scholarly journals ATM orchestrates the DNA-damage response to counter toxic non-homologous end-joining at broken replication forks

2018 ◽  
Author(s):  
Gabriel Balmus ◽  
Domenic Pilger ◽  
Julia Coates ◽  
Mukerrem Demir ◽  
Matylda Sczaniecka-Clift ◽  
...  
Keyword(s):  
Genetic Resistance ◽  
Resistance Mechanisms ◽  
Replication Forks ◽  
End Joining ◽  
Strand Breaks ◽  
Dna Damaging Agents ◽  
Genome Wide ◽  
Topoisomerase Poison ◽  
Recombination Repair ◽  
Non Homologous End Joining

SummaryMutations in the ATM tumor suppressor confer hypersensitivity to DNA-damaging agents. To explore genetic resistance mechanisms, we performed genome-wide CRISPR-Cas9 screens in cells treated with the DNA topoisomerase poison topotecan. Thus, we establish that loss of terminal components of the non-homologous end-joining (NHEJ) machinery or the BRCA1-A complex specifically confers topotecan resistance to ATM-deficient cells. We show that hypersensitivity of ATM-mutant cells to topotecan or the poly-(ADP-ribose) polymerase inhibitor olaparib is due to delayed homologous recombination repair at DNA-replication-fork-associated double-strand breaks (DSBs), resulting in toxic NHEJ-mediated chromosome fusions. Accordingly, restoring legitimate repair in ATM-deficient cells, either by preventing NHEJ DNA ligation or by enhancing DSB-resection by BRCA1-A complex inactivation, markedly suppresses this toxicity. Our work suggests opportunities for patient stratification in ATM-deficient cancers and when using ATM inhibitors in the clinic, and identifies additional therapeutic vulnerabilities that might be exploited when such cancers evolve drug resistance.One Sentence SummaryATM counteracts toxic NHEJ at broken replication forks

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 miR-21-mediated Radioresistance Occurs via Promoting Repair of DNA Double Strand Breaks

2017 ◽  
Vol 292 (8) ◽  
pp. 3531-3540 ◽  
Author(s):  
Baocheng Hu ◽  
Xiang Wang ◽  
Shuofeng Hu ◽  
Xiaomin Ying ◽  
Ping Wang ◽  
...  
Keyword(s):  
Cyclin D1 ◽  
Opposite Effect ◽  
Double Strand Breaks ◽  
Human Tumors ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Recombination Repair ◽  
Non Homologous End Joining ◽  
Novel Target

miR-21, as an oncogene that overexpresses in most human tumors, is involved in radioresistance; however, the mechanism remains unclear. Here, we demonstrate that miR-21-mediated radioresistance occurs through promoting repair of DNA double strand breaks, which includes facilitating both non-homologous end-joining (NHEJ) and homologous recombination repair (HRR). The miR-21-promoted NHEJ occurs through targeting GSK3B (a novel target of miR-21), which affects the CRY2/PP5 pathway and in turn increases DNA-PKcs activity. The miR-21-promoted HRR occurs through targeting both GSK3B and CDC25A (a known target of miR-21), which neutralizes the effects of targeting GSK3B-induced CDC25A increase because GSK3B promotes degradation of both CDC25A and cyclin D1, but CDC25A and cyclin D1 have an opposite effect on HRR. A negative correlation of expression levels between miR-21 and GSK3β exists in a subset of human tumors. Our results not only elucidate miR-21-mediated radioresistance, but also provide potential new targets for improving radiotherapy.

scholarly journals Role of the Mre11 Complex in Preserving Genome Integrity

Genes ◽  
2018 ◽  
Vol 9 (12) ◽  
pp. 589 ◽  
Author(s):  
Julyun Oh ◽  
Lorraine Symington
Keyword(s):  
Cell Cycle Progression ◽  
Genome Integrity ◽  
Replication Forks ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Atm Kinase ◽  
Strand Breaks ◽  
Mre11 Complex ◽  
Non Homologous End Joining ◽  
Dna Ends

