Prior to the introduction of high-throughput sequencing methods, genome-wide studies of DSB formation and processing were largely restricted to meiotic recombination, where frequent DSBs at well-defined sites can be stabilised either before or after end resection and mapped on microarrays. We have a detailed understanding of DSB repair pathways based on decades of research but much less understanding of which pathways are used in a given genomic context in response to particular types of damage. Transferase-Activated End Ligation sequencing TSS,ĭNA double-strand breaks (DSBs) can be caused by exogenous agents (e.g., ionising radiation), defective cellular processes (e.g., replication–transcription collisions or topoisomerase dysfunction), or intentionally by the cell (e.g., in meiosis or immunoglobulin recombination). Terminal deoxynucleotidyl transferase TrAEL-seq, The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Ĭompeting interests: The authors have declared that no competing interests exist. Numerical data is in S1– S7 Data and image in S1 Raw Images.įunding: JH was funded by the Wellcome Trust, JH, PRG and FK by the BBSRC, NK was funded by the MRC and Artios Pharma. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.ĭata Availability: All sequencing files are available from the GEO database (accession number(s) GSE154811. Received: JAccepted: FebruPublished: March 24, 2021Ĭopyright: © 2021 Kara et al. PLoS Biol 19(3):Īcademic Editor: Tanya Paull, The University of Texas at Austin, UNITED STATES Overall, TrAEL-seq provides a flexible and robust methodology with high sensitivity and resolution for studying DNA replication and repair, which will be of significant use in determining mechanisms of genome instability.Ĭitation: Kara N, Krueger F, Rugg-Gunn P, Houseley J (2021) Genome-wide analysis of DNA replication and DNA double-strand breaks using TrAEL-seq. The specificity of TrAEL-seq for DNA 3′ ends also allows accurate detection of double-strand break sites after the initiation of DNA end resection, which we demonstrate by genome-wide mapping of meiotic double-strand break hotspots in a dmc1Δ mutant that is competent for end resection but not strand invasion. Replication maps are similar to those obtained by Okazaki fragment sequencing however, TrAEL-seq is performed on asynchronous populations of wild-type cells without incorporation of labels, cell sorting, or biochemical purification of replication intermediates, rendering TrAEL-seq far simpler and more widely applicable than existing replication fork direction profiling methods. TrAEL-seq labels both DNA breaks and replication forks, providing genome-wide maps of replication fork progression and fork stalling sites in yeast and mammalian cells. Here, we describe Transferase- Activated End Ligation sequencing (TrAEL-seq), a method that captures single-stranded DNA 3′ ends genome-wide and with base pair resolution. Understanding the distribution of sites at which replication forks stall, and the ensuing fork processing events, requires genome-wide methods that profile replication fork position and the formation of recombinogenic DNA ends. Faithful replication of the entire genome requires replication forks to copy large contiguous tracts of DNA, and sites of persistent replication fork stalling present a major threat to genome stability.
0 Comments
Leave a Reply. |