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 sh own that its structure differs significantly from previously characterized classes of DNA-binding domains. Here we report biochemical studies of the D NA-binding properties of this peptide. Binding interference and binding sit e 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 s ynthetic oligonucleotides with single base analogues at each position in th e core sequence. Base analogues that altered the major groove structure red uced or eliminated Mrf-a binding when present in the second, third, and fou rth basepairs of the core sequence, but had little or no effect in the firs t and fifth positions. These results suggest that Mrf-a contacts both the m ajor and minor grooves of its target sequences. (C) 1999 Academic Press.