Evolution of I-SceI derivatives with altered binding and cleavage profiles

Rakesh Joshi, Purdue University

Abstract

Homing endonucleases, expressed from mobile genetic elements, recognize and cleave rare DNA target sites (14-40 bp). Understanding how homing endonucleases tolerate modifications in recognition site specificity will aid endeavors to engineer proteins for gene targeting applications. I-SceI is a monomer of the LAGLIDADG family of homing endonucleases that binds and cleaves an 18-bp target. It allows limited degeneracy in its target site, including substitution of a C/G+4 base-pair for the wild-type A/T+4 base-pair. Plasmid and phagemid libraries encoding randomized amino acids at I-SceI residue positions that contact or are proximal to A/T+4 were used in combination with a bacterial one-hybrid system to select I-SceI derivatives that bind to recognition sites containing either the A/T+4 or C/G+4 base pairs.As expected, isolates encoding wild-type residues at the randomized positions were selected using both A/T+4 and C/G +4 DNA targets. All I-SceI mutants isolated using the C/G+4 recognition site included small side chain substitutions at G100, and either contained or lacked a K86R substitution. Interestingly, the binding affinities of the selected variants for the wild-type A/T+4 target are 4-11-fold lower than that of wild-type I-SceI, whereas those for the C/G+4 target are similar. The increased specificity of the mutant proteins for the mutant C/G+4 target is also conspicuous in binding experiments in vivo. These alterations in binding affinities explain the apparent ∼36-fold switch in recognition site preference between the K86R/G100T and wild-type proteins in competitive DNA cleavage assays. X-ray crystal structures of the wild-type and K86R/G100T I-SceI derivatives bound to a DNA duplex containing the C/G+4 substitution suggest how sequence specificity of a homing enzyme can be maintained or lost. This biochemical and structure-based analysis elucidates one avenue by which target specificity is enhanced for a homing endonuclease. Efforts at employing a negative selection system that filters out promiscuous I-SceI mutants in combination with an I-SceI library containing seven randomized codons (7-var), as well as attempts at co-crystallizing a nicking derivative of I-SceI, with intact and nicked DNA targets, are also briefly described.

Degree

Ph.D.

Advisors

Gimble, Purdue University.

Subject Area

Biochemistry|Biophysics

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