From monomeric to homodimeric endonucleases and back: engineering novel specificity of LAGLIDADG enzymes.
Monomeric homing endonucleases of the LAGLIDADG family recognize DNA in a bipartite manner, reflecting the underlying structural assembly of two protein domains (A and B) related by pseudo 2-fold symmetry. This architecture allows for changes in DNA specificity via the distinct combination of these half-site domains. The key to engineering such hybrid proteins lies in the LAGLIDADG two-helix bundle that forms both the domain interface and the endonuclease active site. In this study, we utilize domain A of the monomeric I-DmoI to demonstrate the feasibility of generating functional homodimeric endonucleases that recognize palindromic DNA sequences derived from the original, non-palindromic target. Wild-type I-DmoI domain A is capable of forming a homodimer (H-DmoA) that binds tightly to, but does not cleave efficiently, its anticipated DNA target. Partial restoration of DNA cleavage ability was obtained by re-engineering the LAGLIDADG dimerization interface (H-DmoC). Upon fusing two copies of H-DmoC via a short peptide linker, a novel, site-specific DNA endonuclease was created (H-DmoC2). Like I-DmoI, H-DmoC2 is thermostable and cleaves the new target DNA to generate the predicted 4 nt 3'-OH overhangs but, unlike I-DmoI, H-DmoC2 retains stringent cleavage specificity when substituting Mn2+ for Mg2+ as co-factor. This novel endonuclease allows speculation regarding specificity of monomeric LAGLIDADG proteins, while it supports the evolutionary genesis of these proteins by a gene duplication event.
Silva
George H
GH
Institut für Biochemie, Justus-Liebig-Universität, Heinrich-Buff-Ring 58, D-35392 Giessen, Germany. George.Silva@chemie.bio.uni-giessen.de
Belfort
Marlene
M
Wende
Wolfgang
W
Pingoud
Alfred
A
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D015253:Deoxyribonucleases, Type I Site-Specific / Q000737:chemistry*; D019281:Dimerization; D008958:Models, Molecular; D016133:Polymerase Chain Reaction; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity
D004247:DNA; C081755:endodeoxyribonuclease I-Dmo I; D015253:Deoxyribonucleases, Type I Site-Specific