DNA deformability in linear and circular DNA: Implications for site-specific recognition

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Sequence-dependent DNA shape, and level of flexibility, is at the core of how DNA binding proteins recognize their binding sites. Protein binding to DNA is important for many aspects of DNA function as a means for storing and transmitting information in a cell. DNA flexibility is studied using both computational and experimental methods, but experimental studies testing computational predictions have lagged, leaving a big gap in our understanding. This study will provide an experimental dataset that will significantly expand our understanding of one type of DNA deformability, contributing to the overall efforts by the DNA mechanics community to rationally predict and map sequence-dependent DNA conformations and deformations. Specifically, the research targets sequence patterns that render DNA highly kinkable, leading to improved rules for recognizing such sites and their protein binding partners. As participants in the research activities, undergraduate and graduate students will be trained in a cross-disciplinary research environment. The PI will contribute to curriculum revisions leading to more quantitative skills for life science majors, and will develop biophysics-related lab activities to be integrated into the curriculum for physics majors. These efforts, together with biophysics courses the PI previously developed, will be integrated into a planned Biophysics major at the PI's institution. Longer range goals are to develop an interdisciplinary graduate program in Molecular and Cellular Biophysics. The PI will work with high school teachers to develop teaching modules at the growing interface between physics and biology, and host high school students in her lab in the summer. The PI will also mentor, through summer research activities, underrepresented students recruited through an outreach program at the PI's institution. This study brings together multifaceted approaches to elucidate the rules that govern DNA sequence-dependent shape and deformability, and to examine how these variations in DNA flexibility influence protein binding. This study will identify highly deformable DNA sequences using in vitro selection of random sequences that bind with high affinity to architectural IHF/HU family of DNA bending proteins that severely kink DNA at two sites. The sequence patterns that enable this high degree of kinking will be important for developing models of sequence-dependent DNA deformability that go well beyond the small base-step deformations described by harmonic potentials. The intrinsic DNA deformability of selected sequences will be investigated using laser temperature-jump spectroscopy, biochemical assays, NMR-probed base pair dynamics, and computational modeling of bending deformations. The researchers will (1) examine the correlation between protein-binding affinities and DNA deformation energies; (2) investigate how the variations in the binding affinities are reflected in the DNA-bending rates to form the complex; and (3) examine the potential for spontaneous bending/kinking of these DNA substrates by (i) measuring base-pair dynamics at kink sites using NMR approaches; (ii) probing their reactivity within the context of minicircles by biochemically detecting kinks and unpaired bases induced by severe bending; (iii) modeling structural dynamics of highly deformable sequences. This study will generate a comprehensive dataset of sequence patterns that underpin DNA dynamics and flexibility. Results from this project will be made available on the website http://ansari.lab.uic.edu/research/ .