Conserved cysteine residues of Oct-2 POU domain confer sensitivity to oxidation but are dispensable for sequence-specific DNA binding

The POU family of proteins, including the Oct-2 transcription factor, is characterized by a highly conserved bipartite DNA binding domain containing a 'POU homeodomain', distantly related to homeodomains of other DNA binding proteins, and a 'POU specific' domain unique to this class of factors. Prompted by the finding that in vitro DNA binding by Oct-2 is reversibly inhibited by oxidation of the protein, we investigated the role of the cysteine residues in the POU domain. All POU homeodomains identified contain a cysteine in the helix 3 region presumed to contact DNA directly; many (including Oct-2) also contain a less-well conserved cysteine residue(s) in the POU specific domain. Replacement of these cysteines with serine residues rendered the DNA binding domain resistant to oxidation but did not appreciably change the binding to a canonical octamer sequence, suggesting that the conserved cysteine residues are not required for sequence-specific DNA contacts, but may be important for another function.