Cohesin regulates homology search during recombinational DNA repair

SummaryHomologous recombination (HR) is a ubiquitous DNA double-strand break (DSB) repair mechanism that promotes cell survival. It entails a potentially genome-wide homology search step, carried out along a conserved RecA/Rad51-ssDNA nucleoprotein filament (NPF) assembled on each DSB ends1–3. This search is subdued to NPF-dsDNA collision probability, dictated in part by chromatin conformation2,4. In contrast to the extensive knowledge about chromatin composition and mobility changes elicited by the DNA damage checkpoint (DDC)5–7, whether, how, and to which extent a DSB impacts spatial chromatin organization, and whether this organization in turns influences the homology search process, remains ill-defined8,9. Here we characterize two layers of spatial chromatin reorganization following DSB formation inS. cerevisiae.While cohesin folds chromosomes into cohesive arrays of 10-20 kb long chromatin loops as cells arrest in G2/M10,11, the DSB-flanking regions locally interact in a resection- and 9-1-1 clamp-dependent manner, independently of cohesin and HR proteins. This local structure blocks cohesin progression, constraining the extending NPF at loop base. Functionally this organization promotes side-specificcisDSB-dsDNA interactions that scales with loop expansion span, and provides a kinetic advantage for identification of intra- over inter-chromosomal homologies. We propose that cohesins regulate homology search by promotingcisdsDNA over-sampling, both upon loop expansion-coupled unidimensional dsDNA scanning, NPF trapping, and chromosome individualization, largely independent of their role in sister chromatid cohesion.

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i-BLESS is an ultra-sensitive method for detection of DNA double-strand breaks

Communications Biology ◽

10.1038/s42003-018-0165-9 ◽

2018 ◽

Vol 1 (1) ◽

Cited By ~ 22

Author(s):

Anna Biernacka ◽

Yingjie Zhu ◽

Magdalena Skrzypczak ◽

Romain Forey ◽

Benjamin Pardo ◽

...

Keyword(s):

Cell Fate ◽

Genome Stability ◽

Strand Break ◽

Double Strand Breaks ◽

Universal Method ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Agarose Beads ◽

Genome Wide ◽

Dna Double Strand Break

AbstractMaintenance of genome stability is a key issue for cell fate that could be compromised by chromosome deletions and translocations caused by DNA double-strand breaks (DSBs). Thus development of precise and sensitive tools for DSBs labeling is of great importance for understanding mechanisms of DSB formation, their sensing and repair. Until now there has been no high resolution and specific DSB detection technique that would be applicable to any cells regardless of their size. Here, we present i-BLESS, a universal method for direct genome-wide DNA double-strand break labeling in cells immobilized in agarose beads. i-BLESS has three key advantages: it is the only unbiased method applicable to yeast, achieves a sensitivity of one break at a given position in 100,000 cells, and eliminates background noise while still allowing for fixation of samples. The method allows detection of ultra-rare breaks such as those forming spontaneously at G-quadruplexes.

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Quantification of DNA double-strand break induced by radiation in cervix cancer cells: in vitro study

Journal of Radiotherapy in Practice ◽

10.1017/s1460396918000456 ◽

2018 ◽

Vol 18 (1) ◽

pp. 52-54

Author(s):

Sothing Vashum ◽

Rabi Raja Singh I ◽

Saikat Das ◽

Mohammed Azharuddin KO ◽

Prabhakaran Vasudevan

Keyword(s):

Cervical Cancer ◽

Cancer Cells ◽

Strand Break ◽

Double Strand Break ◽

Ionising Radiation ◽

Dependent Manner ◽

Survival Fraction ◽

Dna Double Strand Break ◽

Cervical Cancer Cells

AbstractAimDNA double-strand break (DSB) results in the phosphorylation of the protein, H.2AX histone. In this study, the effect of radiotherapy and chemotherapy on DNA DSB in cervical cancer cells is analysed by the phosphorylation of the protein.MethodsThe cervical cancer cells (HeLa cells) were cultured and exposed to ionising radiation. Radiation sensitivity was measured by clonogenic survival fraction after exposing to ionising radiation. Since the phosphorylation of H.2AX declines with time, the DNA damage was quantified at different time points: 1 hour, 3 hours and 1 week after exposed to the radiation. The analysis of γ-H.2AX was done by Western-blot technique. The protein expression was observed at different dose of radiation and combination of both radiation and paclitaxel.ResultsLow-dose hypersensitivity was observed. By 1 week after radiation at 0·5, 0·8 and 2 Gy, there was no expression of phosphorylated H.2AX. Previous experiments on the expression of phosphorylated H.2AX (γ-H.2AX) in terms of foci analysis was found to peak at 1 hour and subsequently decline with time. In cells treated with the DNA damaging agents, the expression of phosphorylated H.2AX decreases in a dose-dependent manner when treated with radiation alone. However, when combined with paclitaxel, at 0·5 Gy, the expression peaked and reduces at 0·8 Gy and slightly elevated at 2 Gy.FindingsIn this study, the peak phosphorylation was observed at 3 hour post irradiation indicating that DSBs are still left unrepaired.

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Resveratrol modulates DNA double-strand break repair pathways in an ATM/ATR–p53- and –Nbs1-dependent manner

Carcinogenesis ◽

10.1093/carcin/bgm283 ◽

2008 ◽

Vol 29 (3) ◽

pp. 519-527 ◽

Cited By ~ 35

Author(s):

Susanne Andrea Gatz ◽

Marlen Keimling ◽

Cindy Baumann ◽

Thilo Dörk ◽

Klaus-Michael Debatin ◽

...

