Monitoring early S-phase origin firing and replication fork movement by sequencing nascent DNA from synchronized cells

Abstract

A better understanding of DNA replication initiation in human cells and how this process is altered upon DNA replication stress requires the ability to study origin firing genome wide. Previously described methods of mapping DNA replication origins in higher eukaryotes rely principally on fractionation of DNA fragments based on their size and, optionally, on the presence of ribonucleotides at their 5ʹ end. Here, we describe a protocol for EdUseq-HU, a method for mapping early S-phase replication origins. Cells, synchronized by mitotic shake-off, are released in medium containing 5-ethynyl-2′-deoxyuridine (EdU; to label nascent DNA) and hydroxyurea (HU; to limit fork progression after origin firing). After using click chemistry to tag the EdU label with a biotin conjugate that is cleavable under mild conditions, the nascent DNA is captured on streptavidin beads. One variant of EdUseq-HU allows mapping of DNA replication origins on the genome at a resolution of 10 kb, and a second variant monitors progression of replication forks. Using EdUseq-HU, the spatiotemporal program of DNA replication in human cell lines can be interrogated in <2 weeks. The protocol requires basic cell culture and molecular biology skills, as well as familiarity with the Perl programming language and the Linux operating system.

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Fig. 1: Outline of the protocol.
Fig. 2: Flow cytometry controls.
Fig. 3: Anticipated results.

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Acknowledgements

We thank present and past laboratory members for helpful discussions, N. Roggli for help with the graphics scripts and M. Docquier and the Genomics Platform of the University of Geneva for scientific input and for performing the high-throughput sequencing. This work was supported by grants from the European Commission (ONIDDAC) and the Swiss Science National Foundation.

Author information

T.D.H. and M.M. designed the experiments and wrote the paper; M.M. performed the experiments; T.D.H. wrote the computer scripts with contributions from M.M.

Correspondence to Thanos D. Halazonetis.

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Key reference using this protocol

Macheret, M. & Halazonetis, T. D. Nature 555, 112–116 (2018): https://doi.org/10.1038/nature25507

Integrated supplementary information

Supplementary Figure 1 Gating strategy.

Typical flow cytometry profiles of U2OS cells (with normal levels of cyclin E) that were released into medium containing EdU (25 µM) for 14 h after mitosis exit. The strategy used to analyse the flow cytometry profiles consists of first gating the cells based on their distribution in the forward scatter/ side scatter plot (gate A, left panel); then, a second gate is applied to eliminate cell doublets on the propidium iodide (PI) peak height versus integral plot (gate B, center-left panel). After gating, the DNA content histograms and the EdU versus DNA content density scatter plots of the cells are plotted.

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