scholarly journals Loss of BRCA1 or BRCA2 markedly increases the rate of base substitution mutagenesis and has distinct effects on genomic deletions

Oncogene ◽  
2016 ◽  
Vol 36 (6) ◽  
pp. 746-755 ◽  
Author(s):  
J Zámborszky ◽  
B Szikriszt ◽  
J Z Gervai ◽  
O Pipek ◽  
Á Póti ◽  
...  
Keyword(s):  
Large Scale ◽  
Dna Lesions ◽  
High Rate ◽  
Base Substitution ◽  
Loss Of Function ◽  
End Joining ◽  
Genomic Deletions ◽  
Non Homologous End Joining ◽  
Risk Of Cancer ◽  
Mutant Cells

Abstract Loss-of-function mutations in the BRCA1 and BRCA2 genes increase the risk of cancer. Owing to their function in homologous recombination repair, much research has focused on the unstable genomic phenotype of BRCA1/2 mutant cells manifest mainly as large-scale rearrangements. We used whole-genome sequencing of multiple isogenic chicken DT40 cell clones to precisely determine the consequences of BRCA1/2 loss on all types of genomic mutagenesis. Spontaneous base substitution mutation rates increased sevenfold upon the disruption of either BRCA1 or BRCA2, and the arising mutation spectra showed strong and specific correlation with a mutation signature associated with BRCA1/2 mutant tumours. To model endogenous alkylating damage, we determined the mutation spectrum caused by methyl methanesulfonate (MMS), and showed that MMS also induces more base substitution mutations in BRCA1/2-deficient cells. Spontaneously arising and MMS-induced insertion/deletion mutations and large rearrangements were also more common in BRCA1/2 mutant cells compared with the wild-type control. A difference in the short deletion phenotypes of BRCA1 and BRCA2 suggested distinct roles for the two proteins in the processing of DNA lesions, as BRCA2 mutants contained more short deletions, with a wider size distribution, which frequently showed microhomology near the breakpoints resembling repair by non-homologous end joining. An increased and prolonged gamma-H2AX signal in MMS-treated BRCA1/2 cells suggested an aberrant processing of stalled replication forks as the cause of increased mutagenesis. The high rate of base substitution mutagenesis demonstrated by our experiments is likely to significantly contribute to the oncogenic effect of the inactivation of BRCA1 or BRCA2.

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 Genome-scale CRISPR screens are efficient in non-homologous end-joining deficient cells

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Joana Ferreira da Silva ◽  
Sejla Salic ◽  
Marc Wiedner ◽  
Paul Datlinger ◽  
Patrick Essletzbichler ◽  
...  
Keyword(s):  
Genome Editing ◽  
Large Scale ◽  
Dependent Manner ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Knock Out ◽  
Genetic Ablation ◽  
Alternative End Joining ◽  
Non Homologous End Joining ◽  
Genome Scale

Abstract The mutagenic repair of Cas9 generated breaks is thought to predominantly rely on non-homologous end-joining (NHEJ), leading to insertions and deletions within DNA that culminate in gene knock-out (KO). In this study, by taking focused as well as genome-wide approaches, we show that this pathway is dispensable for the repair of such lesions. Genetic ablation of NHEJ is fully compensated for by alternative end joining (alt-EJ), in a POLQ-dependent manner, resulting in a distinct repair signature with larger deletions that may be exploited for large-scale genome editing. Moreover, we show that cells deficient for both NHEJ and alt-EJ were still able to repair CRISPR-mediated DNA double-strand breaks, highlighting how little is yet known about the mechanisms of CRISPR-based genome editing.

scholarly journals DNA Strand Break-Sensing Molecule Poly(ADP-Ribose) Polymerase Cooperates with p53 in Telomere Function, Chromosome Stability, and Tumor Suppression

2001 ◽  
Vol 21 (12) ◽  
pp. 4046-4054 ◽  
Author(s):  
Wei-Min Tong ◽  
M. Prakash Hande ◽  
Peter M. Lansdorp ◽  
Zhao-Qi Wang
Keyword(s):  
Strand Break ◽  
Cell Cycle Checkpoints ◽  
High Rate ◽  
Loss Of Function ◽  
Li Fraumeni Syndrome ◽  
Dna Strand Break ◽  
Telomere Function ◽  
Dna Strand ◽  
High Degree ◽  
Mutant Cells

