A Quantitative DNA Fiber Assay to Monitor Replication Fork Progression, Protection, and Restart.

Our genome is duplicated during every round of cell division through the process of DNA replication, but this fundamental process is subjected to various stresses arising from endogenous or exogenous sources. Thus, studying replication dynamics is crucial for understanding the mechanisms underlying genome duplication in physiological and replication stress conditions. Earlier, radioisotope-based autoradiography and density-labeling methods were used to study replication dynamics, which were limited in spatial resolution, representing only average estimates from many DNA samples. Here, we describe a DNA fiber assay that utilizes different thymidine analog incorporation, like 5-chloro-2'-deoxyuridine (CldU) and 5-iodo-2'-deoxyuridine (IdU), into replicating DNA. Such labeled DNA can be stretched and fixed on silanized glass slides, which are denatured with mild acidic treatment to expose the labeled nascent DNA. This DNA can then be visualized by using primary antibodies against CldU and IdU, followed by fluorophore-conjugated secondary antibodies, and observing them using a fluorescence microscope. The DNA fiber assay allows the visualization of individually replicating DNA at a single-molecular resolution and is highly quantitative, high-throughput, and easily reproducible. This technique offers insights into different replication parameters, like rate of DNA synthesis, extent of reversed fork protection, restart of stalled forks, and fork asymmetry under untreated or replication stress conditions at a single-molecule level. Key features • Single-molecule resolution of DNA replication dynamics. • Diverse replication parameters can be quantified with variations of the labeling protocol. • Easily reproducible across different cell lines in any lab with a basic molecular biology setup. • Effective in studying the effects of different genotoxic treatments on replication.