Mutations altering the cleavage specificity of a homing endonuclease

Abstract

The homing endonuclease I-CreI recognizes and cleaves a particular 22 bp DNA sequence. The crystal structure of I-CreI bound to homing site DNA has previously been determined, leading to a number of predictions about specific protein-DNA contacts. We test these predictions by analyzing a set of endonuclease mutants and a complementary set of homing site mutants. We find evidence that all structurally predicted I-CreI/DNA contacts contribute to DNA recognition and show that these contacts differ greatly in terms of their relative importance. We also describe the isolation of a collection of altered specificity I-CreI derivatives. The in vitro DNA-binding and cleavage properties of two such endonucleases demonstrate that our genetic approach is effective in identifying homing endonucleases that recognize and cleave novel target sequences.

Figure 1

Predicted I-CreI/DNA contacts. The 22 bp I-CreI homing site is shown, with palindromic base pairs in bold. Cleavage positions are indicated by vertical lines between bases 13 and 14 on the top strand and 9 and 10 on the bottom. I-CreI amino acids are indicated by their single letter abbreviations, with residues from one monomer of the homodimer in bold and those from the other having primes. Solid lines indicate direct hydrogen bond interactions, with double lines indicating two such interactions. Dashed lines indicate water-mediated interactions. The figure is based upon the structure of Jurica et al. (14).

Figure 2

Escherichia coli-based assays for I-CreI function. (A) The F′cre-kan assay. Plasmid pA-E contains a chloramphenicol resistance gene and expresses I-CreI from an arabinose-inducible promoter. F′cre-kan contains a wild-type I-CreI homing site adjacent to a kanamycin resistance gene. Introduction of pA-E into F′cre-kan-containing cells results in recipient cells being converted from kanamycin-resistant to kanamycin-sensitive. (B) The F′o-cre assay. F′o-cre contains an I-CreI homing site in place of lacO sequences. Introduction of pA-E into F′o-cre-containing cells results in recipient cells being converted from lacZ⁺ to lacZ^–.

Figure 3

Cleavage of the wild-type I-CreI homing site by partially active I-CreI derivatives. The F′o-cre assay with recipients containing F′o-cre with wild-type I-CreI homing sites. The identities of endonucleases are indicated above each panel, with the ‘endo^–’ control corresponding to pACYC184.

Figure 4

Cleavage of mutant homing sites by various I-CreI derivatives. The F′o-cre assay with recipients containing F′o-cre with symmetrical mutant homing sites, as indicated above each set of panels. The identities of endonucleases are indicated next to each set of panels.

Figure 5

In vitro cleavage competition assays. Wild-type I-CreI and endonuclease mutants C33 and K32 were exposed to linearized plasmids containing wild-type and mutant homing sites. The numbers above each gel indicate relative amounts of endonuclease, with 0 corresponding to no enzyme, 1 corresponding to the minimal enzyme concentration sufficient to completely digest each plasmid and subsequent fractions reflecting serial 2-fold enzyme dilutions. Homing site identities are indicated at the sides of each photo and endonuclease identities below.