Keyword(s):

Strand Break ◽

Double Strand Break ◽

Double Strand Break Repair ◽

Dependent Manner ◽

Dna Double Strand Break ◽

Repair Pathways ◽

Break Repair

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Monitoring Homology Search during DNA Double-Strand Break Repair In Vivo

Molecular Cell ◽

10.1016/j.molcel.2013.02.020 ◽

2013 ◽

Vol 50 (2) ◽

pp. 261-272 ◽

Cited By ~ 44

Author(s):

Jörg Renkawitz ◽

Claudio A. Lademann ◽

Marian Kalocsay ◽

Stefan Jentsch

Keyword(s):

Strand Break ◽

Double Strand Break ◽

Double Strand Break Repair ◽

Homology Search ◽

Dna Double Strand Break ◽

Break Repair

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Mechanistic view and genetic control of DNA recombination during meiosis

10.1101/169870 ◽

2017 ◽

Author(s):

Marie-Claude Marsolier-Kergoat ◽

Md Muntaz Khan ◽

Jonathan Schott ◽

Xuan Zhu ◽

Bertrand Llorente

Keyword(s):

Meiotic Recombination ◽

Dna Recombination ◽

Strand Break ◽

Holliday Junctions ◽

Switching Frequency ◽

Template Switching ◽

Genome Wide ◽

Dna Double Strand Break ◽

Strand Annealing ◽

Mechanistic View

ABSTRACTMeiotic recombination is essential for fertility and allelic shuffling. Canonical recombination models fail to capture the observed complexity of meiotic recombinants. Here we revisit these models by analyzing meiotic heteroduplex DNA tracts genome-wide in combination with meiotic DNA double-strand break (DSB) locations. We provide unprecedented support to the synthesis-dependent strand annealing model and establish estimates of its associated template switching frequency and polymerase processivity. We show that resolution of double Holliday junctions (dHJs) is biased toward cleavage of the pair of strands containing newly synthesized DNA near the junctions. The suspected dHJ resolvase Mlh1-3 as well as Mlh1-2, Exo1 and Sgs1 promote asymmetric positioning of crossover intermediates relative to the initiating DSB and bidirectional conversions. Finally, we show that crossover-biased dHJ resolution depends on Mlh1-3, Exo1, Msh5 and to a lesser extent on Sgs1. These properties are likely conserved in eukaryotes containing the ZMM proteins, which includes mammals.

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Ubiquitin-Specific-Processing Protease 47, A Novel 53BP1 regulator

10.1101/2021.01.27.428282 ◽

2021 ◽

Author(s):

Doraid T. Sadideen ◽

Baowei Chen ◽

Manal Basili ◽

Montaser Shaheen

Keyword(s):

Strand Break ◽

Dependent Manner ◽

Dna Double Strand Breaks ◽

End Joining ◽

Strand Breaks ◽

Class Switch ◽

Dna Double Strand Break ◽

Radiation Induced ◽

Non Homologous End Joining ◽

Processing Protease

AbstractDNA double strand breaks (DSBs) are repair by homology-based repair or non-homologous end joining and multiple sub-pathways exist. 53BP1 is a key DNA double strand break repair protein that regulates repair pathway choice. It is key for joining DSBs during immunoglobulin heavy chain class switch recombination. Here we identify USP47 as a deubiquitylase that associates with and regulates 53BP1 function. USP47 loss results in 53BP1 instability in proteasome dependent manner, and defective 53BP1 ionizing radiation induced foci (IRIF). USP47 catalytic activity is required for maintaining 53BP1 protein level. Similar to 53BP1, USP47 depletion results in sensitivity to DNA DSB inducing agents and defective immunoglobulin CSR. Our findings establish a function for USP47 in DNA DSB repair at least partially through 53BP1.

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INO80 requires a polycomb subunit to regulate the establishment of poised chromatin in murine spermatocytes

Development ◽

10.1242/dev.200089 ◽

2022 ◽

Vol 149 (1) ◽

Author(s):

Prabuddha Chakraborty ◽

Terry Magnuson

Keyword(s):

Chromatin Remodeling ◽

Binding Sites ◽

Strand Break ◽

Rna Seq ◽

Genome Wide Analysis ◽

The Core ◽

Chromatin Remodeling Complex ◽

Genome Wide ◽

Dna Double Strand Break ◽

Transcription Program

ABSTRACT INO80 is the catalytic subunit of the INO80-chromatin remodeling complex that is involved in DNA replication, repair and transcription regulation. Ino80 deficiency in murine spermatocytes (Ino80cKO) results in pachytene arrest of spermatocytes due to incomplete synapsis and aberrant DNA double-strand break repair, which leads to apoptosis. RNA-seq on Ino80cKO spermatocytes revealed major changes in transcription, indicating that an aberrant transcription program arises upon INO80 depletion. In Ino80WT spermatocytes, genome-wide analysis showed that INO80-binding sites were mostly promoter proximal and necessary for the regulation of spermatogenic gene expression, primarily of premeiotic and meiotic genes. Furthermore, most of the genes poised for activity, as well as those genes that are active, shared INO80 binding. In Ino80cKO spermatocytes, most poised genes demonstrated de-repression due to reduced H3K27me3 enrichment and, in turn, showed increased expression levels. INO80 interacts with the core PRC2 complex member SUZ12 and promotes its recruitment. Furthermore, INO80 mediates H2A.Z incorporation at the poised promoters, which was reduced in Ino80cKO spermatocytes. Taken together, INO80 is emerging as a major regulator of the meiotic transcription program by mediating poised chromatin establishment through SUZ12 binding.

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