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

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.

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Single-Strand Annealing in Cancer

International Journal of Molecular Sciences ◽

10.3390/ijms22042167 ◽

2021 ◽

Vol 22 (4) ◽

pp. 2167

Author(s):

Janusz Blasiak

Keyword(s):

Dna Repair ◽

Synthetic Lethality ◽

Single Strand ◽

Repair System ◽

Dna Double Strand Breaks ◽

End Joining ◽

Cancer Pathogenesis ◽

Single Strand Annealing ◽

Non Homologous End Joining ◽

Strand Annealing

DNA double-strand breaks (DSBs) are among the most serious forms of DNA damage. In humans, DSBs are repaired mainly by non-homologous end joining (NHEJ) and homologous recombination repair (HRR). Single-strand annealing (SSA), another DSB repair system, uses homologous repeats flanking a DSB to join DNA ends and is error-prone, as it removes DNA fragments between repeats along with one repeat. Many DNA deletions observed in cancer cells display homology at breakpoint junctions, suggesting the involvement of SSA. When multiple DSBs occur in different chromosomes, SSA may result in chromosomal translocations, essential in the pathogenesis of many cancers. Inhibition of RAD52 (RAD52 Homolog, DNA Repair Protein), the master regulator of SSA, results in decreased proliferation of BRCA1/2 (BRCA1/2 DNA Repair Associated)-deficient cells, occurring in many hereditary breast and ovarian cancer cases. Therefore, RAD52 may be targeted in synthetic lethality in cancer. SSA may modulate the response to platinum-based anticancer drugs and radiation. SSA may increase the efficacy of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 (CRISPR associated 9) genome editing and reduce its off-target effect. Several basic problems associated with SSA, including its evolutionary role, interplay with HRR and NHEJ and should be addressed to better understand its role in cancer pathogenesis and therapy.

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Generation of a Mouse Model Lacking the Non-Homologous End-Joining Factor Mri/Cyren

10.20944/preprints201911.0292.v1 ◽

2019 ◽

Cited By ~ 1

Author(s):

Sergio Castañeda-Zegarra ◽

Camilla Huse ◽

Øystein Røsand ◽

Antonio Sarno ◽

Mengtan Xing ◽

...

Keyword(s):

Dna Repair ◽

Class Switch Recombination ◽

Physiological Role ◽

Wild Type ◽

Dna Double Strand Breaks ◽

End Joining ◽

Mouse Development ◽

Class Switch ◽

Accessory Factors ◽

Non Homologous End Joining

Classical non-homologous end joining (NHEJ) is a molecular pathway that detects, processes and ligates DNA double-strand breaks (DSBs) throughout the cell cycle. Mutations in several NHEJ genes result in neurological abnormalities and immunodeficiency both in humans and mice. The NHEJ pathway is required for the V(D)J recombination in developing B and T lymphocytes, and for class switch recombination in mature B cells. Ku heterodimer formed by Ku70 and Ku80 recognizes DSBs and facilitates the recruitment of accessory factors (e.g., DNA-PKcs, Artemis, Paxx and Mri/Cyren) and downstream core factors subunits XLF, XRCC4 and Lig4. Accessory factors might be dispensable for the process depending on the genetic background and DNA lesion type. To determine the physiological role of Mri in DNA repair and development, we introduced frame-shift mutation in the Mri gene in mice. We then analyzed the development of Mri-deficient mice as well as wild type and immunodeficient controls. Mice lacking Mri possessed reduced levels of class switch recombination in B lymphocytes and slow proliferation of neuronal progenitors when compared to wild type littermates. Human cell lines lacking Mri were as sensitive to DSBs as WT controls. Overall, we concluded that Mri/Cyren is largely dispensable for DNA repair and mouse development.

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

Oncogene ◽

10.1038/s41388-019-1028-7 ◽

2019 ◽

Vol 39 (4) ◽

pp. 754-766 ◽

Cited By ~ 7

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.

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Generation of a Mouse Model Lacking the Non-Homologous End-Joining Factor Mri/Cyren

Biomolecules ◽

10.3390/biom9120798 ◽

2019 ◽

Vol 9 (12) ◽

pp. 798 ◽

Cited By ~ 6

Author(s):

Sergio Castañeda-Zegarra ◽

Camilla Huse ◽

Øystein Røsand ◽

Antonio Sarno ◽

Mengtan Xing ◽

...

