Resolving Individual Damage Sites in DNA with AFM using Reengineered Repair Proteins
Resolving Individual Damage Sites in DNA with AFM using Reengineered Repair Proteins
复制标题
使用重新设计的修复蛋白通过 AFM 解决 DNA 中的单个损伤位点
DOI：
10.1016/j.bpj.2015.11.2653
复制
发表时间：
2016
影响因子：
3.4
通讯作者：
Marszalek, Piotr E.
中科院分区：
文献类型：
--
作者：
Fitzgibbon, Christopher J.;Josephs, Eric A.;Marszalek, Piotr E.
紫外线照射可以产生广泛的和化学异质性的损伤，这些损伤可以累积引起疾病，最明显的是黑色素瘤和其他类型的癌症。识别、映射和区分单个DNA分子上的离散损伤位点的能力可以为癌前病症和黑色素瘤的进展和发作提供新的诊断或见解。原子力显微镜（AFM）提供了一个理想的方法来映射个人的损伤位置，因为它产生真实的空间图像的单个DNA分子。然而，UV诱导的损伤和许多可用于标记这些位点的损伤结合蛋白不能直接通过AFM解析。在这里，我们在基因水平上重新设计了DNA损伤蛋白，当与受损DNA结合时，可以通过AFM成像观察到。使用T4核酸内切酶V，嘧啶二聚体特异性碱基切除修复蛋白太小，无法通过AFM成像，作为模型UV损伤修复蛋白，我们在C-末端引入了许多离散的I27结构域，这充分增加了其大小而不干扰天然活性。使用这些新的“结构标签”，我们目前正试图通过单分子AFM成像同时在线性和基因组DNA中观察多个损伤位置。这些结构标记物可以与许多损伤结合蛋白结合，靶向多种损伤位点，为单分子诊断提供了潜在的方法学。
UV irradiation can produce widespread and chemically heterogeneous damage that can accumulate to cause diseases, most notably melanoma and other types of cancers. The ability to identify, map and discriminate discrete damage sites on single DNA molecules can provide new diagnostics or insights into the progression and onset of precancerous conditions and melanoma. Atomic Force Microscopy (AFM) provides an ideal method to map individual damage locations because it produces real space images of single DNA molecules. However, UV-induced damage and many damage-binding proteins that could be used to label these sites are not directly resolvable by AFM. Here we have reengineered DNA damage proteins at the genetic level to be observable by AFM imaging when bound to damaged DNA. Using T4 Endonuclease V, a pyrimidine dimer-specific base excision repair protein too small to imaged by AFM, as a model UV damage repair protein we introduced a number of discrete I27 domains at the C-terminus, which sufficiently increases its size without interfering with native activity. Using these novel “structural labels”, we are currently trying to visualize multiple damage locations simultaneously in both linear and genomic DNA via single molecule AFM imaging. These structural labels which can be adapted to a number of damage-binding proteins to target a variety of damage sites provide a potential methodology for single molecule diagnostics.