Resolving Individual Damage Sites in DNA with AFM using Reengineered Repair Proteins

Abstract

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.