DNA mismatches reveal widespread conformational penalties in protein-DNA recognition

Transcription-factor (TF) proteins recognize specific genomic sequences, despite an overwhelming excess of non-specific DNA, to regulate complex gene expression programs1-3. While there have been significant advances in understanding how DNA sequence and shape contribute to recognition, some fundamental aspects of protein-DNA binding remain poorly understood2,3. Many DNA-binding proteins induce changes in the DNA structure outside the intrinsic B-DNA envelope. How the energetic cost associated with distorting DNA contributes to recognition has proven difficult to study and measure experimentally because the distorted DNA structures exist as low-abundance conformations in the naked B-DNA ensemble4-10. Here, we use a novel high-throughput assay called SaMBA (Saturation Mismatch-Binding Assay) to investigate the role of DNA conformational penalties in TF-DNA recognition. The approach introduces mismatched base-pairs (i.e. mispairs) within TF binding sites to pre-induce a variety of DNA structural distortions much larger than those induced by changes in Watson-Crick sequence. Strikingly, while most mismatches either weakened TF binding (~70%) or had negligible effects (~20%), approximately 10% of mismatches increased binding and at least one mismatch was found that increased the binding affinity for each of 21 examined TFs. Mismatches also converted sites from the non-specific affinity range into specific sites, and high-affinity sites into \"super-sites\" stronger than any known canonical binding site. These findings reveal a complex binding landscape that cannot be explained based on DNA sequence alone. Analysis of crystal structures together with NMR and molecular dynamics simulations revealed that many of the mismatches that increase binding induce distortions similar to those induced by TF binding, thus pre-paying some of the energetic cost to deform the DNA. Our work indicates that conformational penalties are a major determinant of protein-DNA recognition, and reveals mechanisms by which mismatches can recruit TFs and thus modulate replication and repair activities in the cell11,12.