Keyword(s):

Dna Repair ◽

Class Switch Recombination ◽

Physiological Role ◽

Wild Type ◽

Dna Double Strand Breaks ◽

End Joining ◽

Mouse Development ◽

Class Switch ◽

Accessory Factors ◽

Non Homologous End Joining

Classical non-homologous end joining (NHEJ) is a molecular pathway that detects, processes, and ligates DNA double-strand breaks (DSBs) throughout the cell cycle. Mutations in several NHEJ genes result in neurological abnormalities and immunodeficiency both in humans and mice. The NHEJ pathway is required for V(D)J recombination in developing B and T lymphocytes, and for class switch recombination in mature B cells. The Ku heterodimer formed by Ku70 and Ku80 recognizes DSBs and facilitates the recruitment of accessory factors (e.g., DNA-PKcs, Artemis, Paxx and Mri/Cyren) and downstream core factor subunits X-ray repair cross-complementing group 4 (XRCC4), XRCC4-like factor (XLF), and DNA ligase 4 (Lig4). Accessory factors might be dispensable for the process, depending on the genetic background and DNA lesion type. To determine the physiological role of Mri in DNA repair and development, we introduced a frame-shift mutation in the Mri gene in mice. We then analyzed the development of Mri-deficient mice as well as wild type and immunodeficient controls. Mice lacking Mri possessed reduced levels of class switch recombination in B lymphocytes and slow proliferation of neuronal progenitors when compared to wild type littermates. Human cell lines lacking Mri were as sensitive to DSBs as the wild type controls. Overall, we concluded that Mri/Cyren is largely dispensable for DNA repair and mouse development.

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Recent Perspectives in Radiation-Mediated DNA Damage and Repair: Role of NHEJ and Alternative Pathways

10.5772/intechopen.96374 ◽

2021 ◽

Author(s):

Ajay Kumar Sharma ◽

Priyanka Shaw ◽

Aman Kalonia ◽

M.H. Yashavarddhan ◽

Pankaj Chaudhary ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Double Strand Breaks ◽

Single Strand ◽

End Joining ◽

Strand Breaks ◽

Single Strand Annealing ◽

Repair Pathways ◽

Non Homologous End Joining ◽

Alternative Nhej

Radiation is one of the causative agents for the induction of DNA damage in biological systems. There is various possibility of radiation exposure that might be natural, man-made, intentional, or non-intentional. Published literature indicates that radiation mediated cell death is primarily due to DNA damage that could be a single-strand break, double-strand breaks, base modification, DNA protein cross-links. The double-strand breaks are lethal damage due to the breakage of both strands of DNA. Mammalian cells are equipped with strong DNA repair pathways that cover all types of DNA damage. One of the predominant pathways that operate DNA repair is a non-homologous end-joining pathway (NHEJ) that has various integrated molecules that sense, detect, mediate, and repair the double-strand breaks. Even after a well-coordinated mechanism, there is a strong possibility of mutation due to the flexible nature in joining the DNA strands. There are alternatives to NHEJ pathways that can repair DNA damage. These pathways are alternative NHEJ pathways and single-strand annealing pathways that also displayed a role in DNA repair. These pathways are not studied extensively, and many reports are showing the relevance of these pathways in human diseases. The chapter will very briefly cover the radiation, DNA repair, and Alternative repair pathways in the mammalian system. The chapter will help the readers to understand the basic and applied knowledge of radiation mediated DNA damage and its repair in the context of extensively studied NHEJ pathways and unexplored alternative NHEJ pathways.

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ATR inhibition enhances 5-fluorouracil sensitivity independent of non-homologous end-joining and homologous recombination repair pathway

10.1101/2020.04.20.051318 ◽

2020 ◽

Author(s):

Soichiro S. Ito ◽

Yosuke Nakagawa ◽

Masaya Matsubayashi ◽

Yoshihiko M. Sakaguchi ◽

Shinko Kobashigawa ◽

...

