The novel Mrf-2 DNA-binding domain recognizes a five-base core sequence through major and minor-groove contacts

Recent NMR studies of the purified Mrf-a DNA-binding domain peptide have shown that its structure differs significantly from previously characterized classes of DNA-binding domains. Here we report biochemical studies of the DNA-binding properties of this peptide. Binding interference and binding site selection assays indicated that Mrf-2 requires the core sequence AATA(C/T) for high affinity binding. Kinetic analyses of several selected sequences indicated that the core sequence alone is not sufficient for high affinity binding, however. Kinetic analyses were also performed using a series of synthetic oligonucleotides with single base analogues at each position in the core sequence. Base analogues that altered the major groove structure reduced or eliminated Mrf-a binding when present in the second, third, and fourth basepairs of the core sequence, but had little or no effect in the first and fifth positions. These results suggest that Mrf-a contacts both the major and minor grooves of its target sequences. (C) 1999 Academic Press.