scholarly journals DNMT3b Dysfunction Promotes DNA cleavages at Centromeric R-loops to Increase Centromere Instability

2020 ◽  
Author(s):  
Hsueh-Tzu Shih ◽  
Wei-Yi Chen ◽  
Hsin-Yen Wang ◽  
Hsien-Da Huang ◽  
Chih-Hung Chou ◽  
...  
Keyword(s):  
Dna Methyltransferase ◽  
Genome Instability ◽  
Loss Of Function ◽  
Dna Breaks ◽  
End Joining ◽  
Critical Function ◽  
Satellite Sequences ◽  
Mitotic Aberration ◽  
Error Prone Repair ◽  
Non Homologous End Joining

ABSTRACTThis study investigates how DNA methyltransferase 3b (DNMT3b) dysfunction causes genome instability. We showed that in DNMT3b deficient cells, R-loops contribute to prominent γH2AX signal, which was mapped to repetitive satellite sequences including centromere regions. By ChIP and DRIP analyses, our data revealed that centromeric R-loops in DNMT3b deficient cells are removed by XPG/XPF, thus generating DNA breaks in centromeres to increase mitotic aberration. In immunodeficiency-centromeric instability-facial anomalies (ICF) patient cells carrying the loss-of-function mutation at DNMT3b, knockdown of XPG/XPF in ICF cells also reduces DNA breaks in centromere while bringing up centromeric R-loop to the level similar to that in wild-type cells. These results suggest that DNMT3b has a critical function in preventing XPG/XPF-mediated cleavages at centromeric R-loop sites. Finally, we showed the involvement of non-homologous end-joining repair at centromeric sites in ICF cells. Thus, DNA cleavages at centromeric R-loops with error-prone repair undermine centromere stability in ICF cells.

scholarly journals TDP2–Dependent Non-Homologous End-Joining Protects against Topoisomerase II–Induced DNA Breaks and Genome Instability in Cells and In Vivo

PLoS Genetics ◽  
2013 ◽  
Vol 9 (3) ◽  
pp. e1003226 ◽  
Author(s):  
Fernando Gómez-Herreros ◽  
Rocío Romero-Granados ◽  
Zhihong Zeng ◽  
Alejandro Álvarez-Quilón ◽  
Cristina Quintero ◽  
...  
Keyword(s):  
Topoisomerase Ii ◽  
Genome Instability ◽  
Dna Breaks ◽  
End Joining ◽  
Non Homologous End Joining ◽  
In Cells

Haploinsufficiency in non-homologous end joining factor 1 induces ovarian dysfunction in humans and mice

2021 ◽  
pp. jmedgenet-2020-107398
Author(s):  
Guoqing Li ◽  
Xi Yang ◽  
Lingbo Wang ◽  
Yuncheng Pan ◽  
Siyuan Chen ◽  
...  
Keyword(s):  
Ovarian Function ◽  
In Vitro Assays ◽  
Chinese Family ◽  
Common Disease ◽  
Loss Of Function ◽  
End Joining ◽  
Dsb Repair ◽  
Repair Capacity ◽  
Non Homologous End Joining

BackgroundPremature ovarian insufficiency (POI) is a common disease in women that leads to a reduced reproductive lifespan. The aetiology of POI is genetically heterogeneous, with certain double-strand break (DSB) repair genes being implicated in POI. Although non-homologous end joining (NHEJ) is an efficient DSB repair pathway, the functional relationship between this pathway and POI remains unknown.Methods and resultsWe conducted whole-exome sequencing in a Chinese family and identified a rare heterozygous loss-of-function variant in non-homologous end joining factor 1 (NHEJ1): c.532C>T (p.R178*), which co-segregated with POI and irregular menstruation. The amount of NHEJ1 protein in the proband was half of the normal level, indicating a link between NHEJ1 haploinsufficiency and POI. Furthermore, another rare heterozygous NHEJ1 variant c.500A>G (p.Y167C) was identified in one of 100 sporadic POI cases. Both variants were predicted to be deleterious by multiple in silico tools. In vitro assays showed that knock-down of NHEJ1 in human KGN ovarian cells impaired DNA repair capacity. We also generated a knock-in mouse model with a heterozygous Nhej1 variant equivalent to NHEJ1 p.R178* in familial patients. Compared with wild-type mice, heterozygous Nhej1-mutated female mice required a longer time to first birth, and displayed reduced numbers of primordial and growing follicles. Moreover, these mice exhibited higher sensitivity to DSB-inducing drugs. All these phenotypes are analogous to the progressive loss of ovarian function observed in POI.ConclusionsOur observations in both humans and mice suggest that NHEJ1 haploinsufficiency is associated with non-syndromic POI, providing novel insights into genetic counselling and clinical prevention of POI.

