Single-molecule imaging of genome maintenance proteins encountering specific DNA sequences and structures

DNA repair pathways are tightly regulated processes that recognize specific hallmarks of DNA damage and coordinate lesion repair through discrete mechanisms, all within the context of a three-dimensional chromatin landscape. Dysregulation or malfunction of any one of the protein constituents in these pathways can contribute to aging and a variety of diseases. While the collective action of these many proteins is what drives DNA repair on the organismal scale, it is the interactions between individual proteins and DNA that facilitate each step of these pathways. In much the same way that ensemble biochemical techniques have characterized the various steps of DNA repair pathways, single-molecule imaging (SMI) approaches zoom in further, characterizing the individual protein-DNA interactions that compose each pathway step. SMI techniques offer the high resolving power needed to characterize the molecular structure and functional dynamics of individual biological interactions on the nanoscale. In this review, we highlight how our lab has used SMI techniques - traditional atomic force microscopy (AFM) imaging in air, high-speed AFM (HS-AFM) in liquids, and the DNA tightrope assay - over the past decade to study protein-nucleic acid interactions involved in DNA repair, mitochondrial DNA replication, and telomere maintenance. We discuss how DNA substrates containing specific DNA sequences or structures that emulate DNA repair intermediates or telomeres were generated and validated. For each highlighted project, we discuss novel findings made possible by the spatial and temporal resolution offered by these SMI techniques and unique DNA substrates.