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.

scholarly journals A robust CRISPR–Cas9-based fluorescent reporter assay for the detection and quantification of DNA double-strand break repair

2020 ◽  
Vol 48 (21) ◽  
pp. e126-e126
Author(s):  
Rebeka Eki ◽  
Jane She ◽  
Mahmut Parlak ◽  
Mouadh Benamar ◽  
Kang-Ping Du ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
High Sensitivity ◽  
Strand Break ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Dsb Repair ◽  
Fluorescent Reporter ◽  
Homology Directed Repair ◽  
Non Homologous End Joining ◽  
Detection And Quantification

Abstract DNA double-strand breaks (DSBs) are highly cytotoxic lesions that can lead to chromosome rearrangements, genomic instability and cell death. Consequently, cells have evolved multiple mechanisms to efficiently repair DSBs to preserve genomic integrity. We have developed a DSB repair assay system, designated CDDR (CRISPR–Cas9-based Dual-fluorescent DSB Repair), that enables the detection and quantification of DSB repair outcomes in mammalian cells with high precision. CDDR is based on the introduction and subsequent resolution of one or two DSB(s) in an intrachromosomal fluorescent reporter following the expression of Cas9 and sgRNAs targeting the reporter. CDDR can discriminate between high-fidelity (HF) and error-prone non-homologous end-joining (NHEJ), as well as between proximal and distal NHEJ repair. Furthermore, CDDR can detect homology-directed repair (HDR) with high sensitivity. Using CDDR, we found HF-NHEJ to be strictly dependent on DNA Ligase IV, XRCC4 and XLF, members of the canonical branch of NHEJ pathway (c-NHEJ). Loss of these genes also stimulated HDR, and promoted error-prone distal end-joining. Deletion of the DNA repair kinase ATM, on the other hand, stimulated HF-NHEJ and suppressed HDR. These findings demonstrate the utility of CDDR in characterizing the effect of repair factors and in elucidating the balance between competing DSB repair pathways.

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.

Double strand break (DSB) repair pathways in plants and their application in genome engineering

2021 ◽  
pp. 27-62
Author(s):  
Natalja Beying ◽  
◽  
Carla Schmidt ◽  
Holger Puchta ◽  
◽  
...  
Keyword(s):  
Genome Engineering ◽  
Plant Cells ◽  
Strand Break ◽  
End Joining ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Repair Mechanisms ◽  
Repair Pathways ◽  
Non Homologous End Joining ◽  
Underlying Mechanisms

In genome engineering, after targeted induction of double strand breaks (DSBs) researchers take advantage of the organisms’ own repair mechanisms to induce different kinds of sequence changes into the genome. Therefore, understanding of the underlying mechanisms is essential. This chapter will review in detail the two main pathways of DSB repair in plant cells, non-homologous end joining (NHEJ) and homologous recombination (HR) and sum up what we have learned over the last decades about them. We summarize the different models that have been proposed and set these into relation with the molecular outcomes of different classes of DSB repair. Moreover, we describe the factors that have been identified to be involved in these pathways. Applying this knowledge of DSB repair should help us to improve the efficiency of different types of genome engineering in plants.

141 INDICATIONS FOR DNA DOUBLE-STRAND BREAK REPAIR IN EARLY BOVINE EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 188
Author(s):  
A. Brero ◽  
D. Koehler ◽  
T. Cremer ◽  
E. Wolf ◽  
V. Zakhartchenko
Keyword(s):  
Dna Repair ◽  
Homologous Recombination ◽  
Strand Break ◽  
Dna Lesions ◽  
Homologous Recombination Repair ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Male And Female ◽  
Γh2ax Foci ◽  
Recombination Repair

DNA double-strand breaks (DSBs) are considered the most severe type of DNA lesions, because such lesions, if unrepaired, lead to a loss of genome integrity. Soon after induction of DSBs, chromatin surrounding the damage is modified by phosphorylation of the histone variant H2AX, generating so-called γH2AX, which is a hallmark of DSBs (Takahashi et al. 2005 Cancer Lett. 229, 171–179). γH2AX appears to be a signal for the recruitment of proteins constituting the DNA repair machinery. Depending on the type of damage and the cell cycle stage of the affected cell, DSBs are repaired either by nonhomologous end joining or by homologous recombination using the sister chromatid DNA as template (Hoeijmakers 2001 Nature 411, 366–374). We used immunofluorescence to analyze chromatin composition during bovine development and found γH2AX foci in both male and female pronuclei of IVF embryos. The number and size of foci varied considerably between embryos and between the male and female pronuclei. To test whether the observed γH2AX foci represented sites of active DNA repair, we co-stained IVF zygotes for γH2AX and 3 different proteins involved in homologous recombination repair of DSBs: NBS1 (phosphorylated at amino acid serine 343), 53BP1, and Rad51. We found co-localization of γH2AX foci with phosphorylated NBS1 as well as with Rad51 but did not observe the presence of 53BP1 at γH2AX foci in IVF zygotes. Our finding shows the presence of DSBs in IVF zygotes and suggests the capability of homologous recombination repair. The lack of 53BP1, a component of homologous recombination repair, which usually co-localizes with γH2AX foci at exogenously induced DSBs (Schultz et al. 2000 J. Cell. Biol. 151, 1381–1390) poses the possibility that the mechanism present in early embryos differs substantially from that involved in DNA repair of DSBs in somatic cells.