scholarly journals Double-strand DNA breaks recruit the centromeric histone CENP-A

2009 ◽  
Vol 106 (37) ◽  
pp. 15762-15767 ◽  
Author(s):  
Samantha G. Zeitlin ◽  
Norman M. Baker ◽  
Brian R. Chapados ◽  
Evi Soutoglou ◽  
Jean Y. J. Wang ◽  
...  
Keyword(s):  
Dna Cleavage ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Dna Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Double Strand Dna Breaks ◽  
Histone H3 Variant ◽  
Radiation Induced ◽  
Non Homologous End Joining

The histone H3 variant CENP-A is required for epigenetic specification of centromere identity through a loading mechanism independent of DNA sequence. Using multiphoton absorption and DNA cleavage at unique sites by I-SceI endonuclease, we demonstrate that CENP-A is rapidly recruited to double-strand breaks in DNA, along with three components (CENP-N, CENP-T, and CENP-U) associated with CENP-A at centromeres. The centromere-targeting domain of CENP-A is both necessary and sufficient for recruitment to double-strand breaks. CENP-A accumulation at DNA breaks is enhanced by active non-homologous end-joining but does not require DNA-PKcs or Ligase IV, and is independent of H2AX. Thus, induction of a double-strand break is sufficient to recruit CENP-A in human and mouse cells. Finally, since cell survival after radiation-induced DNA damage correlates with CENP-A expression level, we propose that CENP-A may have a function in DNA repair.

scholarly journals cGAS guards against chromosome end-to-end fusions during mitosis and facilitates replicative senescence

2021 ◽  
Author(s):  
Xiaocui Li ◽  
Xiaojuan Li ◽  
Chen Xie ◽  
Sihui Cai ◽  
Mengqiu Li ◽  
...  
Keyword(s):  
Genome Stability ◽  
Mitotic Chromosome ◽  
Replicative Senescence ◽  
Genome Instability ◽  
Strand Break ◽  
End Joining ◽  
Dsb Repair ◽  
End To End ◽  
Non Homologous End Joining ◽  
Sting Pathway

AbstractAs a sensor of cytosolic DNA, the role of cyclic GMP-AMP synthase (cGAS) in innate immune response is well established, yet how its functions in different biological conditions remain to be elucidated. Here, we identify cGAS as an essential regulator in inhibiting mitotic DNA double-strand break (DSB) repair and protecting short telomeres from end-to-end fusion independent of the canonical cGAS-STING pathway. cGAS associates with telomeric/subtelomeric DNA during mitosis when TRF1/TRF2/POT1 are deficient on telomeres. Depletion of cGAS leads to mitotic chromosome end-to-end fusions predominantly occurring between short telomeres. Mechanistically, cGAS interacts with CDK1 and positions them to chromosome ends. Thus, CDK1 inhibits mitotic non-homologous end joining (NHEJ) by blocking the recruitment of RNF8. cGAS-deficient human primary cells are defective in entering replicative senescence and display chromosome end-to-end fusions, genome instability and prolonged growth arrest. Altogether, cGAS safeguards genome stability by controlling mitotic DSB repair to inhibit mitotic chromosome end-to-end fusions, thus facilitating replicative senescence.

Editing livestock genomes with site-specific nucleases

2014 ◽  
Vol 26 (1) ◽  
pp. 74 ◽  
Author(s):  
Daniel F. Carlson ◽  
Wenfang Tan ◽  
Perry B. Hackett ◽  
Scott C. Fahrenkrug
Keyword(s):  
Genetic Alterations ◽  
Large Animal ◽  
Dna Breaks ◽  
Transcription Activator ◽  
End Joining ◽  
Site Specific ◽  
Homology Directed Repair ◽  
Double Strand Dna Breaks ◽  
Non Homologous End Joining ◽  
Inactive Genes

Over the past 5 years there has been a major transformation in our ability to precisely manipulate the genomes of animals. Efficiencies of introducing precise genetic alterations in large animal genomes have improved 100 000-fold due to a succession of site-specific nucleases that introduce double-strand DNA breaks with a specificity of 10–9. Herein we describe our applications of site-specific nucleases, especially transcription activator-like effector nucleases, to engineer specific alterations in the genomes of pigs and cows. We can introduce variable changes mediated by non-homologous end joining of DNA breaks to inactive genes. Alternatively, using homology-directed repair, we have introduced specific changes that support either precise alterations in a gene’s encoded polypeptide, elimination of the gene or replacement by another unrelated DNA sequence. Depending on the gene and the mutation, we can achieve 10%–50% effective rates of precise mutations. Applications of the new precision genetics are extensive. Livestock now can be engineered with selected phenotypes that will augment their value and adaption to variable ecosystems. In addition, animals can be engineered to specifically mimic human diseases and disorders, which will accelerate the production of reliable drugs and devices. Moreover, animals can be engineered to become better providers of biomaterials used in the medical treatment of diseases and disorders.

