Summary: We present CellTool, a stand-alone open source software with a Graphical User Interface for image analysis, optimized for measurement of time-lapse microscopy images. It combines data management, image processing, mathematical modeling and graphical presentation of data in a single package. Multiple image filters, segmentation and particle tracking algorithms, combined with direct visualization of the obtained results make CellTool an ideal application for rapid execution of complex tasks. In addition, the software allows for the fitting of the obtained results to predefined or custom mathematical models. Importantly, CellTool provides a platform for easy implementation of custom image analysis packages written on a variety of programing languages. Availability and Implementation: CellTool is a free software available for MS Windows OS under the terms of the GNU General Public License. Executables and source files, supplementary information and sample data sets are freely available for download at URL: https://dnarepair.bas.bg/software/CellTool/ Contact: danovski@bio21.bas.bg ; stoynov@bio21.bas.bg ; Supplementary information: Supplementary data are available at URL: https://dnarepair.bas.bg/software/CellTool/Program/CellTool_UserGuide.pdf

Replication forks encounter various impediments, which induce fork stalling and threaten genome stability, yet the precise dynamics of fork stalling and restart at the single-cell level remain elusive. Herein, we devise a live-cell microscopy-based approach to follow hydroxyurea-induced fork stalling and subsequent restart at 30 s resolution. We measure two distinct processes during fork stalling. One is rapid PCNA removal, which reflects the drop in DNA synthesis. The other is gradual RPA1 accumulation up to 2400 nt of ssDNA per fork despite an active intra-S checkpoint. Restoring the nucleotide pool enables a prompt restart without post-replicative ssDNA and a smooth cell cycle progression. ATR, but not ATM inhibition, accelerates hydroxyurea-induced RPA1 accumulation nine-fold, leading to RPA1 exhaustion within 20 min. Fork restart under ATR inhibition led to the persistence of ~600 nt ssDNA per fork after S-phase, which reached 2500 nt under ATR/ATM co-inhibition, with both scenarios leading to mitotic catastrophe. MRE11 inhibition had no effect on PCNA/RPA1 dynamics regardless of ATR activity. E3 ligase RAD18 was recruited at stalled replication forks in parallel to PCNA removal. Our results shed light on fork dynamics during nucleotide depletion and provide a valuable tool for interrogating the effects of replication stress-inducing anti-cancer agents.