scholarly journals Microhomology-mediated end joining is the principal mediator of double-strand break repair during mitochondrial DNA lesions

2016 ◽  
Vol 27 (2) ◽  
pp. 223-235 ◽  
Author(s):  
Satish Kumar Tadi ◽  
Robin Sebastian ◽  
Sumedha Dahal ◽  
Ravi K. Babu ◽  
Bibha Choudhary ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Dna Sequences ◽  
Nuclear Dna ◽  
Ex Vivo ◽  
Strand Break ◽  
Mitochondrial Disorders ◽  
Dna Lesions ◽  
End Joining ◽  
Dsb Repair

Mitochondrial DNA (mtDNA) deletions are associated with various mitochondrial disorders. The deletions identified in humans are flanked by short, directly repeated mitochondrial DNA sequences; however, the mechanism of such DNA rearrangements has yet to be elucidated. In contrast to nuclear DNA (nDNA), mtDNA is more exposed to oxidative damage, which may result in double-strand breaks (DSBs). Although DSB repair in nDNA is well studied, repair mechanisms in mitochondria are not characterized. In the present study, we investigate the mechanisms of DSB repair in mitochondria using in vitro and ex vivo assays. Whereas classical NHEJ (C-NHEJ) is undetectable, microhomology-mediated alternative NHEJ efficiently repairs DSBs in mitochondria. Of interest, robust microhomology-mediated end joining (MMEJ) was observed with DNA substrates bearing 5-, 8-, 10-, 13-, 16-, 19-, and 22-nt microhomology. Furthermore, MMEJ efficiency was enhanced with an increase in the length of homology. Western blotting, immunoprecipitation, and protein inhibition assays suggest the involvement of CtIP, FEN1, MRE11, and PARP1 in mitochondrial MMEJ. Knockdown studies, in conjunction with other experiments, demonstrated that DNA ligase III, but not ligase IV or ligase I, is primarily responsible for the final sealing of DSBs during mitochondrial MMEJ. These observations highlight the central role of MMEJ in maintenance of mammalian mitochondrial genome integrity and is likely relevant for deletions observed in many human mitochondrial disorders.

scholarly journals ERCC1-XPF Endonuclease Facilitates DNA Double-Strand Break Repair

2008 ◽  
Vol 28 (16) ◽  
pp. 5082-5092 ◽  
Author(s):  
Anwaar Ahmad ◽  
Andria Rasile Robinson ◽  
Anette Duensing ◽  
Ellen van Drunen ◽  
H. Berna Beverloo ◽  
...  
Keyword(s):  
Gamma Irradiation ◽  
Excision Repair ◽  
Strand Break ◽  
Dna Lesions ◽  
End Joining ◽  
Dsb Repair ◽  
Large Deletions ◽  
Mutant Cells

ABSTRACT ERCC1-XPF endonuclease is required for nucleotide excision repair (NER) of helix-distorting DNA lesions. However, mutations in ERCC1 or XPF in humans or mice cause a more severe phenotype than absence of NER, prompting a search for novel repair activities of the nuclease. In Saccharomyces cerevisiae, orthologs of ERCC1-XPF (Rad10-Rad1) participate in the repair of double-strand breaks (DSBs). Rad10-Rad1 contributes to two error-prone DSB repair pathways: microhomology-mediated end joining (a Ku86-independent mechanism) and single-strand annealing. To determine if ERCC1-XPF participates in DSB repair in mammals, mutant cells and mice were screened for sensitivity to gamma irradiation. ERCC1-XPF-deficient fibroblasts were hypersensitive to gamma irradiation, and γH2AX foci, a marker of DSBs, persisted in irradiated mutant cells, consistent with a defect in DSB repair. Mutant mice were also hypersensitive to irradiation, establishing an essential role for ERCC1-XPF in protecting against DSBs in vivo. Mice defective in both ERCC1-XPF and Ku86 were not viable. However, Ercc1 −/− Ku86 −/− fibroblasts were hypersensitive to gamma irradiation compared to single mutants and accumulated significantly greater chromosomal aberrations. Finally, in vitro repair of DSBs with 3′ overhangs led to large deletions in the absence of ERCC1-XPF. These data support the conclusion that, as in yeast, ERCC1-XPF facilitates DSB repair via an end-joining mechanism that is Ku86 independent.