ABSTRACT Genomic instability is often caused by mutations in genes that are involved in DNA repair and/or cell cycle checkpoints, and it plays an important role in tumorigenesis. Poly(ADP-ribose) polymerase (PARP) is a DNA strand break-sensing molecule that is involved in the response to DNA damage and the maintenance of telomere function and genomic stability. We report here that, compared to single-mutant cells, PARP and p53 double-mutant cells exhibit many severe chromosome aberrations, including a high degree of aneuploidy, fragmentations, and end-to-end fusions, which may be attributable to telomere dysfunction. While PARP−/− cells showed telomere shortening and p53−/− cells showed normal telomere length, inactivation of PARP in p53−/− cells surprisingly resulted in very long and heterogeneous telomeres, suggesting a functional interplay between PARP and p53 at the telomeres. Strikingly, PARP deficiency widens the tumor spectrum in mice deficient in p53, resulting in a high frequency of carcinomas in the mammary gland, lung, prostate, and skin, as well as brain tumors, reminiscent of Li-Fraumeni syndrome in humans. The enhanced tumorigenesis is likely to be caused by PARP deficiency, which facilitates the loss of function of tumor suppressor genes as demonstrated by a high rate of loss of heterozygosity at the p53 locus in these tumors. These results indicate that PARP and p53 interact to maintain genome integrity and identify PARP as a cofactor for suppressing tumorigenesis.

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.

scholarly journals Efficient Generation of Knock-In Zebrafish Models for Inherited Disorders Using CRISPR-Cas9 Ribonucleoprotein Complexes

2021 ◽  
Vol 22 (17) ◽  
pp. 9429
Author(s):  
Erik de Vrieze ◽  
Suzanne E. de Bruijn ◽  
Janine Reurink ◽  
Sanne Broekman ◽  
Vince van de Riet ◽  
...  
Keyword(s):  
Dna Repair ◽  
Cost Effective ◽  
Patient Specific ◽  
Loss Of Function ◽  
Repair Pathway ◽  
End Joining ◽  
Inherited Disorders ◽  
Ribonucleoprotein Complexes ◽  
Cost Effective Method ◽  
Non Homologous End Joining

CRISPR-Cas9-based genome-editing is a highly efficient and cost-effective method to generate zebrafish loss-of-function alleles. However, introducing patient-specific variants into the zebrafish genome with CRISPR-Cas9 remains challenging. Targeting options can be limited by the predetermined genetic context, and the efficiency of the homology-directed DNA repair pathway is relatively low. Here, we illustrate our efficient approach to develop knock-in zebrafish models using two previously variants associated with hereditary sensory deficits. We employ sgRNA-Cas9 ribonucleoprotein (RNP) complexes that are micro-injected into the first cell of fertilized zebrafish eggs together with an asymmetric, single-stranded DNA template containing the variant of interest. The introduction of knock-in events was confirmed by massive parallel sequencing of genomic DNA extracted from a pool of injected embryos. Simultaneous morpholino-induced blocking of a key component of the non-homologous end joining DNA repair pathway, Ku70, improved the knock-in efficiency for one of the targets. Our use of RNP complexes provides an improved knock-in efficiency as compared to previously published studies. Correct knock-in events were identified in 3–8% of alleles, and 30–45% of injected animals had the target variant in their germline. The detailed technical and procedural insights described here provide a valuable framework for the efficient development of knock-in zebrafish models.

scholarly journals Beta Human Papillomavirus 8E6 Attenuates Non-Homologous End Joining by Hindering DNA-PKcs Activity

Cancers ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 2356
Author(s):  
Changkun Hu ◽  
Taylor Bugbee ◽  
Monica Gamez ◽  
Nicholas A. Wallace
Keyword(s):  
Dna Repair ◽  
Viral Infections ◽  
Dna Lesions ◽  
Human Papillomaviruses ◽  
Molecular Evidence ◽  
Repair Pathway ◽  
End Joining ◽  
Dsb Repair ◽  
Dependent Protein ◽  
Non Homologous End Joining

