[Pre-Print] Visualizing the Dynamics of DNA Replication and Repair at the Single-Molecule Molecule Level
October 11, 2022
[Pre-Print] Visualizing the Dynamics of DNA Replication and Repair at the Single-Molecule Molecule Level
Scott Berger1, Gheorghe Chistol1,2,*
1 Biophysics Graduate Program, 2 Chemical and Systems Biology Department, Cancer Biology Graduate Program, Stanford School of Medicine, Stanford CA 94305, * Correspondence should be addressed to chistol@stanford.edu
During cell division, the genome of each eukaryotic cell is copied by thousands of replisomes – large protein complexes consisting of several dozen proteins. Recent studies suggest that the eukaryotic replisome is much more dynamic than previously thought. To directly visualize replisome dynamics in a physiological context, we recently developed a single-molecule approach for imaging replication proteins in Xenopus egg extracts. These extracts contain all the soluble nuclear proteins and faithfully recapitulate DNA replication and repair in vitro, serving as a powerful platform for studying the mechanisms of genome maintenance. Here we present detailed protocols for conducting single-molecule experiments in nuclear egg extracts and preparing key reagents. This workflow can be easily adapted to visualize the dynamics and function of other proteins implicated in DNA replication and repair.
Key Words: single molecule imaging, high throughput imaging, TIRF microscopy, data analysis, DNA replication, DNA repair, Xenopus egg extract, antibody, immuno-depletion, fluorescent labeling, microfluidic flow cell, lambda DNA, PEG, GINS, Cdc45, Mcm2-7, CMG, replisome, DNA-protein crosslinks, KEHRMIT, PhADE, CIDER