Ex vivo zidovudine (AZT) treatment of CD34+ bone marrow progenitors causes decreased steady state mitochondrial DNA (mtDNA) and increased lactate production

2004 ◽  
Vol 23 (4) ◽  
pp. 173-185 ◽  
Author(s):  
L D Lewis ◽  
S Amin ◽  
C I Civin ◽  
P S Lietman
Keyword(s):  
Bone Marrow ◽  
Mitochondrial Dna ◽  
Steady State ◽  
Nuclear Dna ◽  
Ex Vivo ◽  
Lactate Production ◽  
Nuclear Dna Content ◽  
Content Ratio ◽  
The Mean

Haematopoietic suppression is one of the dose-limiting side effects of chronic zidovudine (AZT) therapy. We tested the hypothesis that AZT would reduce mitochondrial DNA (mtDNA) content in haematopoietic progenitors causing impaired haematopoiesis and mitochondrial dysfunction. We studied the effects of AZT 0 / 50 M in vitro, on normal human CD34 / haematopoietic progenitor cells cultured ex vivo for up to 12 days. The mean AZT IC50 for granulocyte (phenotype CD15 / /CD14 /) and erythroid (phenotype glycophorin / /CD45 /) cell proliferation was 2.5 M (SD9 / 0.7) and 0.023 M (SD9 / 0.005), respectively. In myeloid-rich cell cultures, the mean lactate content of the media, compared to untreated controls, increased by 86% (SD9 / 23) at 10 M AZT and in erythroid-rich cultures it increased by 134% (SD9 / 24) in the presence of 0.5 M AZT. In myeloid-rich cultures the AZT IC50 for the reduction in the mitochondrial/nuclear DNA content ratio was 5.6 M, whereas in erythroid rich cultures this AZT IC50 was B / 0.0005 M. AZT produced concentration-dependent inhibition of CD34 / progenitor proliferation into both myeloid and erythroid lineages; erythropoiesis was more sensitive than myelopoiesis. Concurrently, AZT reduced steady state mtDNA content, while increasing lactate production. These findings support the hypothesis that mtDNA is one of the intracellular targets involved in the pathogenesis of AZT-associated bone marrow progenitor cell toxicity.

scholarly journals In VitroExpansion of Bone Marrow Derived Mesenchymal Stem Cells Alters DNA Double Strand Break Repair of Etoposide Induced DNA Damage

2016 ◽  
Vol 2016 ◽  
pp. 1-11 ◽  
Author(s):  
Ian Hare ◽  
Marieta Gencheva ◽  
Rebecca Evans ◽  
James Fortney ◽  
Debbie Piktel ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Ex Vivo ◽  
Strand Break ◽  
Dna Lesions ◽  
Cellular Damage ◽  
Human Mscs ◽  
Dna Breaks ◽  
Double Strand Dna Breaks

Mesenchymal stem cells (MSCs) are of interest for use in diverse cellular therapies.Ex vivoexpansion of MSCs intended for transplantation must result in generation of cells that maintain fidelity of critical functions. Previous investigations have identified genetic and phenotypic alterations of MSCs within vitropassage, but little is known regarding how culturing influences the ability of MSCs to repair double strand DNA breaks (DSBs), the most severe of DNA lesions. To investigate the response to DSB stress with passagein vitro, primary human MSCs were exposed to etoposide (VP16) at various passages with subsequent evaluation of cellular damage responses and DNA repair. Passage number did not affect susceptibility to VP16 or the incidence and repair kinetics of DSBs. Nonhomologous end joining (NHEJ) transcripts showed little alteration with VP16 exposure or passage; however, homologous recombination (HR) transcripts were reduced following VP16 exposure with this decrease amplified as MSCs were passagedin vitro. Functional evaluations of NHEJ and HR showed that MSCs were unable to activate NHEJ repair following VP16 stress in cells after successive passage. These results indicate thatex vivoexpansion of MSCs alters their ability to perform DSB repair, a necessary function for cells intended for transplantation.

