Genome-wide analysis of DNA replication and DNA double strand breaks by TrAEL-seq

Abstract

Understanding the distribution of sites at which replication forks stall, and the ensuing fork processing events, requires genome-wide methods sensitive to both changes in replication fork structure and the formation of recombinogenic DNA ends. Here we describe Tr ansferase- A ctivated E nd L igation seq uencing (TrAEL-seq), a method that captures single stranded DNA 3’ ends genome-wide and with base pair resolution. TrAEL-seq labels DNA breaks, and profiles both stalled and processive replication forks in yeast and mammalian cells. Replication forks stalling at defined barriers and expressed genes are detected by TrAEL-seq with exceptional signal-to-noise, most likely through labelling of DNA 3’ ends exposed during fork reversal. TrAEL-seq also labels unperturbed processive replication forks to yield maps of replication fork direction 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 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 dmc1 Δ mutant. 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.

Stimulation of adaptive gene amplification by origin firing under replication fork constraint

“…We recently developed TrAEL-seq to detect replication fork stalling and replication intermediates (49). Unfortunately, TrAEL-seq is ineffective on copper-treated cells as copper-induced apoptotic fragments generate a high background (49,76) (example data is deposited at GEO GSE154811).…”

“…Plugs were equilibrated once in 100 μl 1x TdT buffer (NEB) for 30 min at room temperature, then incubated for 2 h at 37°C in 100 μl 1x TdT buffer containing 4 μl 10 mM ATP and 1 μl Terminal Transferase (NEB M0315L). Plugs were rinsed with 1 ml tris buffer (10 mM Tris HCl pH 8.0), equilibrated in 100 μl 1x T4 RNA ligase buffer (NEB) containing 40 μl 50% PEG 8000 for 1 hour at room temperature then incubated overnight at 25°C in 100 μl 1x T4 RNA ligase buffer (NEB) containing 40 μl 50% PEG 8000, 1 μl 10 pM/μl TrAEL-seq adaptor 1 (49) and 1 μl T4 RNA ligase 2 truncated KQ (NEB M0373L). Plugs were then rinsed with 1 ml tris buffer, transferred to 15 ml tubes and washed three times in 10 ml tris buffer with rocking at room temperature for 1-2 hours each, then washed again overnight under the same conditions.…”

“…3.5 μl NEBNext Ultra II End Prep buffer, 1 μl 1 ng/μl sonicated salmon sperm DNA (this is used as a carrier) and 1.5 μl NEBNext Ultra II End Prep enzyme were added and reaction incubated 30 min at room temperature and 30 min at 65°C. After cooling, 1.25 μl 10 pM/μl TrAEL-seq adaptor 2 (49), 0.5 μl NEBNext ligation enhancer and 15 μl NEBNext Ultra II ligation mix were added and incubated 30 min at room temperature. The reaction mix was removed and discarded and beads were rinsed with 500 μl wash buffer (5 mM Tris pH 8, 0.5 mM EDTA, 1 M NaCl) then washed twice with 1 ml wash buffer for 10 min on wheel at room temperature and once for 10 min with 1 ml 0.1x TE.…”

“…TrAEL-seq reads accumulate at sites of replication fork stalling, including at the endogenous GAL1 promoter when transcriptionally active, providing a measure of disruption to replication fork progression (49). GAL1 promoter activity is exceptionally strong under normal 2% galactose induction, leading to saturating levels of Sac3-and Thp1-independent CNV in 10 generations that probably result from direct collisions between RNA polymerase and the replication fork ( Supplementary Figure S3A).…”

“…Formation of a replication bubble at a replication origin creates two replication forks that move in opposite directions, and replication origins are therefore detectable as sites of sharp changes in replication fork direction. Replication fork direction is revealed by the polarity of TrAEL-seq reads (49), and TrAEL-seq data from BY4741 wild-type cells with 13 copies of CUP1 shows a change in polarity from negative to positive at the CUP1 origin indicative of ARS activity ( Figure 5A upper panel, highlighted in orange). This signal is weak compared to other ARS elements in the region and becomes undetectable in 3xCUP1 cells (Supplementary Figure 5A), consistent with the CUP1 ARS elements being functional but rarely active.…”