Cutaneous viral infections occur in a background of near continual exposure to environmental genotoxins, like UV radiation in sunlight. Failure to repair damaged DNA is an established driver of tumorigenesis and substantial cellular resources are devoted to repairing DNA lesions. Beta-human papillomaviruses (β-HPVs) attenuate DNA repair signaling. However, their role in human disease is unclear. Some have proposed that β-HPV promotes tumorigenesis, while others suggest that β-HPV protects against skin cancer. Most of the molecular evidence that β-HPV impairs DNA repair has been gained via characterization of the E6 protein from β-HPV 8 (β-HPV 8E6). Moreover, β-HPV 8E6 hinders DNA repair by binding and destabilizing p300, a transcription factor for multiple DNA repair genes. By reducing p300 availability, β-HPV 8E6 attenuates a major double strand DNA break (DSB) repair pathway, homologous recombination. Here, β-HPV 8E6 impairs another DSB repair pathway, non-homologous end joining (NHEJ). Specifically, β-HPV 8E6 acts by attenuating DNA-dependent protein kinase (DNA-PK) activity, a critical NHEJ kinase. This includes DNA-PK activation and the downstream of steps in the pathway associated with DNA-PK activity. Notably, β-HPV 8E6 inhibits NHEJ through p300 dependent and independent means. Together, these data expand the known genome destabilizing capabilities of β-HPV 8E6.

scholarly journals ZNF281 is recruited on DNA breaks to facilitate DNA repair by non-homologous end joining

Oncogene ◽  
2019 ◽  
Vol 39 (4) ◽  
pp. 754-766 ◽  
Author(s):  
Sara Nicolai ◽  
Robert Mahen ◽  
Giuseppe Raschellà ◽  
Alberto Marini ◽  
Marco Pieraccioli ◽  
...  
Keyword(s):  
Dna Damage ◽  
Zinc Finger ◽  
Zinc Finger Protein ◽  
Dna Lesions ◽  
Repair Pathway ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
End Joining ◽  
Nhej Repair ◽  
Non Homologous End Joining

Abstract Efficient repair of DNA double-strand breaks (DSBs) is of critical importance for cell survival. Although non-homologous end joining (NHEJ) is the most used DSBs repair pathway in the cells, how NHEJ factors are sequentially recruited to damaged chromatin remains unclear. Here, we identify a novel role for the zinc-finger protein ZNF281 in participating in the ordered recruitment of the NHEJ repair factor XRCC4 at damage sites. ZNF281 is recruited to DNA lesions within seconds after DNA damage through a mechanism dependent on its DNA binding domain and, at least in part, on poly-ADP ribose polymerase (PARP) activity. ZNF281 binds XRCC4 through its zinc-finger domain and facilitates its recruitment to damaged sites. Consequently, depletion of ZNF281 impairs the efficiency of the NHEJ repair pathway and decreases cell viability upon DNA damage. Survival analyses from datasets of commonly occurring human cancers show that higher levels of ZNF281 correlate with poor prognosis of patients treated with DNA-damaging therapies. Thus, our results define a late ZNF281-dependent regulatory step of NHEJ complex assembly at DNA lesions and suggest additional possibilities for cancer patients’ stratification and for the development of personalised therapeutic strategies.

scholarly journals CDCA7 and HELLS suppress DNA:RNA hybrid-associated DNA damage at pericentromeric repeats

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Motoko Unoki ◽  
Jafar Sharif ◽  
Yuichiro Saito ◽  
Guillaume Velasco ◽  
Claire Francastel ◽  
...  
Keyword(s):  
Dna Damage ◽  
Autosomal Recessive ◽  
Ectopic Expression ◽  
Autosomal Recessive Disorder ◽  
End Joining ◽  
Icf Syndrome ◽  
Centromeric Instability ◽  
Non Homologous End Joining ◽  
Genomic Regions ◽  
Mutant Cells

