FMRP bridges R-loops and DHX9 through direct interactions

2021 ◽  
Author(s):  
Arijita Chakraborty ◽  
Arijit Dutta ◽  
Leonardo G. Dettori ◽  
Leticia Gonzalez ◽  
Xiaoyu Xue ◽  
...  
Keyword(s):  
Rna Binding ◽  
Fragile X ◽  
Dependent Manner ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Amino Terminal ◽  
Intrinsically Disordered ◽  
Genome Wide ◽  
Loop Detection ◽  
Carboxy Terminal

ABSTRACTFragile X Syndrome (FXS) occurs when mutations in the FMR1 gene cause the absence or dysfunction of FMRP, known mainly as a translation repressor. We recently showed that FXS cells suffer genome-wide DNA double-strand breaks near R-loops under replication stress. The expression of FMRP, and not an FMRP-I304N mutant of the K-homology 2 RNA-binding domain, suppresses the R-loop-induced DNA breakage. These observations led us to hypothesize that FMRP safeguards the genome through promotion of R-loop detection and/or resolution. Here, we demonstrate that FMRP directly binds R-loops through multivalent interactions between the carboxy-terminal intrinsically disordered region and the R-loop sub-structures. We also show that the amino-terminal folded domain of FMRP directly binds DHX9, an R-loop resolvase, in a KH2-dependent manner. The FMRP-DHX9 interaction is recapitulated by co-immunoprecipitation in human cells. Our findings are consistent with a model in which FMRP recruits DHX9 to R-loop forming sites by bridging their interaction through its amino- and carboxy-termini, respectively.

scholarly journals DDX17 is required for efficient DSB repair at DNA:RNA hybrid deficient loci

2021 ◽  
Author(s):  
Aldo S Bader ◽  
Janna Luessing ◽  
Ben R Hawley ◽  
George L Skalka ◽  
Wei-Ting Lu ◽  
...  
Keyword(s):  
Dna Repair ◽  
Rna Binding ◽  
Meta Analysis ◽  
Binding Activity ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Proteomic Data ◽  
Genome Wide ◽  
Repair Factor

Proteins with RNA-binding activity are increasingly being implicated in DNA damage responses (DDR). Additionally, DNA:RNA-hybrids are rapidly generated around DNA double-strand breaks (DSBs), and are essential for effective repair. Here, using a meta-analysis of proteomic data, we identify novel DNA repair proteins and characterise a novel role for DDX17 in DNA repair. We found DDX17 to be required for both cell survival and DNA repair in response to numerous agents that induce DSBs. Analysis of DSB repair factor recruitment to damage sites suggested a role for DDX17 early in the DSB ubiquitin cascade. Genome-wide mapping of R-loops revealed that while DDX17 promotes the formation of DNA:RNA-hybrids around DSB sites, this role is specific to loci that are naturally deficient for DNA:RNA-hybrids. We propose that DDX17 facilitates DSB repair at loci that are inefficient at forming DNA:RNA-hybrids by catalysing the formation of DSB-induced hybrids, thereby allowing propagation of the damage response.

scholarly journals Genome-wide mapping of DNA double-strand breaks from eukaryotic cell cultures using Break-seq

2021 ◽  
Vol 2 (2) ◽  
pp. 100554
Author(s):  
Ishita Joshi ◽  
Jenna DeRycke ◽  
Megan Palmowski ◽  
Robert LeSuer ◽  
Wenyi Feng
Keyword(s):  
Cell Cultures ◽  
Eukaryotic Cell ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Genome Wide

scholarly journals Autophosphorylation of DNA-PKCS regulates its dynamics at DNA double-strand breaks

2007 ◽  
Vol 177 (2) ◽  
pp. 219-229 ◽  
Author(s):  
Naoya Uematsu ◽  
Eric Weterings ◽  
Ken-ichi Yano ◽  
Keiko Morotomi-Yano ◽  
Burkhard Jakob ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Laser System ◽  
Double Strand Breaks ◽  
Dependent Manner ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Dna Ends

