Optimal Oct-2 affinity for an extended DNA site and the effect of GST fusion on site preference

The regulator of immunoglobulin expression Oct-2 and the related widely exp ressed transcription factor Oct-1 have been shown to interact with DNA sequ ences containing an "octamer" motif, ATGC(A)/(T)AAT. To better understand O ct-2 function we have used random oligonucleotide selection and competition assays to define the optimal recognition site for this protein. The select ed site contains an extended sequence that is remarkably similar to octamer -heptamer sequences found in immunoglobulin heavy-chain regulatory sequence s, and the affinity of Oct-2 for this site is at least 50-fold greater than for sites containing the octamer motif alone. Fusion to glutathione S-tran sferase, a widely used model for protein-DNA and protein-protein interactio n, does not alter the optimal Oct-2 recognition site, but inhibits Oct-2 PO U-domain dimerization, slows the dissociation rate of the GST-Oct-2/DNA com plex, and increases the relative importance of the heptamer domain for Oct- 2 binding. These data advance our ability to identify in vivo targets of PO U-factor regulation and also suggest that GST-fusion proteins should be use d with caution in DNA-binding studies. (C) 2001 Academic Press.