Monitoring 5′-End Resection at Site-Specific Double-Strand Breaks by Southern Blot Analysis

Peng, Haoyang; Zhang, Simin; Chen, Xuefeng

doi:10.1007/978-1-0716-0868-5_20

Cited by 8 publications

(5 citation statements)

References 25 publications

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“…We first tested whether the HO-induced DSB is efficiently resected into 3' ssDNA tails in late mitosis. Resection is necessary to form the protruding 3' nucleofilaments that invade the donor homologous sequence to restore the break by HR (Peng et al, 2021;Symington, 2016).…”

Section: Resultsmentioning

confidence: 99%

“…We first tested whether the HO-induced DSB is efficiently resected into 3’ ssDNA tails in late mitosis. Resection is necessary to form the protruding 3’ nucleofilaments that invade the donor homologous sequence to restore the break by HR (Peng et al ., 2021; Symington, 2016). To confirm and quantitate the formation of ssDNA flanking the HOcs , we used a qPCR approach whereby primers can only amplify a target DNA cut with the restriction enzyme StyI if it has been rendered single-stranded by resection (Fig 1a, b) (Gnügge et al ., 2018; Zierhut and Diffley, 2008).…”

Section: Resultsmentioning

confidence: 99%

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Cohesin still drives homologous recombination repair of DNA double-strand breaks in late mitosis

Ayra-Plasencia,

Symington,

Machín

2023

Preprint

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The cohesin complex maintains sister chromatid cohesion from S phase to anaphase onset. Cohesin also plays roles in chromosome structure and DNA repair. At anaphase onset, the cohesin subunit Scc1 is cleaved to allow segregation in an orderly manner, although some residual cohesin subunits remain to maintain chromosome structure. Efficient DNA double-strand break (DSB) repair by homologous recombination (HR) with the sister chromatid also depends on cohesin. Here, we have tested whether residual cohesin is important during DSB repair in anaphase/telophase (late mitosis). Using the well-established MAT switching system, we first show that HR is molecularly functional in late mitosis, and then show that cohesin is required for its efficiency. During DSBs in late mitosis, the segregated sister loci get closer and have episodes of coalescence, which may favour repair by HR. Here, we also show that these cytological phenotypes also depend on cohesin. Finally, full-length Scc1 returns after DSBs in late mitosis, suggesting thatde novowidespread cohesin complexes can support these retrograde behaviours.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Cohesin still drives homologous recombination repair of DNA double-strand breaks in late mitosis

Ayra-Plasencia,

Symington,

Machín

2023

Preprint

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“…To Monitor 5’-end resection assays, quantitative PCR (qPCR) or Southern blot methods were employed with some modifications (Gnugge et al ., 2018, Peng et al ., 2021a). DSB was induced by adding 2% galactose to logarithmic phase cells grown in the pre-induction medium (YP + Raffinose).…”

Section: Methodsmentioning

confidence: 99%

Di- and tri-methylation of histone H3K36 play distinct roles in DNA double-strand break repair

Chen,

Zhao,

et al. 2023

Preprint

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Histone H3 Lys36 (H3K36) methylation and its associated modifiers are crucial for DNA double-strand break (DSB) repair, but the mechanism governing whether and how different H3K36 methylation forms impact repair pathways is unclear. Here, we unveil the distinct roles of H3K36 dimethylation (H3K36me2) and H3K36 trimethylation (H3K36me3) in DSB repair via non-homologous end joining (NHEJ) or homologous recombination (HR). Yeast cells lacking H3K36me2 or H3K36me3 exhibit reduced NHEJ or HR efficiency. yKu70 and Rfa1 bind H3K36me2- or H3K36me3-modified peptides and chromatin, respectively. Disrupting these interactions impairs yKu70 and Rfa1 recruitment to damaged H3K36me2- or H3K36me3-rich loci, increasing DNA damage sensitivity and decreasing repair efficiency. Conversely, H3K36me2-enriched intergenic regions and H3K36me3-enriched gene bodies independently recruit yKu70 or Rfa1 under DSB stress. Importantly, human KU70 and RPA1, the homologs of yKu70 and Rfa1, exclusively associate with H3K36me2 and H3K36me3 in a conserved manner. These findings provide valuable insights into how H3K36me2 and H3K36me3 regulate distinct DSB repair pathways, highlighting H3K36 methylation as a critical element in the choice of DSB repair pathway.

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“…Evidence directly supporting such highly precise models of resection is lacking for mitotic DSBs in vivo. Most current methods used to monitor resection in in vivo typically use qPCR-or Southern Blot-based approaches to quantify ssDNA appearance beginning 100s of bp from the DSB end (Peng et al, 2021;Zhou et al, 2014), outside of the window examined by in vitro studies. These methods are highly informative regarding resection kinetics but lack the resolution needed to understand the exact sequence of events during resection initiation, specifically regarding Mre11 and Exo1 nuclease activity and the factors that promote or constrain their patterns of action, including the precise locations of Mre11 incision points.…”

Section: Introductionmentioning

confidence: 99%

Mapping yeast mitotic 5’ resection at base resolution reveals the sequence and positional dependence of nucleasesin vivo

Bazzano

Lomonaco

Wilson

2021

Preprint

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Resection of the 5'-terminated strand at DNA double strand breaks (DSBs) is the critical regulated step in the transition to homologous recombination. Biochemical and genetic studies have led to a multi-step model of DSB resection in which endonucleolytic cleavage mediated by Mre11 in partnership with Sae2 is coupled with exonucleolytic cleavage mediated by redundant pathways catalyzed by Exo1 and Sgs1/Dna2. These models have not been well tested at mitotic DSBs in vivo because most methods commonly used to monitor resection cannot precisely map early cleavage events. Here we report resection monitoring with next-generation sequencing in which unique molecular identifiers allow exact counting of cleaved 5' ends at base pair resolution. Mutant strains, including exo1Δ, mre11-H125N, exo1Δ and exo1Δ sgs1Δ, revealed a major Mre11-dependent cleavage position 60 to 70 bp from the DSB end whose exact position depended on local sequence and tracked an apparent motif. They further revealed an Exo1-dependent pause point approximately 200 bp from the DSB. Suppressing resection extension in exo1Δ sgs1Δ yeast exposed a footprint of regions where cleavage was restricted within 119 bp of the DSB and near the Exo1 pause point and where it was much less restrained. These results provide detailed in vivo support of prevailing models of DSB resection and extend them to show the combined influence of sequence specificity and access restrictions on Mre11 and Exo1 nucleases.

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Monitoring 5′-End Resection at Site-Specific Double-Strand Breaks by Southern Blot Analysis

Cited by 8 publications

References 25 publications

Cohesin still drives homologous recombination repair of DNA double-strand breaks in late mitosis

Cohesin still drives homologous recombination repair of DNA double-strand breaks in late mitosis

Di- and tri-methylation of histone H3K36 play distinct roles in DNA double-strand break repair

Mapping yeast mitotic 5’ resection at base resolution reveals the sequence and positional dependence of nucleasesin vivo

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