Different DNA contact schemes are used by two winged helix proteins to recognize a DNA binding sequence

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Abstract
The hepatocyte nuclear factor 3 (HNF-3)/fork head (fkh) family contains a large number of transcription factors which recognize divergent DNA sequences via a winged-helix binding motif. In this report we present studies on the DNA binding properties of winged-helix HNF-3/fkh homologues 1 (HFH-1) and 2 (HFH-2) which recognize a shared DNA binding site with different affinities. To explore how HFH-1 and HFH-2 proteins recognize this DNA binding sequence, the binding affinities of these two HFH proteins toward a series of DNA sites containing a single strand trimer loop insertion at different positions were studied. This insertion induces a bend of approximately 80 degrees in the DNA binding site (prebending). HFH-1 and HFH-2 were shown to recognize DNA sites prebent at many nucleotide positions on both strands of the DNA sequence. Both HFH-1 and HFH-2 were more sensitive to mismatch insertions on the sense strand of the DNA binding site, especially within the AAAATAAC sequence. Our data suggest that the recognition helix (helix 3) recognizes the AAAATAAC sequence and that the helix 3/DNA interaction results in bending of the DNA which narrows the major groove in the AAAATAAC sequence. Furthermore, the binding affinities of HFH-1 and HFH-2 toward DNA binding sites with base-pair reversion in the AAAATAAC sequence was also investigated. Different patterns of response from HFH-1 and HFH-2 to both prebent and base-pair reverted binding sites was observed. Our results demonstrate that even two highly conserved members of the winged-helix family may contact the same DNA sequence differently.