Chimeric recombinases with designed DNA sequence recognition

Site-specific recombination typically occurs only between DNA sequences that have co-evolved with a natural recombinase enzyme to optimize sequence recognition, catalytic efficiency, and regulation. Here, we show that the sequence recognition and the catalysis functions of a recombinase can be specified by unrelated protein domains. We describe chimeric recombinases with a catalytic domain from an activated multiple mutant of the bacterial enzyme Tn3 resolvase, fused to a DNA recognition domain from the mouse transcription factor Zif268. These proteins catalyze efficient recombination specifically at synthetic target sites recognized by two Zif268 domains. Our results demonstrate the functional autonomy of the resolvase catalytic domain and open the way to creating “custom-built” recombinases that act at chosen natural target sequences.

[1]  F. Neidhart.  Escherichia coli and Salmonella. , 1996 .

[2]  J. S. Kim,et al.  Getting a handhold on DNA: design of poly-zinc finger proteins with femtomolar dissociation constants. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[3]  T. Steitz,et al.  Cleavage of the site-specific recombination protein gamma delta resolvase: the smaller of two fragments binds DNA specifically. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[4]  J. Park,et al.  A novel mutant loxP containing part of long terminal repeat of HIV-1 in spacer region: presentation of possible target site for antiviral strategy using site-specific recombinase. , 1998, Biochemical and biophysical research communications.

[5]  Dana Carroll,et al.  Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases. , 2002, Genetics.

[6]  F. Guo,et al.  Structure of Cre recombinase complexed with DNA in a site-specific recombination synapse , 1997, Nature.

[7]  Frank Buchholz,et al.  Alteration of Cre recombinase site specificity by substrate-linked protein evolution , 2001, Nature Biotechnology.

[8]  Peter G Schultz,et al.  Directed evolution of the site specificity of Cre recombinase , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[9]  P. Dröge,et al.  Site‐specific recombination in mammalian cells catalyzed by γδ resolvase mutants: implications for the topology of episomal DNA , 2000 .

[10]  F. Schneider,et al.  A DNA-binding domain swap converts the invertase gin into a resolvase. , 2000, Journal of molecular biology.

[11]  N. Grindley.  Analysis of a nucleoprotein complex: the synaptosome of gamma delta resolvase. , 1993, Science.

[12]  A. Klug,et al.  A rapid, generally applicable method to engineer zinc fingers illustrated by targeting the HIV-1 promoter , 2001, Nature Biotechnology.

[13]  Wei Yang,et al.  Crystal structure of the site-specific recombinase γδ resolvase complexed with a 34 by cleavage site , 1995, Cell.

[14]  N. Grindley,et al.  Mutants of Tn3 resolvase which do not require accessory binding sites for recombination activity , 1999, The EMBO journal.

[15]  Margaret C. M. Smith,et al.  Diversity in the serine recombinases , 2002, Molecular microbiology.

[16]  C. Pabo,et al.  Zif268 protein-DNA complex refined at 1.6 A: a model system for understanding zinc finger-DNA interactions. , 1996, Structure.

[17]  C. Gorman,et al.  Site-specific gene targeting for gene expression in eukaryotes. , 2000, Current opinion in biotechnology.

[18]  Andras Nagy,et al.  Cre recombinase: The universal reagent for genome tailoring , 2000, Genesis.

[19]  N. Grindley,et al.  Architecture of the γδ Resolvase Synaptosome Oriented Heterodimers Identify Interactions Essential for Synapsis and Recombination , 1998, Cell.

[20]  D. Sherratt,et al.  Determinants of correct res site alignment in site-specific recombination by Tn3 resolvase. , 1990, Genes & development.

[21]  B. Thyagarajan,et al.  Directed evolution of a recombinase for improved genomic integration at a native human sequence. , 2001, Nucleic acids research.

[22]  S. Halford,et al.  DNA binding by mutants of Tn21 resolvase with DNA recognition functions from Tn3 resolvase. , 1990, Journal of molecular biology.

[23]  D. Sherratt,et al.  Cooperative binding of Tn3 resolvase monomers to a functionally asymmetric binding site , 1995, Current Biology.