Zinc-finger recombinase activities in vitro

Zinc-finger recombinases (ZFRs) are chimaeric pro-teins comprising a serine recombinase catalytic do-main linked to a zinc-finger DNA binding domain. ZFRs can be tailored to promote site-specific recombination at diverse ‘Z-sites’, which each com-prise 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 reac-tion conditions, ZFRs can catalyse Z-site-specific double-strand DNA cleavage. ZFR recombination activity in Escherichia coli and in vitro is highly de-pendent 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 vari-ations in Z-site length. We discuss the causes of these phenomena, and their implications for prac-tical applications of ZFRs