scholarly journals Mechanistic and genetic basis of single-strand templated repair at Cas12a-induced DNA breaks in Chlamydomonas reinhardtii

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Aron Ferenczi ◽  
Yen Peng Chew ◽  
Erika Kroll ◽  
Charlotte von Koppenfels ◽  
Andrew Hudson ◽  
...  
Keyword(s):  
Dna Repair ◽  
Chlamydomonas Reinhardtii ◽  
Nuclear Dna ◽  
Model Organism ◽  
Single Strand ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
End Joining ◽  
Non Homologous End Joining ◽  
Underlying Mechanisms

AbstractSingle-stranded oligodeoxynucleotides (ssODNs) are widely used as DNA repair templates in CRISPR/Cas precision genome editing. However, the underlying mechanisms of single-strand templated DNA repair (SSTR) are inadequately understood, constraining rational improvements to precision editing. Here we study SSTR at CRISPR/Cas12a-induced DNA double-strand breaks (DSBs) in the eukaryotic model green microalga Chlamydomonas reinhardtii. We demonstrate that ssODNs physically incorporate into the genome during SSTR at Cas12a-induced DSBs. This process is genetically independent of the Rad51-dependent homologous recombination and Fanconi anemia pathways, is strongly antagonized by non-homologous end-joining, and is mediated almost entirely by the alternative end-joining enzyme polymerase θ. These findings suggest differences in SSTR between C. reinhardtii and animals. Our work illustrates the promising potentially of C. reinhardtii as a model organism for studying nuclear DNA repair.

scholarly journals Loss of Cdc13 causes genome instability by a deficiency in replication-dependent telomere capping

2019 ◽  
Author(s):  
Rachel E Langston ◽  
Dominic Palazzola ◽  
Erin Bonnell ◽  
Raymund J. Wellinger ◽  
Ted Weinert
Keyword(s):  
Homologous Recombination ◽  
Genome Stability ◽  
Genome Instability ◽  
Chromosome Instability ◽  
S Phase ◽  
Temperature Sensitive ◽  
End Joining ◽  
Telomere Capping ◽  
Non Homologous End Joining

AbstractIn budding yeast, Cdc13, Stn1, and Ten1 form a telomere binding heterotrimer dubbed CST. Here we investigate the role of Cdc13/CST in maintaining genome stability, using a Chr VII disome system that can generate recombinants, loss, and enigmatic unstable chromosomes. In cells expressing a temperature sensitive CDC13 allele, cdc13F684S, unstable chromosomes frequently arise due to problems in or near a telomere. Hence, when Cdc13 is defective, passage through S phase causes Exo1-dependent ssDNA and unstable chromosomes, which then are the source for whole chromosome instability events (e.g. recombinants, chromosome truncations, dicentrics, and/or loss). Specifically, genome instability arises from a defect in Cdc13’s replication-dependent telomere capping function, not Cdc13s putative post-replication telomere capping function. Furthermore, the unstable chromosomes form without involvement of homologous recombination nor non-homologous end joining. Our data suggest that a Cdc13/CST defect in semi-conservative replication near the telomere leads to ssDNA and unstable chromosomes, which then are lost or subject to complex rearrangements. This system defines a links between replication-dependent chromosome capping and genome stability in the form of unstable chromosomes.

scholarly journals Unleashing a Novel Function of Endonuclease G in Mitochondrial Genome Instability

2021 ◽  
Author(s):  
Sumedha Dahal ◽  
Humaira Siddiqua ◽  
Shivangi Sharma ◽  
Ravi K Babu ◽  
Meghana Manjunath ◽  
...  
Keyword(s):  
Mitochondrial Genome ◽  
Nuclear Dna ◽  
Genome Instability ◽  
Dna Breaks ◽  
End Joining ◽  
Endonuclease G ◽  
Whole Process ◽  
G4 Dna ◽  
Mtdna Deletion

