ALTERATION OF ZIF268 ZINC-FINGER MOTIFS GIVES RISE TO NONNATIVE ZINC-CO-ORDINATION SITES BUT PRESERVES WILD-TYPE DNA RECOGNITION

Zinc fingers are among the major structural motifs found in proteins t hat are involved in eukaryotic gene regulation. Many of these zinc-fin ger domains are involved in DNA binding. This study investigated wheth er the zinc-co-ordinating (Cys)(2)(His)(2) motif found in the three zi nc fingers of zif268 could be replaced by a (Cys)(4) motif while still preserving DNA recognition. (Cys)(2)(His)(2)-to-(Cys)(4) mutations we re generated in each of the three zinc fingers of zif268 individually, as well as in fingers 1 and 3, and fingers 2 and 3 together. Whereas finger 1 and finger 3 tolerate the switch, such an alteration in finge r 2 renders the polypeptide incapable of DNA recognition. The protein- DNA interaction was examined in greater detail by using a methylation- interference assay. The mutant polypeptides containing the (Cys), moti f in fingers 1 or 3 recognize DNA in a manner identical to the wild-ty pe protein, suggesting that the (Cys), motif appears to give rise to a properly folded finger. Additional results indicate that a zif268 var iant containing a (Cys)(2)(His)(Ala) arrangement in finger 1 is also c apable of DNA recognition in a manner identical to the wild-type polyp eptide. This appears to be the first time that such alterations, in th e context of an intact DNA-binding domain, have still allowed for spec ific DNA recognition. Taken together, the work presented here enhances our understanding of the relationship between metal ligation and DNA- binding by zinc fingers.