Localized DNA flexibility contributes to target site selection by DNA-bending proteins.
Certain DNA-binding proteins function as architectural elements by bending DNA. We have studied the binding of three such proteins, the prokaryotic HU and integration host factor (IHF) and the eukaryotic HMG1, to DNA in which flexibility is enhanced by tandem mismatches and by substituting 5-hydroxymethyluracil (hmU) for thymine (T). IHF and HU have higher affinity for DNA with two 4-nt loops than for perfect duplex DNA with a sequence that corresponds to a binding site for the phage-encoded homolog, TF1. HU has a high affinity for DNA with 4-nt loops separated by 9 bp (Kd = 3.5 nM), with suboptimal binding for other loop separations. IHF-binding is optimal when 4-nt loops are 8 to 9 bp apart; optimal complex formation with DNA representing the specific IHF-binding site H' requires that loops do not disrupt the consensus sequence and that one 4-nt loop borders the dyad axis-proximal block of consensus sequence (Kd = 0.3 nM, approximately tenfold lower than for H' perfect duplex DNA). HMG1 also binds preferentially to DNA with loops. All three proteins bind more tightly to DNA in which thymine is replaced with hmU. IHF has a tenfold higher affinity for hmU-DNA without a consensus IHF site (Kd = 7.6 nM) than for the corresponding T-DNA but does exhibit site-selectivity in hmU-DNA; Kd = 0.6 nM for the hmU-containing version of H'. Tighter binding to hmU-DNA is consistent with greater flexibility, and the distinct influence of loop position on complex formation suggests that sequence-dependent variations in flexibility of duplex DNA play a significant role in target-site selection by these DNA-bending proteins.
Grove
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Department of Biology, University of California, San Diego 92093-0634, USA.
Galeone
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Mayol
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Geiduschek
E P
EP
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006609:High Mobility Group Proteins / Q000737:chemistry / Q000378:metabolism*; D034982:Integration Host Factors; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D006609:High Mobility Group Proteins; D034982:Integration Host Factors; D009838:Oligodeoxyribonucleotides; C040330:histone-like protein HU, bacteria; D004247:DNA