Having its own genome makes mitochondria a unique and semiautonomous organelle within cells. Mammalian mitochondrial DNA (mtDNA) is double-stranded closed circular molecule of about 16 kb coding 37 genes. Mutations, including deletions in the mitochondrial genome can culminate in different human diseases. Mapping of the deletion junctions suggests that the breakpoints are generally seen at hotspots. '9-bp deletion' (8271-8281), seen in the intergenic region of cytochrome c oxidase II/tRNALys, is the most common mitochondrial deletion. While it is associated with several diseases like myopathy, dystonia, and hepatocellular carcinoma, it has also been used as an evolutionary marker. However, the mechanism responsible for its fragility is unclear. In the current study, we show that Endonuclease G, a mitochondrial nuclease responsible for nonspecific cleavage of nuclear DNA during apoptosis, can induce breaks at sequences associated with '9-bp deletion', when it is present on a plasmid or in the mitochondrial genome. Through a series of in vitro and intracellular studies, we show that Endonuclease G binds to G-quadruplex structures formed at the hotspot and induces DNA breaks. Besides, we reconstitute the whole process of '9-bp deletion' using purified Endonuclease G to induce breaks in mtDNA, followed by mitochondrial extract-mediated DSB repair and establish that microhomology-mediated end joining is responsible for the generation of mtDNA deletion. Finally, we show that the whole process is regulated by different stress conditions, which may modulate release of Endonuclease G to the mitochondrial matrix. Therefore, we uncover a new role for Endonuclease G in generating deletions, which is dependent on the formation of G4 DNA within the mitochondrial genome. Thus, in this study we identify a novel property of Endonuclease G, besides its role in apoptosis, and the recently described 'elimination of paternal mitochondria during fertilization.

scholarly journals DNA barcoding reveals that injected transgenes are predominantly processed by homologous recombination in mouse zygote

2019 ◽  
Author(s):  
Alexander Smirnov ◽  
Anastasia Yunusova ◽  
Alexey Korablev ◽  
Irina Serova ◽  
Veniamin Fishman ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Dna Molecules ◽  
Dna Breaks ◽  
End Joining ◽  
Exogenous Dna ◽  
Transgenic Organisms ◽  
Mouse Zygote ◽  
Non Homologous End Joining ◽  
Transgenic Embryos ◽  
Double Holliday Junction

AbstractMechanisms that ensure repair of double-stranded DNA breaks play a key role in the integration of foreign DNA into the genome of transgenic organisms. After pronuclear microinjection, exogenous DNA is usually found in the form of concatemer consisting of multiple co-integrated transgene copies. Here we investigated contribution of various DSB repair pathways to the concatemer formation. We injected a pool of linear DNA molecules carrying unique barcodes at both ends into mouse zygotes and obtained 10 transgenic embryos with transgene copy number ranging from 1 to 300 copies. Sequencing of the barcodes allowed us to assign relative positions to the copies in concatemers and to detect recombination events that happened during integration. Cumulative analysis of approximately 1000 integrated copies revealed that more than 80% of copies underwent recombination when their linear ends were processed by SDSA or DSBR. We also observed evidence of double Holliday junction (dHJ) formation and crossing-over during the formation of concatemers. Additionally, sequencing of indels between copies showed that at least 10% of the DNA molecules introduced into the zygote are ligated by non-homologous end joining (NHEJ). Our barcoding approach documents high activity of homologous recombination after exogenous DNA injection in mouse zygote.

scholarly journals A CRISPR/Cas9 Method Facilitates Efficient Oligo-Mediated Gene Editing in Debaryomyces Hansenii

2021 ◽  
Author(s):  
Tomas Strucko ◽  
Niklas L Andersen ◽  
Mikkel R Mahler ◽  
José L Martínez ◽  
Uffe H Mortensen
Keyword(s):  
Gene Editing ◽  
Point Mutations ◽  
Debaryomyces Hansenii ◽  
Cell Factory ◽  
Full Potential ◽  
Dna Breaks ◽  
End Joining ◽  
Dna Oligonucleotides ◽  
Non Homologous End Joining ◽  
Basic And Applied Research

Abstract The halophilic and osmotolerant yeast Debaryomyces hansenii has a high potential for cell factory applications due to its resistance to harsh environmental factors and compatibility with a wide substrate range. However, currently available genetic techniques does not allow the full potential of D. hansenii as a cell factory to be harnessed. Moreover, most of the currently available tools rely on the use of auxotrophic markers that are not suitable in wild-type prototrophic strains. In addition, the preferred non-homologous end-joining (NHEJ) DNA damage repair mechanism pose further challenges when precise gene targeting is required. In this study, we present a novel plasmid based CRISPRCUG/Cas9 method for easy and efficient gene editing of the prototrophic strains of D. hansenii. Our toolset design is based on a dominant marker and facilitates quick assembly of the vectors expressing Cas9 and single or multiple sgRNAs that provides possibility for multiplex gene engineering even in prototrophic strains. Moreover, we have constructed an NHEJ deficient D. hansenii that enable our CRISPRCUG/Cas9 tools to support highly efficient introduction of point mutations and single/double gene deletions. Importantly, we also demonstrate that 90-nt single stranded DNA oligonucleotides are sufficient to direct repair of DNA breaks induced by sgRNA-Cas9 resulting in precise edits reaching 100% efficiencies. In conclusion, tools developed in this study will greatly advance basic and applied research in D. hansenii. In addition, we envision that our tools can be rapidly adapted for gene editing of other non-conventional yeast species including the ones belonging to the CUG clade.

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