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Investigation of energetics of sharp DNA bending

DNA急剧弯曲的能量学研究

基本信息

批准号：
10251343
负责人：
Harold D Kim
金额：
$ 28.59万
依托单位：
GEORGIA INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-03-06 至 2024-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10251343
关键词：
3-Dimensional Affect Affinity Base Pair Mismatch Base Pairing Base Sequence Binding Biological Assay Biological Models Biology Biophysics Cell physiology Cells Chromatin Loop Clustered Regularly Interspaced Short Palindromic Repeats Conflict (Psychology)Confusion Coupling Cyclization DNA DNA Binding DNA Sequence DNA Structure Defect Dependence Disease Entropy Enzyme Kinetics Enzymes Face Fluorescence Resonance Energy Transfer Freezing Funding Genes Genetic Carriers Genetic Code Genetic Transcription Genetic Variation Genome Genomics Geometry Goals Grain Individual Investigation Kinetics Laws Lead Length Ligation Light Link Major Groove Measurement Measures Mechanics Methods Minor Groove Mismatch Repair Modeling Molecular Conformation Monitor Mutation Paper Physics Polymers Predisposition Probability Process Property Proteins Proxy RNA Reaction Regulation Research Proposals Rotation Shapes Signal Transduction Site Statistical Mechanics Stress Structure Surface System Testing Thermodynamics Time base design genetic information human disease innovation insight mechanical behavior mechanical properties molecular dynamics novel novel strategies nuclease repaired single molecule single-molecule FRET tool transcription factor

项目摘要

Genetic information is carried by DNA, a polymeric molecule governed by the laws of physics. Strongly bent and twisted DNA is associated with many genomic processes including packaging, transcription, repair, and editing, which suggests that these processes are aided by the intrinsic deformability of DNA. Hence, altered deformability of DNA due to damage or mutation can perturb the regulatory state of the genome, thus increasing the susceptibility to disease. Understanding how deformability of DNA changes with base sequence can thus provide a missing link between genetic variation and cell physiology. DNA is a double helical ladder of base pair steps with the major and minor grooves. This groove asymmetry confers DNA with asymmetric bendability and bend- twist coupling, properties truly unique to DNA, but these properties have not been thoroughly investigated by experimental means. Extreme bending or twisting of a single base pair step can lead to large changes in the three-dimensional DNA conformation, but the thermodynamics and sequence dependence of extreme bendability and twistability remain largely unknown due to the lack of experimental methods. Deformed base pair steps will likely affect how enzymes and transcription factors interact with DNA, but testing this idea requires fine control of base-pair step deformation. The PI has investigated the thermodynamics of strong DNA bending by the combined use of short DNA with sticky ends and surface-based single-molecule assays. During the first funding period of this R01, looping and unlooping rates were measured from DNA molecules of different lengths and base sequences including mismatched bases. The results from these studies elucidated the kinetics of loop formation and helped us to design new approaches to quantifying asymmetric bendability and bend-twist coupling of DNA. Furthermore, they revealed DNA loop geometries that enable measurement of the bending and twist stiffness of individual base pair steps. Building upon these key results and insights from the first R01, this proposal will measure extreme deformability of DNA and investigate its consequence on the kinetics of a DNA targeting protein. The experimental approach is to combine singe-molecule FRET with small DNA loops of different geometries. Four specific aims are proposed: Aim 1, quantifying the bending asymmetry and bend-twist coupling of DNA; Aim 2, quantifying bending stiffness of mismatched base pairs at different bending angles; Aim 3, measuring coaxial stacking and unstacking rates of individual base pair steps; and Aim 4, measuring the reaction kinetics of Cas12, an RNA-guided DNA targeting protein of the CRISPR system, on curved and twisted DNA substrates. These studies will shed light on the generic mechanics-function relationship of DNA.

遗传信息由DNA携带，DNA是一种受物理定律支配的聚合分子。强烈弯曲