Abstract Immunodeficiency, centromeric instability, facial anomalies (ICF) syndrome is a rare autosomal recessive disorder that is caused by mutations in either DNMT3B, ZBTB24, CDCA7, HELLS, or yet unidentified gene(s). Previously, we reported that the CDCA7/HELLS chromatin remodeling complex facilitates non-homologous end-joining. Here, we show that the same complex is required for the accumulation of proteins on nascent DNA, including the DNMT1/UHRF1 maintenance DNA methylation complex as well as proteins involved in the resolution or prevention of R-loops composed of DNA:RNA hybrids and ssDNA. Consistent with the hypomethylation state of pericentromeric repeats, the transcription and formation of aberrant DNA:RNA hybrids at the repeats were increased in ICF mutant cells. Furthermore, the ectopic expression of RNASEH1 reduced the accumulation of DNA damage at a broad range of genomic regions including pericentromeric repeats in these cells. Hence, we propose that hypomethylation due to inefficient DNMT1/UHRF1 recruitment at pericentromeric repeats by defects in the CDCA7/HELLS complex could induce pericentromeric instability, which may explain a part of the molecular pathogenesis of ICF syndrome.

scholarly journals Genome-wide microhomologies enable precise template-free editing of biologically relevant deletion mutations

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Janin Grajcarek ◽  
Jean Monlong ◽  
Yoko Nishinaka-Arai ◽  
Michiko Nakamura ◽  
Miki Nagai ◽  
...  
Keyword(s):  
Loss Of Function ◽  
End Joining ◽  
Protein Coding ◽  
Strand Breaks ◽  
Deletion Mutations ◽  
Biologically Relevant ◽  
Genome Wide ◽  
Template Free ◽  
Non Homologous End Joining ◽  
Induced Pluripotent

Abstract The functional effect of a gene edit by designer nucleases depends on the DNA repair outcome at the targeted locus. While non-homologous end joining (NHEJ) repair results in various mutations, microhomology-mediated end joining (MMEJ) creates precise deletions based on the alignment of flanking microhomologies (µHs). Recently, the sequence context surrounding nuclease-induced double strand breaks (DSBs) has been shown to predict repair outcomes, for which µH plays an important role. Here, we survey naturally occurring human deletion variants and identify that 11 million or 57% are flanked by µHs, covering 88% of protein-coding genes. These biologically relevant mutations are candidates for precise creation in a template-free manner by MMEJ repair. Using CRISPR-Cas9 in human induced pluripotent stem cells (hiPSCs), we efficiently create pathogenic deletion mutations for demonstrable disease models with both gain- and loss-of-function phenotypes. We anticipate this dataset and gene editing strategy to enable functional genetic studies and drug screening.

scholarly journals Polo-like kinase 3 regulates CtIP during DNA double-strand break repair in G1

2014 ◽  
Vol 206 (7) ◽  
pp. 877-894 ◽  
Author(s):  
Olivia Barton ◽  
Steffen C. Naumann ◽  
Ronja Diemer-Biehs ◽  
Julia Künzel ◽  
Monika Steinlage ◽  
...  
Keyword(s):  
Large Scale ◽  
Strand Break ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Interacting Protein ◽  
Genomic Deletions ◽  
Cellular Processes ◽  
Dna Double Strand Break ◽  
Radiation Induced ◽  
Alternative Nhej

DNA double-strand breaks (DSBs) are repaired by nonhomologous end joining (NHEJ) or homologous recombination (HR). The C terminal binding protein–interacting protein (CtIP) is phosphorylated in G2 by cyclin-dependent kinases to initiate resection and promote HR. CtIP also exerts functions during NHEJ, although the mechanism phosphorylating CtIP in G1 is unknown. In this paper, we identify Plk3 (Polo-like kinase 3) as a novel DSB response factor that phosphorylates CtIP in G1 in a damage-inducible manner and impacts on various cellular processes in G1. First, Plk3 and CtIP enhance the formation of ionizing radiation-induced translocations; second, they promote large-scale genomic deletions from restriction enzyme-induced DSBs; third, they are required for resection and repair of complex DSBs; and finally, they regulate alternative NHEJ processes in Ku−/− mutants. We show that mutating CtIP at S327 or T847 to nonphosphorylatable alanine phenocopies Plk3 or CtIP loss. Plk3 binds to CtIP phosphorylated at S327 via its Polo box domains, which is necessary for robust damage-induced CtIP phosphorylation at S327 and subsequent CtIP phosphorylation at T847.

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