Keyword(s):

Homologous Recombination ◽

Homologous Recombination Repair ◽

Dna Double Strand Breaks ◽

Dna Breaks ◽

End Joining ◽

Drug Induced ◽

Nucleotide Synthesis ◽

Double Strand Dna Breaks ◽

Recombination Repair ◽

Non Homologous End Joining

ABSTRACTThe anticancer agent, 5-fluorouracil (5-FU), is typically applied in the treatment of various types of cancers because of its properties. Thought to be an inhibitor of the enzyme thymidylate synthase which plays a role in nucleotide synthesis, 5-FU has been found to induce single- and double-strand DNA breaks. The activation of ATR occurs as a reaction to UV- and chemotherapeutic drug-induced replication stress. In this study, we examined the effect of ATR inhibition on 5-FU sensitivity. Using western blotting, we found that 5-FU treatment led to the phosphorylation of ATR. Surviving fractions were remarkably decreased in 5-FU with ATR inhibitor (ATRi) compared to 5-FU with other major DNA repair kinases inhibitors. ATR inhibition enhanced induction of DNA double-strand breaks and apoptosis in 5-FU-treated cells. Using gene expression analysis, we found that 5-FU could induce the activation of intra-S checkpoint. Surprisingly, BRCA2-deficient cells were sensitive to 5-FU in the presence of ATRi. In addition, ATR inhibition enhanced the efficacy of 5-FU treatment, independent of non-homologous end-joining and homologous recombination repair pathways. Findings from the present study suggest ATR as a potential therapeutic target for 5-FU chemotherapy.

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Meiotic DNA repair in the nucleolus employs a non-homologous end joining mechanism

10.1101/553529 ◽

2019 ◽

Cited By ~ 1

Author(s):

Jason Sims ◽

Gregory P. Copenhaver ◽

Peter Schlögelhofer

Keyword(s):

Genome Stability ◽

Dependent Manner ◽

Dna Double Strand Breaks ◽

Dna Breaks ◽

End Joining ◽

Strand Breaks ◽

Meiotic Dsbs ◽

Dna Elements ◽

Highly Repetitive Dna ◽

Non Homologous End Joining

AbstractRibosomal RNA genes are arranged in large arrays with hundreds of rDNA units in tandem. These highly repetitive DNA elements pose a risk to genome stability since they can undergo non-allelic exchanges. During meiosis DNA double strand breaks (DSBs) are induced as part of the regular program to generate gametes. Meiotic DSBs initiate homologous recombination (HR) which subsequently ensures genetic exchange and chromosome disjunction.In Arabidopsis thaliana we demonstrate that all 45S rDNA arrays become transcriptionally active and are recruited into the nucleolus early in meiosis. This shields the rDNA from acquiring canonical meiotic chromatin modifications, meiotic cohesin and meiosis-specific DSBs. DNA breaks within the rDNA arrays are repaired in a RAD51-independent, but LIG4-dependent manner, establishing that it is non-homologous end joining (NHEJ) that maintains rDNA integrity during meiosis. Utilizing ectopically integrated rDNA repeats we validate our findings and demonstrate that the rDNA constitutes a HR-refractory genome environment.

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Impact of hypoxia on the double-strand break repair after photon and carbon ion irradiation of radioresistant HNSCC cells

Scientific Reports ◽

10.1038/s41598-020-78354-7 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Anne-Sophie Wozny ◽

Gersende Alphonse ◽

Audrey Cassard ◽

Céline Malésys ◽

Safa Louati ◽

...

Keyword(s):

Dna Repair ◽

Ion Irradiation ◽

Strand Break ◽

Carbon Ions ◽

Dna Double Strand Breaks ◽

End Joining ◽

Dsb Repair ◽

Photon Irradiation ◽

Hypoxic Conditions ◽

Non Homologous End Joining

AbstractDNA double-strand breaks (DSBs) induced by photon irradiation are the most deleterious damage for cancer cells and their efficient repair may contribute to radioresistance, particularly in hypoxic conditions. Carbon ions (C-ions) act independently of the oxygen concentration and trigger complex- and clustered-DSBs difficult to repair. Understanding the interrelation between hypoxia, radiation-type, and DNA-repair is therefore essential for overcoming radioresistance. The DSBs signaling and the contribution of the canonical non-homologous end-joining (NHEJ-c) and homologous-recombination (HR) repair pathways were assessed by immunostaining in two cancer-stem-cell (CSCs) and non-CSCs HNSCC cell lines. Detection and signaling of DSBs were lower in response to C-ions than photons. Hypoxia increased the decay-rate of the detected DSBs (γH2AX) in CSCs after photons and the initiation of DSB repair signaling (P-ATM) in CSCs and non-CSCs after both radiations, but not the choice of DSB repair pathway (53BP1). Additionally, hypoxia increased the NHEJ-c (DNA-PK) and the HR pathway (RAD51) activation only after photons. Furthermore, the involvement of the HR seemed to be higher in CSCs after photons and in non-CSCs after C-ions. Taken together, our results show that C-ions may overcome the radioresistance of HNSCC associated with DNA repair, particularly in CSCs, and independently of a hypoxic microenvironment.

