An AFM Study of DNA Damage and Repair

本研究的目的是通过阐明DNA损伤和修复过程与DNA纳米力学之间的关系，以及利用原子力显微镜技术显示DNA上修复蛋白质的活动，来加深对DNA损伤和修复的理解。目标是：1)优化单分子DNA测量的AFM平台；2)检查单个DNA分子中的各种损伤，并在AFM中跟踪它们被DNA修复酶直接逆转的情况；3)使用AFM成像和力光谱来可视化大肠杆菌的错配修复反应。为了实现这些目标，需要对AFM平台进行优化，以将力误差减少到1皮牛顿，并利用超小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，并将被AFM成像在与DNA的复合体中，以定位和识别损伤位置。力光谱测量将确定紫外线和伽马辐射造成的各种类型DNA损伤的机械指纹，并将在修复活动存在的情况下直接进行损伤逆转。该项目还将使用大肠杆菌修复活动以及原子力显微镜成像和力光谱技术来检查和可视化DNA错配修复反应。该项目将为工程学和生物学之间的三名研究生提供一个令人兴奋的教育和研究机会，并可能开发新的超灵敏DNA损伤检测方法，这对生物学的许多领域都具有重要意义。

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.