The DNA-dependent protein kinase catalytic subunit (DNA-PKCS) plays an important role during the repair of DNA double-strand breaks (DSBs). It is recruited to DNA ends in the early stages of the nonhomologous end-joining (NHEJ) process, which mediates DSB repair. To study DNA-PKCS recruitment in vivo, we used a laser system to introduce DSBs in a specified region of the cell nucleus. We show that DNA-PKCS accumulates at DSB sites in a Ku80-dependent manner, and that neither the kinase activity nor the phosphorylation status of DNA-PKCS influences its initial accumulation. However, impairment of both of these functions results in deficient DSB repair and the maintained presence of DNA-PKCS at unrepaired DSBs. The use of photobleaching techniques allowed us to determine that the kinase activity and phosphorylation status of DNA-PKCS influence the stability of its binding to DNA ends. We suggest a model in which DNA-PKCS phosphorylation/autophosphorylation facilitates NHEJ by destabilizing the interaction of DNA-PKCS with the DNA ends.

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

2018 ◽  
Vol 1 (1) ◽  
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.

scholarly journals Genome-wide mapping implicates R-loops in lineage-specific processes and formation of DNA break hotspots in neural stem/progenitor cells

2021 ◽  
Author(s):  
Supawat Thongthip ◽  
Annika Carlson ◽  
Madzia P. Crossley ◽  
Bjoern Schwer
Keyword(s):  
Progenitor Cells ◽  
Cluster Formation ◽  
Strand Break ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Neural Genes ◽  
Genome Wide ◽  
Gc Skew ◽  
Neural Stem Progenitor Cells ◽  
Dna Break

ABSTRACTRecent work has revealed classes of recurrent DNA double-strand breaks (DSBs) in neural stem/progenitor cells, including transcription-associated, promoter-proximal breaks and recurrent DSB clusters in late-replicating, long neural genes. However, the mechanistic factors promoting these different classes of DSBs in neural stem/progenitor cells are not understood. Here, we elucidated the genome-wide landscape of DNA:RNA hybrid structures called “R-loops” in primary neural stem/progenitor cells in order to assess their contribution to the different classes of DNA break “hotspots”. We report that R-loops in neural stem/progenitor cells are associated primarily with transcribed regions that replicate early and genes that show GC skew in their promoter region. Surprisingly, the majority of genes with recurrent DSB clusters in long, neural genes does not show substantial R-loop accumulation. We implicate R-loops in promoter-proximal DNA break formation in highly transcribed, early replicating regions and conclude that R-loops are not a driver of recurrent double-strand break cluster formation in most long, neural genes. Together, our study provides an understanding of how R-loops may contribute to DNA break hotspots and affect lineage-specific processes in neural stem/progenitor cells.

scholarly journals Emerging Technologies for Genome-Wide Profiling of DNA Breakage

2021 ◽  
Vol 11 ◽  
Author(s):  
Matthew J. Rybin ◽  
Melina Ramic ◽  
Natalie R. Ricciardi ◽  
Philipp Kapranov ◽  
Claes Wahlestedt ◽  
...  
Keyword(s):  
Genome Instability ◽  
Dna Double Strand Breaks ◽  
Single Nucleotide ◽  
Strand Breaks ◽  
Single Strand Breaks ◽  
Genome Wide ◽  
A Genome ◽  
Wide Scale ◽  
Nucleotide Resolution ◽  
Genomic Regions

Genome instability is associated with myriad human diseases and is a well-known feature of both cancer and neurodegenerative disease. Until recently, the ability to assess DNA damage—the principal driver of genome instability—was limited to relatively imprecise methods or restricted to studying predefined genomic regions. Recently, new techniques for detecting DNA double strand breaks (DSBs) and single strand breaks (SSBs) with next-generation sequencing on a genome-wide scale with single nucleotide resolution have emerged. With these new tools, efforts are underway to define the “breakome” in normal aging and disease. Here, we compare the relative strengths and weaknesses of these technologies and their potential application to studying neurodegenerative diseases.

