Spatiotemporal Visualization of DNA Replication Dynamics

Reinhart, Marius; Casas-Delucchi, Corella S.; Cardoso, M. Cristina

doi:10.1007/978-1-62703-526-2_15

Cited by 3 publications

(1 citation statement)

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“… 26 modified from Reinhart et al . 69 ( d ) HeLa Kyoto cells stably expressing mCherry-PCNA were labelled with modified nucleotides (20 μm EdU) for 15 min before fixation. Wide-field images of cells going through different S-phase stages show that, while the overall level of PCNA is rather constant, the total amount of incorporated nucleotides is clearly lower in cells going through early S-phase, indicating a lower synthesis rate.…”

Section: Figurementioning

confidence: 99%

3D replicon distributions arise from stochastic initiation and domino-like DNA replication progression

et al. 2016

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DNA replication dynamics in cells from higher eukaryotes follows very complex but highly efficient mechanisms. However, the principles behind initiation of potential replication origins and emergence of typical patterns of nuclear replication sites remain unclear. Here, we propose a comprehensive model of DNA replication in human cells that is based on stochastic, proximity-induced replication initiation. Critical model features are: spontaneous stochastic firing of individual origins in euchromatin and facultative heterochromatin, inhibition of firing at distances below the size of chromatin loops and a domino-like effect by which replication forks induce firing of nearby origins. The model reproduces the empirical temporal and chromatin-related properties of DNA replication in human cells. We advance the one-dimensional DNA replication model to a spatial model by taking into account chromatin folding in the nucleus, and we are able to reproduce the spatial and temporal characteristics of the replication foci distribution throughout S-phase.

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Section: Figurementioning

confidence: 99%

3D replicon distributions arise from stochastic initiation and domino-like DNA replication progression

et al. 2016

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High-Resolution Analysis of Mammalian DNA Replication Units

Chagin

Reinhart

Cardoso

2015

Methods in Molecular Biology

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Genomic DNA of a eukaryotic cell is replicated once during the S-phase of the cell cycle to precisely maintain the complete genetic information. In the course of S-phase, semiconservative DNA synthesis is sequentially initiated and performed at thousands of discrete patches of the DNA helix termed replicons. At any given moment of S-phase, multiple replicons are active in parallel in different parts of the genome. In the last decades, tools and methods to visualize DNA synthesis inside cells have been developed. Pulse labeling with nucleotides as well as detecting components of the replication machinery yielded an overall picture of multiple discrete sites of active DNA synthesis termed replication foci (RFi) and forming spatiotemporal patterns within the cell nucleus. Recent advances in fluorescence microscopy and digital imaging in combination with computational image analysis allow a comprehensive quantitative analysis of RFi and provide valuable insights into the organization of the genomic DNA replication process and also of the genome itself. In this chapter, we describe in detail protocols for the visualization and quantification of RFi at different levels of optical and physical resolution.

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