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

Kara, Neesha; Krueger, Felix; Rugg‐Gunn, Peter J.; Houseley, Jonathan

doi:10.1101/2020.08.10.243931

Cited by 9 publications

(32 citation statements)

References 84 publications

(107 reference statements)

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“…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).…”

Section: Resultsmentioning

confidence: 99%

“…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.…”

Section: Methodsmentioning

confidence: 99%

“…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.…”

Section: Methodsmentioning

confidence: 99%

“…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).…”

Section: Homologous Recombination Occurs At Cup1 Without Extensive Fomentioning

confidence: 99%

“…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.…”

Section: Late Firing Replication Origins Rather Than Late Replicationmentioning

confidence: 99%

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

Aj¹,

King²,

Rm³

et al. 2021

Preprint

Self Cite

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Adaptive mutations can cause drug resistance in cancers and pathogens, and increase the tolerance of agricultural pests and diseases to chemical treatment. When and how adaptive mutations form is often hard to discern, but we have shown that adaptive copy number amplification of the copper resistance gene CUP1 occurs in response to environmental copper due to CUP1 transcriptional activation. Here we dissect the mechanism by which CUP1 transcription in budding yeast stimulates copy number variation (CNV). We show that transcriptionally stimulated CNV requires TREX-2 and Mediator, such that cells lacking TREX-2 or Mediator respond normally to copper but cannot acquire increased resistance. Mediator and TREX-2 cause replication stress by tethering transcribed loci to nuclear pores, a process known as gene gating, and transcription at the CUP1 locus causes a TREX-2-dependent accumulation of replication forks indicative of replication fork stalling. TREX-2-dependent CUP1 gene amplification occurs by a Rad52 and Rad51-mediated homologous recombination mechanism that is enhanced by histone H3K56 acetylation and repressed by Pol32, factors known to alter the frequency of template switching during break induced replication (BIR). CUP1 amplification is also critically dependent on late firing replication origins present in the CUP1 repeats, and mutations that remove or inactivate these origins strongly suppress the acquisition of copper resistance. We propose that replicative stress imposed by nuclear pore association causes replication bubbles from these origins to collapse soon after firing, leaving an epigenetic scar of H3K56 acetylation that promotes template switching during later break induced replication events. The capacity for inefficient replication origins to promote copy number variation renders certain genomic regions more fragile than others, and therefore more likely to undergo adaptive evolution through de novo gene amplification.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Homologous Recombination Occurs At Cup1 Without Extensive Fomentioning

confidence: 99%

Section: Late Firing Replication Origins Rather Than Late Replicationmentioning

confidence: 99%

See 3 more Smart Citations

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

Aj¹,

King²,

Rm³

et al. 2021

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

Transcription as source of genetic heterogeneity in budding yeast

Piguet,

Houseley

2024

Yeast

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Transcription presents challenges to genome stability both directly, by altering genome topology and exposing single‐stranded DNA to chemical insults and nucleases, and indirectly by introducing obstacles to the DNA replication machinery. Such obstacles include the RNA polymerase holoenzyme itself, DNA‐bound regulatory factors, G‐quadruplexes and RNA‐DNA hybrid structures known as R‐loops. Here, we review the detrimental impacts of transcription on genome stability in budding yeast, as well as the mitigating effects of transcription‐coupled nucleotide excision repair and of systems that maintain DNA replication fork processivity and integrity. Interactions between DNA replication and transcription have particular potential to induce mutation and structural variation, but we conclude that such interactions must have only minor effects on DNA replication by the replisome with little if any direct mutagenic outcome. However, transcription can significantly impair the fidelity of replication fork rescue mechanisms, particularly Break Induced Replication, which is used to restart collapsed replication forks when other means fail. This leads to de novo mutations, structural variation and extrachromosomal circular DNA formation that contribute to genetic heterogeneity, but only under particular conditions and in particular genetic contexts, ensuring that the bulk of the genome remains extremely stable despite the seemingly frequent interactions between transcription and DNA replication.

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

Whale

King

Hull

et al. 2022

Nucleic Acids Research

Self Cite

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Adaptive mutations can cause drug resistance in cancers and pathogens, and increase the tolerance of agricultural pests and diseases to chemical treatment. When and how adaptive mutations form is often hard to discern, but we have shown that adaptive copy number amplification of the copper resistance gene CUP1 occurs in response to environmental copper due to CUP1 transcriptional activation. Here we dissect the mechanism by which CUP1 transcription in budding yeast stimulates copy number variation (CNV). We show that transcriptionally stimulated CNV requires TREX-2 and Mediator, such that cells lacking TREX-2 or Mediator respond normally to copper but cannot acquire increased resistance. Mediator and TREX-2 can cause replication stress by tethering transcribed loci to nuclear pores, a process known as gene gating, and transcription at the CUP1 locus causes a TREX-2-dependent accumulation of replication forks indicative of replication fork stalling. TREX-2-dependent CUP1 gene amplification occurs by a Rad52 and Rad51-mediated homologous recombination mechanism that is enhanced by histone H3K56 acetylation and repressed by Pol32 and Pif1. CUP1 amplification is also critically dependent on late-firing replication origins present in the CUP1 repeats, and mutations that remove or inactivate these origins strongly suppress the acquisition of copper resistance. We propose that replicative stress imposed by nuclear pore association causes replication bubbles from these origins to collapse soon after activation, leaving a tract of H3K56-acetylated chromatin that promotes secondary recombination events during elongation after replication fork re-start events. The capacity for inefficient replication origins to promote copy number variation renders certain genomic regions more fragile than others, and therefore more likely to undergo adaptive evolution through de novo gene amplification.

show abstract

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

Cited by 9 publications

References 84 publications

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

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

Transcription as source of genetic heterogeneity in budding yeast

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

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