An AFM Study of DNA Damage and Repair

本研究的目的是通过阐明这些过程与DNA纳米力学之间的关系，并通过使用原子力显微镜技术可视化DNA上的修复蛋白活性，进一步了解DNA损伤和修复。目标是：1)优化单分子DNA测量的AFM平台；2)检查单个DNA分子中的各种病变，并在AFM中通过DNA修复酶直接逆转它们；3)使用AFM成像和力谱可视化大肠杆菌错配修复反应。为了实现这些目标，AFM平台需要进行优化，以将力误差降低到单皮牛顿，并通过使用超小型悬臂技术加速图像采集。DNA修复蛋白如光解酶、内切酶和连接酶将被用作损伤标记物，并将在与DNA的复合物中通过AFM成像以定位和识别损伤位点。力谱测量将确定由紫外线和伽马辐射引起的各种类型DNA损伤的机械指纹，并将直接跟踪修复活动中存在的损伤逆转。该项目还将使用大肠杆菌修复活性、AFM成像和力谱技术来检测和可视化DNA错配修复反应。该项目将为工程学和生物学交叉领域的三名研究生提供令人兴奋的教育和研究机会，并可能开发出对生物学许多领域具有重要意义的新型超灵敏DNA损伤检测方法。

The purpose of this research is to further the understanding of DNA damage and repair by elucidating the relationship between these processes and DNA nanomechanics and by visualizing repair protein activities on DNA using atomic force microscopy techniques. The objectives are: 1) optimize the AFM platform for single-molecule DNA measurements 2) examine various lesions in individual DNA molecules and follow in the AFM their direct reversal by DNA repair enzymes 3) use AFM imaging and force spectroscopy to visualize the mismatch repair reaction of E. coli. To achieve these objectives the AFM platform needs to be optimized to reduce force errors to single piconewtons and to accelerate image acquisition by using the ultra-small cantilever technology. DNA repair proteins such as photolyases, endonucleases and ligases will be used as damage markers and will be imaged in complexes with DNA by AFM in order to locate and identify the damage sites. Force spectroscopy measurements will determine the mechanical fingerprints of various types of DNA damage caused by UV and gamma radiation and will directly follow damage reversal in the presence of the repair activities. This project will also examine and visualize the DNA mismatch repair reaction using E. coli repair activities and AFM imaging and force spectroscopy technologies. This project will provide an exciting education and research opportunity for three graduate students at the interface between engineering and biology and will likely develop new ultra sensitive assays for DNA damage detection that is of significance to many areas of biology.