Single-Molecule Dynamics and Localization of DNA Repair Proteins in Cells
Direct observation of individual protein molecules in their native environment, at nanometer resolution, in a living cell, in motion is not only fascinating but also uniquely informative. Several recent major technological advances in genomic engineering, protein and synthetic fluorophore development, and light microscopy have dramatically increased the accessibility of this approach. This chapter describes the procedures for modifying endogenous genomic loci to producing fluorescently tagged proteins, their high-resolution visualization, and analysis of their dynamics in mammalian cells, using DNA repair proteins BRCA2 and RAD51 as an example.
|BRCA2, CRISPR/Cas9, Genetic engineering, Homologous recombination, Live-cell imaging, RAD51, Single-particle tracking, Super-resolution microscopy|
|Methods in Enzymology|
|Organisation||Erasmus MC: University Medical Center Rotterdam|
Paul, M.W, Zelensky, A, Wyman, C, & Kanaar, R. (2018). Single-Molecule Dynamics and Localization of DNA Repair Proteins in Cells. Methods in Enzymology. doi:10.1016/bs.mie.2017.11.015