Role of the interdomain linker in distance determination for remote cleavage by homing endonuclease I-TevI.
J Mol Biol
; 379(5): 1094-106, 2008 Jun 20.
Article
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| MEDLINE
| ID: mdl-18499124
ABSTRACT
I-TevI is a modular intron-encoded endonuclease, consisting of an N-terminal catalytic domain and a C-terminal DNA-binding domain, joined by a 75 amino acid linker. This linker can be divided into three regions, starting at the N terminus the deletion-intolerant (DI) region; the deletion-tolerant (DT) region; and a zinc finger, which acts as a distance determinant for cleavage. To further explore linker function, we generated deletion and substitution mutants that were tested for their preference to cleave at a particular distance or at the correct sequence. Our results demonstrate that the I-TevI linker is multi-functional, a property that sets it apart from junction sequences in most other proteins. First, the linker DI region has a role in I-TevI cleavage activity. Second, the DT linker region participates in distance determination, as evident from DT mutants that display a phenotype similar to that of the zinc-finger mutants in their selection of a cleavage site. Finally, NMR analysis of a freestanding 56 residue linker segment showed an unstructured stretch corresponding to the DI region and a portion of the DT region, followed by a beta-strand corresponding to the remainder of the DT region and containing a key distance-determining arginine, R129. Mutation of this arginine to alanine abolished distance determination and disrupted the beta-strand, indicating that the structure of the DT linker region has a role in cleavage at a fixed distance.
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Endodesoxirribonucleasas
Idioma:
En
Revista:
J Mol Biol
Año:
2008
Tipo del documento:
Article
País de afiliación:
Estados Unidos