andBiochemistry,SchoolofBiologicalSciences,UniversityofCalifornia,Irvine, Califoria 92697

Integration host factor (IHF) is a bacterial histone-like pro- tein whose primary biological role is to condense the bacterial nucleoid and to constrain DNA supercoils. It does so by binding in a sequence-independent manner throughout the genome. However, unlike other structurally related bacterial histone-like proteins, IHF has evolved a sequence-dependent, high affinity DNA-binding motif. The high affinity binding sites are impor- tant for the regulation of a wide range of cellular processes. A remarkable feature of IHF is that it employs an indirect readout mechanism to bind and wrap DNA at both the nonspecific and high affinity (sequence-dependent) DNA sites. In this study we assessed the contributions of pre-formed and protein-induced DNA conformations to the energetics of IHF binding. Binding energies determined experimentally were compared with ener- gies predicted for the IHF-induced deformation of the DNA helix (DNA deformation energy) in the IHF-DNA complex. Combinatorial sets of de novo DNA sequences were designed to systematically evaluate the influence of sequence-dependent structural characteristics of the conserved IHF recognition ele- ments of the consensus DNA sequence. We show that IHF rec- ognizes pre-formed conformational characteristics of the con- sensus DNA sequence at high affinity sites, whereas at all other sites relative affinity is determined by the deformational energy required for nearest-neighbor base pairs to adopt the DNA structure of the bound DNA-IHF complex. Site-specific DNA binding by regulatory proteins is a feature of the regulatory processes that maintain, expand, and express genetic information such as replication, recombination, trans- position, and transcription. The chemical and physical mecha- nisms that underlie sequence-specific recognition of regulatory elements by cognate DNA-binding proteins are typically clas- sified as direct versus indirect readout. The former refers pri- marily to hydrogen bonds between proteins and the unique extra-cyclic substituents at C-4 of pyrimidines, C-6 of purines, and N-7 of purines. These groups provide a base pair-specific pattern of hydrogen bond donors and acceptors in the major groove of DNA that can be directly read by a complementary pattern of amino acid side chain donors and acceptors. Indirect readout refers to recognition of aspects of DNA structure such as intrinsic curvature, topology of major and minor grooves, ordered water structures, local geometry of backbone phos- phates, and flexibility or deformability. Because both the local DNA structure and energy to deform DNA are themselves intrinsic sequence-dependent properties, the conserved sequences that distinguish binding sites necessarily include contributions from both direct and indirect mechanisms. Con- sequently, although the contribution from indirect mecha- nisms is expected to be significant in protein-DNA complexes that feature substantial DNA deformation, it has proven diffi- cult to evaluate these contributions quantitatively. A protein that relies exclusively, or primarily, on indirect readout would clearly be advantageous for this purpose.