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Volume 272, Number 20, Issue of May 16, 1997 pp. 13084-13087
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Twin Hydroxymethyluracil-A Base Pair Steps Define the Binding Site for the DNA-bending Protein TF1

(Received for publication, February 12, 1997)

Anne Grove , Marxa L. Figueiredo , Aldo Galeone ^¶ , Luciano Mayol ^¶ and E. Peter Geiduschek

From the  Department of Biology and Center for Molecular Genetics, University of California, San Diego, La Jolla, California 92093-0634 and ^¶ Dipartimento di Chimica delle Sostanze Naturali, Università degli Studi di Napoli Federico II, Via Domenico Montesano, 49 80131 Napoli, Italy

The DNA-bending protein TF1 is the Bacillus subtilis bacteriophage SPO1-encoded homolog of the bacterial HU proteins and the Escherichia coli integration host factor. We recently proposed that TF1, which binds with high affinity (K_d was ~3 nM) to preferred sites within the hydroxymethyluracil (hmU)-containing phage genome, identifies its binding sites based on sequence-dependent DNA flexibility. Here, we show that two hmU-A base pair steps coinciding with two previously proposed sites of DNA distortion are critical for complex formation. The affinity of TF1 is reduced 10-fold when both of these hmU-A base pair steps are replaced with A-hmU, G-C, or C-G steps; only modest changes in affinity result when substitutions are made at other base pairs of the TF1 binding site. Replacement of all hmU residues with thymine decreases the affinity of TF1 greatly; remarkably, the high affinity is restored when the two hmU-A base pair steps corresponding to previously suggested sites of distortion are reintroduced into otherwise T-containing DNA. T-DNA constructs with 3-base bulges spaced apart by 9 base pairs 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 largely, if not entirely, on sequence-dependent DNA flexibility.

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