engleski

Analysis of Replication Fork Progression by Elongation Assay and 3D Microscopy

During S-phase, a typical eukaryotic cell has to replicate all of its genome once and only once. Given that replication occurs from multiple sites, replication from these sites has to be coordinated and controlled. The labelling with thymidine analogues enabled to visualize activation of origins of replication and revealed distinct replication patterns, which reflect a defined spatiotemporal organization. In early S-phase nuclei of mammalian cells, hundreds of replication foci corresponding to numerous forks progress synchronously. To monitor fork progression, elongation assays have been developed using labelling with two subsequent nucleotide pulses, which are separated by increasing chase times. In this way, a decrease of co-localization of the corresponding signal can provide a convenient measure of fork progression. We have developed tools and methods to exploit this approach and will present here a comprehensive protocol for an elongation assay in mammalian cells. In particular, we will present our immunodetection assay to reveal replication foci labelled with dNTP analogues. We will discuss the methods of choice for image acquisition and processing, as well as for co-localization analysis of replication foci. Given that noise due to background and the high density of foci preclude commonly used intensity-based analysis methods, we propose a novel object-based method to evaluate co-localization of replication foci, using fully processed 3D image stacks. The complete and user-friendly approach for the quantification of fork progression in early S-phase replicating nuclei at the level of a single cell should prove useful to address the impact of components of the replication machinery, nuclear and chromatin environment or DNA damage on DNA replication.

DNA replication; fork progression; elongation assay; mammalian cells; 3D microscopy

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