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

序列依赖的DNA形状和灵活性水平是DNA结合蛋白如何识别其结合位点的核心。蛋白质与DNA的结合对于DNA作为细胞中储存和传递信息的手段的功能的许多方面是重要的。 DNA的灵活性是使用计算和实验方法来研究的，但是测试计算预测的实验研究已经滞后，在我们的理解中留下了很大的差距。 这项研究将提供一个实验数据集，将显着扩大我们对一种类型的DNA变形能力的理解，有助于DNA力学界的整体努力，合理地预测和映射序列依赖的DNA构象和变形。具体来说，这项研究的目标是使DNA高度扭结的序列模式，从而改进了识别这些位点及其蛋白质结合伴侣的规则。作为研究活动的参与者，本科生和研究生将在跨学科的研究环境中接受培训。PI将有助于课程修订，从而为生命科学专业提供更多的定量技能，并将开发与生物制药相关的实验室活动，以融入物理专业的课程。这些努力，连同PI以前开发的生物物理学课程，将被整合到PI机构计划的生物物理学专业。更长远的目标是在分子和细胞生物物理学发展一个跨学科的研究生课程。PI将与高中教师合作，在物理学和生物学之间不断增长的界面上开发教学模块，并在夏天在她的实验室里接待高中学生。PI还将通过夏季研究活动指导通过PI机构的外展计划招募的代表性不足的学生。这项研究汇集了多方面的方法来阐明管理DNA序列依赖的形状和变形性的规则，并研究DNA灵活性的这些变化如何影响蛋白质结合。本研究将使用体外选择的随机序列鉴定高度可变形的DNA序列，所述随机序列以高亲和力结合DNA弯曲蛋白的结构IHF/HU家族，所述DNA弯曲蛋白在两个位点严重地扭结DNA。序列模式，使这种高度的扭结将是重要的序列依赖性DNA变形模型的发展远远超出了小的基本步骤的变形所描述的谐波电位。所选序列的内在DNA变形性将使用激光温度跳跃光谱，生化分析，NMR探测的碱基对动力学和弯曲变形的计算建模进行研究。研究人员将（1）检查蛋白质结合亲和力和DNA变形能之间的相关性;（2）研究结合亲和力的变化如何反映在DNA弯曲率中以形成复合物;（3）通过（i）使用NMR方法测量扭结位点的碱基对动力学来检查这些DNA底物自发弯曲/扭结的可能性;（ii）通过生物化学检测严重弯曲引起的扭结和不成对碱基来探测它们在小环背景下的反应性;（iii）对高度可变形序列的结构动力学进行建模。这项研究将产生一个全面的序列模式数据集，支持DNA动态和灵活性。该项目的结果将在网站http://ansari.lab.uic.edu/research/上公布。

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/ .