Abstract
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. 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. 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. 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. 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. 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. 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.
Highlights
DNA double-strand breaks (DSBs) can be caused by exogenous agents, defective cellular processes, or intentionally by the cell [1,2,3]
TrAEL-seq adaptor 1 is a hairpin that primes conversion of single-stranded ligation products to double-stranded DNA suitable for library construction, incorporates a biotin moiety flanked by deoxyuracil residues that allows selective purification and elution of ligation products, and includes an 8-nucleotide unique molecular identifier (UMI) for bioinformatic removal of PCR duplicates (Fig 1A)
The 30 ends detected by TrAEL-seq correspond to the leading rather than the lagging strand, despite the fact that many more 30 ends occur on the lagging strand, and we suggest that these 30 ends are exposed by replication fork reversal occurring either in vivo or during sample processing
Summary
DNA 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) [1,2,3]. We have a detailed understanding of DSB repair pathways based on decades of research [4,5,6] but much less understanding of which pathways are used in a given genomic context in response to particular types of damage. Prior to the introduction of high-throughput sequencing methods, genome-wide studies of DSB formation and processing were largely restricted to meiotic recombination, where. Analysis of replication and double strand breaks by TrAEL-seq
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