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

• Published in: Nature protocols Vol. 14; no. 1; pp. 51 - 67
• Main Authors: Macheret, Morgane, Halazonetis, Thanos D
• Format: Journal Article
• Language: English
• Published: England Nature Publishing Group 01.01.2019
• Subjects: Beads; Biological research; Biotin; Cell culture; Cell lines; Cell physiology; Chemical synthesis; Deoxyribonucleic acid; DNA; DNA biosynthesis; DNA replication; DNA sequencing; Eukaryotes; Familiarity; Firing; Fractionation; Gene mapping; Genomes; High-throughput screening (Biochemical assaying); Hydroxyurea; Mapping; Methods; Molecular biology; Organic chemistry; Origins; Perl; Programming languages; Replication; Replication forks; Replication initiation; Replication origins; Ribonucleotides; Streptavidin; Yeast; Switzerland
• ISSN: 1754-2189; 1750-2799
• DOI: 10.1038/s41596-018-0081-y

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 5' 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.