scholarly journals Mapping the Genetic Landscape of DNA Double-strand Break Repair

2021 ◽  
Author(s):  
Jeffrey A Hussmann ◽  
Jia Ling ◽  
Purnima Ravisankar ◽  
Jun Yan ◽  
Ann Cirincione ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Strand Break ◽  
Dna Lesions ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Dsb Repair ◽  
Homology Directed Repair ◽  
Genetic Perturbations ◽  
Genetic Landscape ◽  
Repair Mechanisms

Cells repair DNA double-strand breaks (DSBs) through a complex set of pathways that are critical for maintaining genomic integrity. Here we present Repair-seq, a high-throughput screening approach that measures the effects of thousands of genetic perturbations on the distribution of mutations introduced at targeted DNA lesions. Using Repair-seq, we profiled DSB repair outcomes induced by two programmable nucleases (Cas9 and Cas12a) after knockdown of 476 genes involved in DSB repair or associated processes in the presence or absence of oligonucleotides for homology-directed repair (HDR). The resulting data enabled principled, data-driven inference of DSB end joining and HDR pathways and demonstrated that repair outcomes with superficially similar sequence architectures can have markedly different genetic dependencies. Systematic interrogation of these dependencies then uncovered unexpected relationships among DSB repair genes and isolated incompletely characterized repair mechanisms. This work provides a foundation for understanding the complex pathways of DSB repair and for optimizing genome editing across modalities.

scholarly journals Regulatory and Functional Involvement of Long Non-Coding RNAs in DNA Double-Strand Break Repair Mechanisms

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1506
Author(s):  
Angelos Papaspyropoulos ◽  
Nefeli Lagopati ◽  
Ioanna Mourkioti ◽  
Andriani Angelopoulou ◽  
Spyridon Kyriazis ◽  
...  
Keyword(s):  
Therapeutic Potential ◽  
Strand Break ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Dsb Repair ◽  
Repair Mechanisms ◽  
Non Coding Rnas ◽  
Repair Pathways ◽  
Non Homologous End Joining ◽  
Living Organisms

Protection of genome integrity is vital for all living organisms, particularly when DNA double-strand breaks (DSBs) occur. Eukaryotes have developed two main pathways, namely Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR), to repair DSBs. While most of the current research is focused on the role of key protein players in the functional regulation of DSB repair pathways, accumulating evidence has uncovered a novel class of regulating factors termed non-coding RNAs. Non-coding RNAs have been found to hold a pivotal role in the activation of DSB repair mechanisms, thereby safeguarding genomic stability. In particular, long non-coding RNAs (lncRNAs) have begun to emerge as new players with vast therapeutic potential. This review summarizes important advances in the field of lncRNAs, including characterization of recently identified lncRNAs, and their implication in DSB repair pathways in the context of tumorigenesis.

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.

Precise assignment of the heavy-strand promoter of mouse mitochondrial DNA: cognate start sites are not required for transcriptional initiation

1986 ◽  
Vol 6 (9) ◽  
pp. 3262-3267
Author(s):  
D D Chang ◽  
D A Clayton
Keyword(s):  
Mitochondrial Dna ◽  
Dna Sequences ◽  
High Rate ◽  
In Vitro Assays ◽  
Regulatory Sequences ◽  
Sufficient Information ◽  
Mitochondrial Rna ◽  
Loop Region ◽  
Heavy Strand

