Single-Molecule Dynamics and Localization of DNA Repair Proteins in Cells

Maarten W Paul; Alex N Zelensky; Claire Wyman; Roland Kanaar

doi:10.1016/bs.mie.2017.11.015

Single-Molecule Dynamics and Localization of DNA Repair Proteins in Cells

Methods Enzymol. 2018:600:375-406. doi: 10.1016/bs.mie.2017.11.015. Epub 2018 Feb 1.

Authors

Maarten W Paul¹, Alex N Zelensky¹, Claire Wyman², Roland Kanaar³

Affiliations

¹ Department of Molecular Genetics, Cancer Genomics Center Netherlands, Erasmus University Medical Center, Rotterdam, The Netherlands.
² Department of Molecular Genetics, Cancer Genomics Center Netherlands, Erasmus University Medical Center, Rotterdam, The Netherlands; Department of Radiation Oncology, Erasmus University Medical Center, Rotterdam, The Netherlands.
³ Department of Molecular Genetics, Cancer Genomics Center Netherlands, Erasmus University Medical Center, Rotterdam, The Netherlands. Electronic address: r.kanaar@erasmusmc.nl.

PMID: 29458767
DOI: 10.1016/bs.mie.2017.11.015

Abstract

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.

Keywords: BRCA2; CRISPR/Cas9; Genetic engineering; Homologous recombination; Live-cell imaging; RAD51; Single-particle tracking; Super-resolution microscopy.

MeSH terms

Animals
BRCA2 Protein / analysis*
BRCA2 Protein / chemistry
BRCA2 Protein / metabolism
CRISPR-Cas Systems / genetics
Cell Culture Techniques / instrumentation
Cell Culture Techniques / methods*
Cells, Cultured
DNA Breaks, Double-Stranded
Fluorescence Recovery After Photobleaching / instrumentation
Fluorescence Recovery After Photobleaching / methods
Gene Editing / methods
Green Fluorescent Proteins / chemistry
Intravital Microscopy / instrumentation
Intravital Microscopy / methods*
Luminescent Agents / chemistry
Mice
Microscopy, Fluorescence / instrumentation
Microscopy, Fluorescence / methods
Mouse Embryonic Stem Cells
Protein Binding
Rad51 Recombinase / analysis*
Rad51 Recombinase / chemistry
Rad51 Recombinase / metabolism
Recombinational DNA Repair*
Single Molecule Imaging / instrumentation
Single Molecule Imaging / methods*

Substances

BRCA2 Protein
BRCA2 protein, mouse
Luminescent Agents
Green Fluorescent Proteins
Rad51 Recombinase
Rad51 protein, mouse