zinc finger recombinases with adaptable dna sequence specificity锌指可与适应性的dna序列特异性.pdfVIP

Zinc Finger Recombinases with Adaptable DNA Sequence Specificity 1 1 2 1 Chris Proudfoot , Arlene L. McPherson , Andreas F. Kolb , W. Marshall Stark * 1 College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, Scotland, United Kingdom, 2 Nutrition and Epigenetics Group, Life Long Health Division, Rowett Institute of Nutrition and Health, University of Aberdeen, Aberdeen, Scotland, United Kingdom Abstract Site-specific recombinases have become essential tools in genetics and molecular biology for the precise excision or integration of DNA sequences. However, their utility is currently limited to circumstances where the sites recognized by the recombinase enzyme have been introduced into the DNA being manipulated, or natural ‘pseudosites’ are already present. Many new applications would become feasible if recombinase activity could be targeted to chosen sequences in natural genomic DNA. Here we demonstrate efficient site-specific recombination at several sequences taken from a 1.9 kilobasepair locus of biotechnological interest (in the bovine b-casein gene), mediated by zinc finger recombinases (ZFRs), chimaeric enzymes with linked zinc finger (DNA recognition) and recombinase (catalytic) domains. In the Z-sites tested here, 22 bp casein gene sequences are flanked by 9 bp motifs recognized by zinc finger domains. Asymmetric Z-sites were recombined by the concomitant action of two ZFRs with different zinc finger DNA-binding specificities, and could be recombined with a heterologous site in the presence of a third recombinase. Our results show that engineered ZFRs may be designed to promote site-specific recombination at many natural DNA sequences. Citation: Proudfoot C, McPherson AL, Kolb AF