Transcription of the heavy strand of mouse mitochondrial DNA starts from two closely spaced, distinct sites located in the displacement loop region of the genome. We report here an analysis of regulatory sequences required for faithful transcription from these two sites. Data obtained from in vitro assays demonstrated that a 51-base-pair region, encompassing nucleotides -40 to +11 of the downstream start site, contains sufficient information for accurate transcription from both start sites. Deletion of the 3' flanking sequences, including one or both start sites to -17, resulted in the initiation of transcription by the mitochondrial RNA polymerase from alternative sites within vector DNA sequences. This feature places the mouse heavy-strand promoter uniquely among other known mitochondrial promoters, all of which absolutely require cognate start sites for transcription. Comparison of the heavy-strand promoter with those of other vertebrate mitochondrial DNAs revealed a remarkably high rate of sequence divergence among species.

scholarly journals The Transcriptional Histone Acetyltransferase Cofactor TRRAP Associates with the MRN Repair Complex and Plays a Role in DNA Double-Strand Break Repair

2006 ◽  
Vol 26 (2) ◽  
pp. 402-412 ◽  
Author(s):  
Flavie Robert ◽  
Sara Hardy ◽  
Zita Nagy ◽  
Céline Baldeyron ◽  
Rabih Murr ◽  
...  
Keyword(s):  
Cell Cycle Progression ◽  
Histone Acetyltransferase ◽  
Gel Filtration ◽  
Embryonic Stem ◽  
Strand Break ◽  
Mitotic Checkpoint ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Mrn Complex

ABSTRACT Transactivation-transformation domain-associated protein (TRRAP) is a component of several multiprotein histone acetyltransferase (HAT) complexes implicated in transcriptional regulation. TRRAP was shown to be required for the mitotic checkpoint and normal cell cycle progression. MRE11, RAD50, and NBS1 (product of the Nijmegan breakage syndrome gene) form the MRN complex that is involved in the detection, signaling, and repair of DNA double-strand breaks (DSBs). By using double immunopurification, mass spectrometry, and gel filtration, we describe the stable association of TRRAP with the MRN complex. The TRRAP-MRN complex is not associated with any detectable HAT activity, while the isolated other TRRAP complexes, containing either GCN5 or TIP60, are. TRRAP-depleted extracts show a reduced nonhomologous DNA end-joining activity in vitro. Importantly, small interfering RNA knockdown of TRRAP in HeLa cells or TRRAP knockout in mouse embryonic stem cells inhibit the DSB end-joining efficiency and the precise nonhomologous end-joining process, further suggesting a functional involvement of TRRAP in the DSB repair processes. Thus, TRRAP may function as a molecular link between DSB signaling, repair, and chromatin remodeling.

scholarly journals RAP80 suppresses the vulnerability of R-loops during DNA double-strand break repair

2021 ◽  
Author(s):  
Takaaki Yasuhara ◽  
Reona Kato ◽  
Motohiro Yamauchi ◽  
Yuki Uchihara ◽  
Lee Zou ◽  
...  
Keyword(s):  
Active Regions ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Critical Component ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Chromosome Translocations ◽  
Dna Double Strand Break

AbstractR-loops, consisting of ssDNA and DNA-RNA hybrids, are potentially vulnerable unless they are appropriately processed. Recent evidence suggests that R-loops can form in the proximity of DNA double-strand breaks (DSBs) within transcriptionally active regions. Yet, how the vulnerability of R-loops is overcome during DSB repair remains unclear. Here, we identify RAP80 as a factor suppressing the vulnerability of ssDNA in R-loops and chromosome translocations and deletions during DSB repair. Mechanistically, RAP80 prevents unscheduled nucleolytic processing of ssDNA in R-loops by CtIP. This mechanism promotes efficient DSB repair via transcription-associated end-joining dependent on BRCA1, Polθ, and LIG1/3. Thus, RAP80 suppresses the vulnerability of R-loops during DSB repair, thereby precluding genomic abnormalities in a critical component of the genome caused by deleterious R-loop processing.

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