Premium
Intron‐encoded endonuclease I‐TevI binds as a monomer to effect sequential cleavage via conformational changes in the td homing site.
Author(s) -
Mueller J. E.,
Smith D.,
Bryk M.,
Belfort M.
Publication year - 1995
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1002/j.1460-2075.1995.tb00259.x
Subject(s) - biology , homing endonuclease , cleavage (geology) , endonuclease , intron , homing (biology) , biophysics , monomer , binding site , microbiology and biotechnology , genetics , dna , gene , paleontology , ecology , chemistry , organic chemistry , fracture (geology) , polymer
I‐TevI, the intron‐encoded endonuclease from the thymidylate synthase (td) gene of bacteriophage T4, binds its DNA substrate across the minor groove in a sequence‐tolerant fashion. We demonstrate here that the 28 kDa I‐TevI binds the extensive 37 bp td homing site as a monomer and significantly distorts its substrate. In situ cleavage assays and phasing analyses indicate that upon nicking the bottom strand of the td homing site, I‐TevI induces a directed bend of 38 degrees towards the major groove near the cleavage site. Formation of the bent I‐TevI‐DNA complex is proposed to promote top‐strand cleavage of the homing site. Furthermore, reductions in the degree of distortion and in the efficiency of binding base‐substitution variants of the td homing site indicate that sequences flanking the cleavage site contribute to the I‐TevI‐induced conformational change. These results, combined with genetic, physical and computer‐modeling studies, form the basis of a model, wherein I‐TevI acts as a hinged monomer to induce a distortion that widens the minor groove, facilitating access to the top‐strand cleavage site. The model is compatible with both unmodified DNA and glucosylated hydroxymethylcytosine‐containing DNA, as exists in the T‐even phages.