DNA damage recognition in linear and supercoiled DNA

我们细胞中的特殊蛋白质已经进化到通过识别和修复由环境污染物、太阳紫外线或DNA复制时引入的错配引起的DNA损伤来维持基因组完整性。这些蛋白质需要快速扫描DNA，但在潜在的“麻烦点”慢下来，以识别损伤，开始修复。这个过程是复杂的，因为细胞内的DNA是环状和超螺旋的（解开或过度缠绕）。该项目旨在揭示DNA循环和超螺旋如何影响DNA修复蛋白与受损DNA的相互作用。这些研究中开发的发现和技术将为理解其他蛋白质（如调节基因表达的蛋白质）如何与超螺旋DNA结合打开大门。该项目将通过为不同的本科生和研究生提供参与跨学科前沿研究的机会来产生教育影响;开发生物物理学跨学科本科课程;与高中教师合作开发物理学和生物学之间接口的教学模块;并在夏季在实验室接待高中生。公众和外展活动将包括每周一次的“星期六早晨科学”，让高中生谈论科学。缺乏对DNA损伤识别的分子水平的理解，在很大程度上是因为在与询问和识别相关的微米至毫秒的时间尺度上测量蛋白质-DNA构象动力学是困难的，因此很少。一些证据表明，损伤感应蛋白感知局部DNA波动和变形能力的差异，以区分受损部位和未受损部位。然而，表征DNA动力学和灵活性一直是该领域的主要挑战。关于DNA拓扑结构（循环和超螺旋）如何影响这些动力学的研究更是凤毛麟角。使用创新的荧光方法，包括激光温度跳变，荧光寿命为基础的FRET，荧光相关光谱，该项目将建立在PI实验室以前的研究，揭示了蛋白质-DNA构象动力学的损伤识别途径Rad 4，酵母直向同源物着色性干皮病C蛋白在核苷酸切除修复途径。错配修复途径中MutS蛋白的错配识别研究将测量不同错配的DNA动力学，可视化MutS如何与错配位点结合，并确定不同错配的错配询问/识别率是否与MutS介导的修复效率相关。用受控的超螺旋度设计的DNA微环将用于研究成环和超螺旋对DNA波动的影响，以及对Rad 4/MutS损伤识别的热力学和动力学的影响。 这些研究的极高灵敏度和时间分辨率将能够测量Rad 4/MutS介导的扭结、解旋、在错配位点形成识别复合物的核苷酸翻转动力学，揭示当DNA超螺旋时这些动力学是如何改变的，并为内在DNA波动如何停滞和传递“遇险信号”以破坏传感蛋白提供答案。该项目是共同的，该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Special proteins in our cells have evolved to maintain genome integrity by recognizing and repairing damage in DNA caused by environmental pollutants, UV-rays from the sun, or mismatches introduced when DNA is replicated. These proteins need to rapidly scan DNA and yet slow down at potential “trouble spots” to recognize damage to begin repair. This process is complicated by the fact that DNA inside cells is looped and supercoiled (unwound or overwound). This project aims to uncover how DNA looping and supercoiling impact the interactions of DNA repair proteins with damaged DNA. The findings and the technologies developed in these studies will open doors for understanding how other proteins, such as those that regulate gene expression, engage with supercoiled DNA. The project will have educational impacts by providing opportunities for diverse undergraduate and graduate students to take part in cross-disciplinary cutting-edge research; developing an interdisciplinary undergraduate program in biophysics; engaging with high school teachers to develop teaching modules at the interface between physics and biology; and hosting high school students in their laboratories in the summer. Public and outreach activities will include a weekly “Saturday Morning Science” to bring in high school students to talk about science. A molecular-level understanding of DNA damage recognition is lacking, in large part because measurements of protein-DNA conformational dynamics on micro-to-milliseconds timescales relevant for interrogation and recognition are difficult and, thus, rare. Several lines of evidence suggest that damage sensing proteins sense differences in local DNA fluctuations and deformability to distinguish damaged from undamaged sites. Characterizing DNA dynamics and flexibility, however, has been a major challenge in the field. Even rarer are studies on how DNA topology (looping and supercoiling) – conserved across the domains of life – influence these dynamics. Using innovative fluorescence approaches that include laser temperature-jump, fluorescence lifetime-based FRET, and fluorescence correlation spectroscopy, this project will build on previous studies in the PI’s laboratory that unveiled protein-DNA conformational dynamics en route to damage recognition by Rad4, yeast ortholog of xeroderma pigmentosum C protein in the nucleotide excision repair pathway. Studies for mismatch recognition by MutS protein in the mismatch repair pathway will measure DNA dynamics for different mismatches, visualize how MutS engages with the mismatched sites, and ascertain whether mismatch interrogation/recognition rates for different mismatches correlate with MutS-mediated repair efficiencies. DNA minicircles designed with controlled superhelicity will be used to investigate the effect of looping and supercoiling on DNA fluctuations and what impact that has on the thermodynamics and kinetics of damage recognition by Rad4/MutS. The exquisitely high sensitivity and temporal resolution of these studies will enable measurements of Rad4/MutS-mediated kinking, unwinding, nucleotide-flipping kinetics at mismatched sites to form the recognition complex, shed light on how these dynamics are altered when DNA is supercoiled, and provide answers to how intrinsic DNA fluctuations stall and communicate “distress signals” to damage sensing proteins.This project is co-funded by the Genetic Mechanisms and the Molecular Biophysics Programs in the Division of Molecular and Cellular Biosciences in the Directorate for Biological Sciences.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.