A Live-Cell Imaging Approach for Measuring DNA Replication Rates
Daniel Dovrat,
Danielle Dahan,
Shachar Sherman,
Ioannis Tsirkas,
Natalie Elia,
Amir Aharoni
Affiliations
Daniel Dovrat
Department of Life Sciences and the National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Be’er Sheva 8410501, Israel
Danielle Dahan
Department of Life Sciences and the National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Be’er Sheva 8410501, Israel
Shachar Sherman
Department of Life Sciences and the National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Be’er Sheva 8410501, Israel
Ioannis Tsirkas
Department of Life Sciences and the National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Be’er Sheva 8410501, Israel
Natalie Elia
Department of Life Sciences and the National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Be’er Sheva 8410501, Israel; Corresponding author
Amir Aharoni
Department of Life Sciences and the National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Be’er Sheva 8410501, Israel; Corresponding author
Summary: We describe a simple and direct approach to measure the progression of single DNA replication forks in living cells by monitoring two fluorescently labeled loci downstream of an origin of replication. We employ this approach to investigate the roles of several leading and lagging strand factors in overall replisome function and show that fork progression is strongly dependent on proper maturation of Okazaki fragments. We also demonstrate how related cellular phenotypes, such as cell-cycle progression and the dynamics of sister chromatid cohesion, may be simultaneously monitored and correlated to DNA replication at the single-cell level. : Dovrat et al. develop a simple approach based on live-cell imaging to measure the progression rates of single DNA replication forks at specific loci while simultaneously examining cell-cycle progression and sister chromatid cohesion. The rate of DNA replication is shown to be strongly dependent on full maturation of Okazaki fragments. Keywords: DNA replication, live-cell imaging, FROS, Okazaki fragment maturation, sister chromatid cohesion
