Measure for measure: a zinc finger ruler

Abstract

Intervening sequences encoding introns or inteins are transferred laterally between homologous alleles in a mechanism termed ‘homing’. Homing endonucleases are the class of enzymes that trigger homing by catalyzing double-strand DNA breaks at target sequences. Over 250 homing endonucleases have been identified; they are encoded by open reading frames (ORFs) within the mobile intervening sequences.In contrast to restriction endonucleases, which typically function as dimers and cleave palindromic DNA sequences of 4–8 bp, homing endonucleases are small monomeric proteins that recognize longer more variable sequences. For example, the homing endonuclease I-TevI, encoded by the td intron of phage T4, is a 28 kD protein that recognizes a target site of 37 bp. Double-strand cleavage occurs 23–25 bp upstream from this homing site. I-TevI contains two functionally distinct domains, an N-terminal catalytic domain and a C-terminal binding domain, joined by a long flexible linker. Limited insertions and deletions between the recognition sequence and cleavage site are tolerated, indicating that the linker extends and retracts to locate the cleavage site. A crystal structure of the DNA-binding domain bound to its target sequence revealed an unexpected Cys-4 zinc finger subdomain. As demonstrated by Marlene Belfort and colleagues, this zinc finger acts as a molecular ruler between the homing and cleavage sites on the target DNA [1xZinc finger as distance determinant in the flexible linker of intron endonuclease I-TevI. Dean, A.B. et al. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8554–8561Crossref | PubMed | Scopus (37)See all References][1].Although previously classified as part of the C-terminal binding domain, the zinc finger is dispensable for binding. To determine its contribution to endonuclease function, Belfort et al. constructed mutants of I-TevI in which the zinc finger was deleted or zinc chelation impaired by substituting one or more cysteines with alanine. DNA variants were also constructed with insertions or deletions between the homing and cleavage sites. The wild-type protein compensates for small insertions or deletions, but primarily cleaves the DNA a fixed distance from the homing site. By contrast, protein variants lacking an intact zinc finger cleave at the wild-type DNA sequence with less regard for distance. The protein does not simply use linker length to determine cleavage position. A mutant lacking the entire zinc finger (a 19 aa deletion) cleaves at the wild-type sequence even when it is further from the binding site owing to nucleotide insertions; that is, the linker ‘stretches out’ to find the preferred cleavage sequence. In the presence of the zinc finger, distance dominates, but without this motif, sequence overrides distance. The authors present a model in which the zinc finger organizes the linker between the catalytic and binding domains of the endonuclease.Zinc fingers typically bind in the major groove of DNA, as in the sequential Cys-2/His-2 zinc fingers of Zif268. The zinc finger of I-TevI, however, does not contribute significantly to binding energy. Indeed, the major groove of T4 DNA is effectively blocked by glycosylation of 5-hydroxymethylcytosine. Zinc fingers also mediate protein–protein interactions, although, to date, there is no evidence that other proteins are required for endonuclease activity. This function of a zinc finger as a distance determinant appears novel even among closely related homing endonucleases; it will be interesting to see whether zinc fingers exist in such a role in other modular proteins.