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ATM antagonizes NHEJ proteins assembly and DNA-ends synapsis at single-ended DNA double strand breaks

Nucleic Acids Research ◽

10.1093/nar/gkaa723 ◽

2020 ◽

Vol 48 (17) ◽

pp. 9710-9723

Author(s):

Sébastien Britton ◽

Pauline Chanut ◽

Christine Delteil ◽

Nadia Barboule ◽

Philippe Frit ◽

...

Keyword(s):

Dna Repair ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

End Joining ◽

Strand Breaks ◽

Repair Pathways ◽

Non Homologous End Joining ◽

Dna Strand ◽

Dna Ends

Abstract Two DNA repair pathways operate at DNA double strand breaks (DSBs): non-homologous end-joining (NHEJ), that requires two adjacent DNA ends for ligation, and homologous recombination (HR), that resects one DNA strand for invasion of a homologous duplex. Faithful repair of replicative single-ended DSBs (seDSBs) is mediated by HR, due to the lack of a second DNA end for end-joining. ATM stimulates resection at such breaks through multiple mechanisms including CtIP phosphorylation, which also promotes removal of the DNA-ends sensor and NHEJ protein Ku. Here, using a new method for imaging the recruitment of the Ku partner DNA-PKcs at DSBs, we uncover an unanticipated role of ATM in removing DNA-PKcs from seDSBs in human cells. Phosphorylation of DNA-PKcs on the ABCDE cluster is necessary not only for DNA-PKcs clearance but also for the subsequent MRE11/CtIP-dependent release of Ku from these breaks. We propose that at seDSBs, ATM activity is necessary for the release of both Ku and DNA-PKcs components of the NHEJ apparatus, and thereby prevents subsequent aberrant interactions between seDSBs accompanied by DNA-PKcs autophosphorylation and detrimental commitment to Lig4-dependent end-joining.

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NHEJ pathway is involved in post-integrational DNA repair due to Ku70 binding to HIV-1 integrase

Retrovirology ◽

10.1186/s12977-019-0492-z ◽

2019 ◽

Vol 16 (1) ◽

Cited By ~ 4

Author(s):

Ekaterina Knyazhanskaya ◽

Andrey Anisenko ◽

Olga Shadrina ◽

Anastasia Kalinina ◽

Timofei Zatsepin ◽

...

Keyword(s):

Dna Repair ◽

Wild Type Virus ◽

Dna Breaks ◽

End Joining ◽

Double Stranded Dna ◽

Repair Efficiency ◽

Non Homologous End Joining ◽

Cellular Dna ◽

Successful Replication ◽

Hiv 1

Abstract Background HIV-1 integration results in genomic DNA gaps that are repaired by cellular DNA repair pathways. This step of the lentiviral life cycle remains poorly understood despite its crucial importance for successful replication. We and others reported that Ku70 protein of the non-homologous end joining pathway (NHEJ) directly binds HIV-1 integrase (IN). Here, we studied the importance of this interaction for post-integrational gap repair and the recruitment of NHEJ factors in this process. Results We engineered HIV-based pseudovirus with mutant IN defective in Ku70 binding and generated heterozygous Ku70, Ku80 and DNA-PKcs human knockout (KO) cells using CRISPR/Cas9. KO of either of these proteins or inhibition of DNA-PKcs catalytic activity substantially decreased the infectivity of HIV-1 with native IN but not with the mutant one. We used a recently developed qPCR assay for the measurement of gap repair efficiency to show that HIV-1 with mutant IN was defective in DNA post-integrational repair, whereas the wild type virus displayed such a defect only when NHEJ system was disrupted in any way. This effect was present in CRISPR/Cas9 modified 293T cells, in Jurkat and CEM lymphoid lines and in primary human PBMCs. Conclusions Our data provide evidence that IN recruits DNA-PK to the site of HIV-1 post-integrational repair due to Ku70 binding—a novel finding that explains the involvement of DNA-PK despite the absence of free double stranded DNA breaks. In addition, our data clearly indicate the importance of interactions between HIV-1 IN and Ku70 in HIV-1 replication at the post-integrational repair step.

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