scholarly journals TOPOVIBL-REC114 interaction regulates meiotic DNA double-strand breaks

2021 ◽  
Author(s):  
Alexandre Nore ◽  
Ariadna B Juarez-Martinez ◽  
Julie AJ Clement ◽  
Christine Brun ◽  
Bouboub Diagouraga ◽  
...  
Keyword(s):  
Dna Cleavage ◽  
Point Mutations ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Catalytic Complex ◽  
Strand Breaks ◽  
Genome Wide ◽  
Dna Cleavage Activity ◽  
Meiotic Dsbs

Meiosis requires the formation of programmed DNA double strand breaks (DSBs), essential for fertility and for generating genetic diversity. In male and female meiotic cells, DSBs are induced by the catalytic activity of the TOPOVIL complex formed by SPO11 and TOPOVIBL. To ensure genomic integrity, DNA cleavage activity is tightly regulated, and several accessory factors (REC114, MEI4, IHO1, and MEI1) are needed for DSB formation in mice. How and when these proteins act is not understood. Here, we show that REC114 is a direct partner of TOPOVIBL, and identified their conserved interacting domains by structural analysis. We then analysed the role of this interaction by monitoring meiotic DSBs in female and male mice carrying point mutations in TOPOVIBL that decrease or disrupt its binding to REC114. In these mutants, DSB activity was strongly reduced genome-wide in oocytes, but only in sub-telomeric regions in spermatocytes. In addition, in mutant spermatocytes, DSB activity was delayed in autosomes. These results provide evidence that REC114 is a key member of the TOPOVIL catalytic complex, and that the REC114/TOPOVIBL interaction ensures the efficiency and timing of DSB activity by integrating specific chromosomal features.

scholarly journals Wortmannin, a specific inhibitor of phosphatidylinositol-3-kinase, induces accumulation of DNA double-strand breaks

2020 ◽  
Vol 61 (2) ◽  
pp. 171-176 ◽  
Author(s):  
Makoto Ihara ◽  
Kazuko Shichijo ◽  
Satoshi Takeshita ◽  
Takashi Kudo
Keyword(s):  
Dna Damage ◽  
Specific Inhibitor ◽  
Double Strand Breaks ◽  
Dependent Manner ◽  
Dna Double Strand Breaks ◽  
Phosphatidylinositol 3 Kinase ◽  
Strand Breaks ◽  
Γ Ray ◽  
Scid Cells ◽  
Phosphatidylinositol 3

Abstract Wortmannin, a fungal metabolite, is a specific inhibitor of the phosphatidylinositol 3-kinase (PI3K) family, which includes double-stranded DNA dependent protein kinase (DNA-PK) and ataxia telangiectasia mutated kinase (ATM). We investigated the effects of wortmannin on DNA damage in DNA-PK-deficient cells obtained from severe combined immunodeficient mice (SCID cells). Survival of wortmannin-treated cells decreased in a concentration-dependent manner. After treatment with 50 μM wortmannin, survival decreased to 60% of that of untreated cells. We observed that treatment with 20 and 50 μM wortmannin induced DNA damage equivalent to that by 0.37 and 0.69 Gy, respectively, of γ-ray radiation. The accumulation of DNA double-strand breaks (DSBs) in wortmannin-treated SCID cells was assessed using pulsed-field gel electrophoresis. The maximal accumulation was observed 4 h after treatment. Moreover, the presence of DSBs was confirmed by the ability of nuclear extracts from γ-ray-irradiated SCID cells to produce in vitro phosphorylation of histone H2AX. These results suggest that wortmannin induces cellular toxicity by accumulation of spontaneous DSBs through inhibition of ATM.

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