Assessing the plasticity of DNA target site recognition of the PI-SceI homing endonuclease using a bacterial two-hybrid selection system.
The PI-SceI protein from Saccharomyces cerevisiae is a member of the LAGLIDADG family of homing endonucleases that have been used in genomic engineering. To assess the flexibility of the PI-SceI-binding interaction and to make progress towards the directed evolution of homing endonucleases that cleave specified DNA targets, we applied a two-hybrid method to select PI-SceI variants from a randomized expression library that bind to different DNA substrates. In particular, the codon for Arg94, which is located in the protein splicing domain and makes essential contacts to two adjacent base-pairs, and the codons for four proximal residues were randomized. There is little conservation of the wild-type amino acid residues at the five randomized positions in the variants that were selected to bind to the wild-type site, yet one of the purified derivatives displays DNA-binding specificity and DNA endonuclease activity that is similar to that of the wild-type enzyme. A spectrum of DNA-binding behaviors ranging from partial relaxation of specificity to marked shifts in target site recognition are present in variants selected to bind to sites containing mutations at the two base-pairs. Our results illustrate the inherent plasticity of the PI-SceI/DNA interface and demonstrate that selection based on DNA binding is an effective means of altering the DNA cleavage specificity of homing endonucleases. Furthermore, it is apparent that homing endonuclease target specificity derives, in part, from constraints on the flexibility of DNA contacts imposed by hydrogen bonds to proximal residues.
Gimble
Frederick S
FS
Center for Genome Research, Institute of Biosciences and Technology, Texas A & M University System Health Science Center, 2121 W. Holcombe Blvd., Houston, TX 77030, USA. fgimble@ibt.tamushsc.edu
Moure
Carmen M
CM
Posey
Karen L
KL
D001483:Base Sequence; D017931:DNA Primers; D004269:DNA, Bacterial / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D020798:Two-Hybrid System Techniques
D017931:DNA Primers; D004269:DNA, Bacterial; D015252:Deoxyribonucleases, Type II Site-Specific