Target-site overlap

In a zinc finger protein, certain sequences of amino acid residues are able to recognise and bind to an extended target-site of four or even five nucleotides (Wolfe et al., 2001). When this occurs in a ZFP in which the three-nucleotide subsites are contiguous, one zinc finger interferes with the target-site of the zinc finger adjacent to it, a situation known as target-site overlap. For example, a zinc finger containing arginine at position -1 and aspartic acid at position 2 along its alpha-helix will recognise an extended sequence of four nucleotides of the sequence 5'-NNG(G/T)-3'. The hydrogen bond between Asp² and the N4 of either a cytosine or adenine base paired to the guanine or thymine, respectively defines these two nucleotides at the 3' position, defining a sequence that overlaps into the subsite of any zinc finger that may be attached N-terminally (Beerli et al., 1998; Segal et al., 1999).

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Target-site overlap limits the modularity of those zinc fingers which exhibit it, by restricting the number of situations to which they can be applied. If some of the zinc fingers are restricted in this way, then a larger repertoire is required to address the situations in which those zinc fingers cannot be used (Segal et al., 1999). Target-site overlap may also affect the selection of zinc fingers during by display, in cases where amino acids on a non-randomised finger, and the bases of its associated subsite, influence the binding of residues on the adjacent finger which contains the randomised residues. Indeed, attempts to derive zinc finger proteins targeting the 5'-(A/T)NN-3' family of sequences by site-directed mutagenesis of finger two of the C7 protein were unsuccessful due to the Asp² of the third finger of said protein (Dreier et al., 2005).

The extent to which target-site overlap occurs is largely unknown, with a variety of amino acids having shown involvement in such interactions (Wolfe et al., 2001). When interpreting the zinc finger repertoires presented by investigations using ZFP phage display, it is important to appreciate the effects that the rest of the zinc finger framework may have had in these selections (Segal et al., 1999). Since the problem only appears to occur in a limited number of cases, the issue is nullified in most situations in which there are a variety of suitable targets to choose from (Beerli and Barbas, 2002) and only becomes a real issue if binding to a specific DNA sequence is required (e.g. blocking binding by endogenous DNA-binding proteins).

References

• Beerli, R. R. and C. F. Barbas, 3rd (2002). "Engineering polydactyl zinc-finger transcription factors." Nature Biotechnology 20(2): 135-41.
• Beerli, R. R., D. J. Segal, B. Dreier and C. F. Barbas, 3rd (1998). "Toward controlling gene expression at will: specific regulation of the erbB-2/HER-2 promoter by using polydactyl zinc finger proteins constructed from modular building blocks." Proceedings of the National Academy of Sciences USA 95(25): 14628-33.
• Dreier, B., R. P. Fuller, D. J. Segal, C. V. Lund, P. Blancafort, A. Huber, B. Koksch and C. F. Barbas, 3rd (2005). "Development of zinc finger domains for recognition of the 5'-CNN-3' family DNA sequences and their use in the construction of artificial transcription factors." Journal of Biological Chemistry 280(42): 35588-97.
• Segal, D. J., B. Dreier, R. R. Beerli and C. F. Barbas, 3rd (1999). "Toward controlling gene expression at will: selection and design of zinc finger domains recognizing each of the 5'-GNN-3' DNA target sequences." Proceedings of the National Academy of Sciences USA 96(6): 2758-63.
• Wolfe, S. A., R. A. Grant, M. Elrod-Erickson and C. O. Pabo (2001). "Beyond the "recognition code": structures of two Cys2His2 zinc finger/TATA box complexes." Structure 9(8): 717-23.

See also

• Zinc finger chimera
• Zinc finger protein