TWIN HYDROXYMETHYLURACIL-A BASE-PAIR STEPS DEFINE THE BINDING-SITE FOR THE DNA-BENDING PROTEIN TF1

The DNA bending protein TF1 is the Bacillus subtilis bacteriophage SPO 1-encoded homolog of the bacterial HU proteins and the Escherichia col i integration host factor, We recently proposed that TF1, which binds with high affinity (K-d was similar to 3 nM) to preferred sites within the hydroxymethyluracil (hmU)-containing phage genome, identifies its binding sites based on sequence-dependent DNA flexibility, Here, we s how that two hmU-A base pair steps coinciding with two previously prop osed sites of DNA distortion are critical for complex formation, The a ffinity of TF1 is reduced 10-fold when both of these hmU-A base pair s teps are replaced with A-hmU, G-C, or C-G steps; only modest changes i n affinity result when substitutions are made at other base pairs of t he TP1 binding site, Replacement of all hmU residues with thymine decr eases the affinity of TF1 greatly; remarkably, the high affinity is re stored when the two hmU-A base pair steps corresponding to previously suggested sites of distortion are reintroduced into otherwise T-contai ning DNA. T-DNA constructs with 3-base bulges spaced apart by 9 base p airs of duplex also generate nM affinity of TF1. We suggest that twin hmU-A base pair steps located at the proposed sites of distortion are key to target site selection by TF1 and that recognition is based larg ely, if not entirely, on sequence-dependent DNA flexibility.