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The roles of certain amino acids within zinc finger peptides of the Cys2His2
type in determining DNA-binding site specificity were investigated. A variety of
three domain peptides designed using a consensus sequence framework were
prepared with different potential DNA-contacting residues and tested in a
recently developed specificity determination assay. Proteins with recognition
helix sequence Q-1S1S2N3L4Q5K6 and QSSDLQK prefer binding subsites 5'-GAA-3' and
5'-(G/T)C(A/G)-3', respectively. Changing the Ser residues in position 2 to Ala
in these proteins did not significantly affect the DNA binding site preferences,
indicating that Ser residues, which occur frequently in position 2 in known zinc
finger proteins are not, in general, responsible for determining binding site
preferences. Examination of proteins with recognition helices HSSNLQK and
ASSNLQK revealed considerable loss of binding site discrimination throughout the
binding subsite. These observations provide further evidence for the importance
of residues in positions -1 and 3 in determining DNA binding specificity.
Moreover, these results illustrate the effects of changes of one residue in the
recognition helix on binding site preferences throughout the recognition subsite.
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