Olorunniji
authors.forename: Aram
authors.initials: A
authors.lastname: Akopian
authors.forename: Jan-Gero
authors.initials: JG
authors.lastname: Schloetel
authors.forename: Adèle
authors.initials: A
authors.lastname: Hannigan
authors.forename: Arlene L
authors.initials: AL
authors.lastname: McPherson
authors.forename: W Marshall
authors.initials: WM
authors.lastname: Stark
mesh_terms: D000595:Amino Acid Sequence; D053837:DNA Cleavage; D008969:Molecular Sequence Data; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D045522:Recombinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic; D016335:Zinc Fingers
chemical_list: D011993:Recombinant Fusion Proteins; D045522:Recombinases