DNA double-strand breaks (DSBs) are hazardous lesions that threaten genome integrity and cell survival. The DNA damage response (DDR) safeguards the genome by sensing DSBs, halting cell cycle progression and promoting repair through either non-homologous end joining (NHEJ) or homologous recombination (HR). The Mre11-Rad50-Xrs2/Nbs1 (MRX/N) complex is central to the DDR through its structural, enzymatic, and signaling roles. The complex tethers DNA ends, activates the Tel1/ATM kinase, resolves protein-bound or hairpin-capped DNA ends, and maintains telomere homeostasis. In addition to its role at DSBs, MRX/N associates with unperturbed replication forks, as well as stalled replication forks, to ensure complete DNA synthesis and to prevent chromosome rearrangements. Here, we summarize the significant progress made in characterizing the MRX/N complex and its various activities in chromosome metabolism.

scholarly journals Structural basis for proficient oxidized ribonucleotide insertion in double strand break repair

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Joonas A. Jamsen ◽  
Akira Sassa ◽  
Lalith Perera ◽  
David D. Shock ◽  
William A. Beard ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Time Lapse ◽  
Strand Break ◽  
Structural Basis ◽  
End Joining ◽  
Strand Breaks ◽  
Nucleotide Pools ◽  
Nucleotide Insertion ◽  
Non Homologous End Joining

AbstractReactive oxygen species (ROS) oxidize cellular nucleotide pools and cause double strand breaks (DSBs). Non-homologous end-joining (NHEJ) attaches broken chromosomal ends together in mammalian cells. Ribonucleotide insertion by DNA polymerase (pol) μ prepares breaks for end-joining and this is required for successful NHEJ in vivo. We previously showed that pol μ lacks discrimination against oxidized dGTP (8-oxo-dGTP), that can lead to mutagenesis, cancer, aging and human disease. Here we reveal the structural basis for proficient oxidized ribonucleotide (8-oxo-rGTP) incorporation during DSB repair by pol μ. Time-lapse crystallography snapshots of structural intermediates during nucleotide insertion along with computational simulations reveal substrate, metal and side chain dynamics, that allow oxidized ribonucleotides to escape polymerase discrimination checkpoints. Abundant nucleotide pools, combined with inefficient sanitization and repair, implicate pol μ mediated oxidized ribonucleotide insertion as an emerging source of widespread persistent mutagenesis and genomic instability.

scholarly journals Genetic dissection of vertebrate 53BP1: A major role in non-homologous end joining of DNA double strand breaks

DNA Repair ◽  
2006 ◽  
Vol 5 (6) ◽  
pp. 741-749 ◽  
Author(s):  
Kyoko Nakamura ◽  
Wataru Sakai ◽  
Takuo Kawamoto ◽  
Ronan T. Bree ◽  
Noel F. Lowndes ◽  
...  
Keyword(s):  
Double Strand Breaks ◽  
Genetic Dissection ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Non Homologous End Joining

scholarly journals Beyond DNA Repair: DNA-PKcs in Tumor Metastasis, Metabolism and Immunity

Cancers ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 3389
Author(s):  
Haitang Yang ◽  
Feng Yao ◽  
Thomas M. Marti ◽  
Ralph A. Schmid ◽  
Ren-Wang Peng
Keyword(s):  
Tumor Metastasis ◽  
Immune Escape ◽  
Critical Role ◽  
Large Body ◽  
Tumor Development ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Dependent Protein ◽  
Non Homologous End Joining

The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a key component of the DNA-PK complex that has a well-characterized function in the non-homologous end-joining repair of DNA double-strand breaks. Since its identification, a large body of evidence has demonstrated that DNA-PKcs is frequently overexpressed in cancer, plays a critical role in tumor development and progression, and is associated with poor prognosis of cancer patients. Intriguingly, recent studies have suggested novel functions beyond the canonical role of DNA-PKcs, which has transformed the paradigm of DNA-PKcs in tumorigenesis and has reinvigorated the interest to target DNA-PKcs for cancer treatment. In this review, we update recent advances in DNA-PKcs, in particular the emerging roles in tumor metastasis, metabolic dysregulation, and immune escape. We further discuss the possible molecular basis that underpins the pleiotropism of DNA-PKcs in cancer. Finally, we outline the biomarkers that may predict the therapeutic response to DNA-PKcs inhibitor therapy. Understanding the functional repertoire of DNA-PKcs will provide mechanistic insights of DNA-PKcs in malignancy and, more importantly, may revolutionize the design and utility of DNA-PKcs-based precision cancer therapy.

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