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 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 DNA Substrate Dependence of p53-Mediated Regulation of Double-Strand Break Repair

2002 ◽  
Vol 22 (17) ◽  
pp. 6306-6317 ◽  
Author(s):  
Nuray Akyüz ◽  
Gisa S. Boehden ◽  
Silke Süsse ◽  
Andreas Rimek ◽  
Ute Preuss ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Strand Break ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Dsb Repair ◽  
P53 Expression ◽  
Strand Breaks ◽  
Single Strand Breaks ◽  
Multistep Process ◽  
Non Homologous End Joining

ABSTRACT DNA double-strand breaks (DSBs) arise spontaneously after the conversion of DNA adducts or single-strand breaks by DNA repair or replication and can be introduced experimentally by expression of specific endonucleases. Correct repair of DSBs is central to the maintenance of genomic integrity in mammalian cells, since errors give rise to translocations, deletions, duplications, and expansions, which accelerate the multistep process of tumor progression. For p53 direct regulatory roles in homologous recombination (HR) and in non-homologous end joining (NHEJ) were postulated. To systematically analyze the involvement of p53 in DSB repair, we generated a fluorescence-based assay system with a series of episomal and chromosomally integrated substrates for I-SceI meganuclease-triggered repair. Our data indicate that human wild-type p53, produced either stably or transiently in a p53-negative background, inhibits HR between substrates for conservative HR (cHR) and for gene deletions. NHEJ via microhomologies flanking the I-SceI cleavage site was also downregulated after p53 expression. Interestingly, the p53-dependent downregulation of homology-directed repair was maximal during cHR between sequences with short homologies. Inhibition was minimal during recombination between substrates that support reporter gene reconstitution by HR and NHEJ. p53 with a hotspot mutation at codon 281, 273, 248, 175, or 143 was severely defective in regulating DSB repair (frequencies elevated up to 26-fold). For the transcriptional transactivation-inactive variant p53(138V) a defect became apparent with short homologies only. These results suggest that p53 plays a role in restraining DNA exchange between imperfectly homologous sequences and thereby in suppressing tumorigenic genome rearrangements.

scholarly journals Rapid Recruitment of BRCA1 to DNA Double-Strand Breaks Is Dependent on Its Association with Ku80

2008 ◽  
Vol 28 (24) ◽  
pp. 7380-7393 ◽  
Author(s):  
Leizhen Wei ◽  
Li Lan ◽  
Zehui Hong ◽  
Akira Yasui ◽  
Chikashi Ishioka ◽  
...  
Keyword(s):  
Susceptibility Gene ◽  
Repair Process ◽  
Strand Break ◽  
Missense Mutations ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Dsb Repair ◽  
Nhej Pathway ◽  
C Terminus ◽  
N Terminus

ABSTRACT BRCA1 is the first susceptibility gene to be linked to breast and ovarian cancers. Although mounting evidence has indicated that BRCA1 participates in DNA double-strand break (DSB) repair pathways, its precise mechanism is still unclear. Here, we analyzed the in situ response of BRCA1 at DSBs produced by laser microirradiation. The amino (N)- and carboxyl (C)-terminal fragments of BRCA1 accumulated independently at DSBs with distinct kinetics. The N-terminal BRCA1 fragment accumulated immediately after laser irradiation at DSBs and dissociated rapidly. In contrast, the C-terminal fragment of BRCA1 accumulated more slowly at DSBs but remained at the sites. Interestingly, rapid accumulation of the BRCA1 N terminus, but not the C terminus, at DSBs depended on Ku80, which functions in the nonhomologous end-joining (NHEJ) pathway, independently of BARD1, which binds to the N terminus of BRCA1. Two small regions in the N terminus of BRCA1 independently accumulated at DSBs and interacted with Ku80. Missense mutations found within the N terminus of BRCA1 in cancers significantly changed the kinetics of its accumulation at DSBs. A P142H mutant failed to associate with Ku80 and restore resistance to irradiation in BRCA1-deficient cells. These might provide a molecular basis of the involvement of BRCA1 in the NHEJ pathway of the DSB repair process.

scholarly journals Cdc14A and Cdc14B Redundantly Regulate DNA Double-Strand Break Repair

2015 ◽  
Vol 35 (21) ◽  
pp. 3657-3668 ◽  
Author(s):  
Han Lin ◽  
Kyungsoo Ha ◽  
Guojun Lu ◽  
Xiao Fang ◽  
Ranran Cheng ◽  
...  
Keyword(s):  
Dna Repair ◽  
Strand Break ◽  
Mitotic Exit ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Dsb Repair ◽  
Downstream Target ◽  
Damage Repair ◽  
Repair Pathways ◽  
Repair Defect

Cdc14 is a phosphatase that controls mitotic exit and cytokinesis in budding yeast. In mammals, the two Cdc14 homologues, Cdc14A and Cdc14B, have been proposed to regulate DNA damage repair, whereas the mitotic exit and cytokinesis rely on another phosphatase, PP2A-B55α. It is unclear if the two Cdc14s work redundantly in DNA repair and which repair pathways they participate in. More importantly, their target(s) in DNA repair remains elusive. Here we report that Cdc14B knockout (Cdc14B−/−) mouse embryonic fibroblasts (MEFs) showed defects in repairing ionizing radiation (IR)-induced DNA double-strand breaks (DSBs), which occurred only at late passages when Cdc14A levels were low. This repair defect could occur at early passages if Cdc14A levels were also compromised. These results indicate redundancy between Cdc14B and Cdc14A in DSB repair. Further, we found that Cdc14B deficiency impaired both homologous recombination (HR) and nonhomologous end joining (NHEJ), the two major DSB repair pathways. We also provide evidence that Cdh1 is a downstream target of Cdc14B in DSB repair.

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