scholarly journals The (Lack of) DNA Double-Strand Break Repair Pathway Choice During V(D)J Recombination

2022 ◽  
Vol 12 ◽  
Author(s):  
Alice Libri ◽  
Timea Marton ◽  
Ludovic Deriano
Keyword(s):  
Dna Repair ◽  
Gene Diversity ◽  
Receptor Gene ◽  
Strand Break ◽  
Repair Pathway ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Homology Directed Repair ◽  
Pathway Choice ◽  
Non Homologous End Joining

DNA double-strand breaks (DSBs) are highly toxic lesions that can be mended via several DNA repair pathways. Multiple factors can influence the choice and the restrictiveness of repair towards a given pathway in order to warrant the maintenance of genome integrity. During V(D)J recombination, RAG-induced DSBs are (almost) exclusively repaired by the non-homologous end-joining (NHEJ) pathway for the benefit of antigen receptor gene diversity. Here, we review the various parameters that constrain repair of RAG-generated DSBs to NHEJ, including the peculiarity of DNA DSB ends generated by the RAG nuclease, the establishment and maintenance of a post-cleavage synaptic complex, and the protection of DNA ends against resection and (micro)homology-directed repair. In this physiological context, we highlight that certain DSBs have limited DNA repair pathway choice options.

scholarly journals FAM35A co-operates with REV7 to coordinate DNA double-strand break repair pathway choice

2018 ◽  
Author(s):  
Steven Findlay ◽  
John Heath ◽  
Vincent M. Luo ◽  
Abba Malina ◽  
Théo Morin ◽  
...  
Keyword(s):  
Dna Repair ◽  
Strand Break ◽  
Dependent Manner ◽  
Repair Pathway ◽  
Dna Double Strand Breaks ◽  
Uncharacterized Protein ◽  
Antibody Diversification ◽  
Pathway Choice ◽  
Dna End Resection ◽  
Non Homologous End Joining

SUMMARYDNA double-strand breaks (DSBs) can be repaired by two major pathways: non-homologous end-joining (NHEJ) and homologous recombination (HR). DNA repair pathway choice is governed by the opposing activities of 53BP1, in complex with its effectors RIF1 and REV7, and BRCA1. However, it remains unknown how the 53BP1/RIF1/REV7 complex stimulates NHEJ and restricts HR to the S/G2 phases of the cell cycle. Using a mass spectrometry (MS)-based approach, we identify 11 high-confidence REV7 interactors and elucidate the role of a previously undescribed factor, FAM35A/SHDL2, as a novel effector of REV7 in the NHEJ pathway. FAM35A depletion impairs NHEJ-mediated DNA repair and compromises antibody diversification by class switch recombination (CSR) in B-cells. FAM35A accumulates at DSBs in a 53BP1-, RIF1- and REV7-dependent manner and antagonizes HR by limiting DNA end resection. In fact, FAM35A is part of a larger complex composed of REV7 and another previously uncharacterized protein, C20orf196/SHDL1, which promotes NHEJ and limits HR. Together, these results establish FAM35A as a novel effector of REV7 in controlling the decision-making process during DSB repair.

scholarly journals Multifunctional properties of Nej1XLF C-terminus promote end-joining and impact DNA double-strand break repair pathway choice

2022 ◽  
Author(s):  
Aditya Mojumdar ◽  
Nancy Adam ◽  
Jennifer A Cobb
Keyword(s):  
Strand Break ◽  
Double Strand Break ◽  
Repair Pathway ◽  
End Joining ◽  
C Terminus ◽  
Dna Double Strand Break ◽  
Canonical Pathways ◽  
Pathway Choice ◽  
Non Homologous End Joining ◽  
End Resection

A DNA double strand break (DSB) is primarily repaired by one of two canonical pathways, non-homologous end-joining (NHEJ) and homologous recombination (HR). NHEJ requires no or minimal end processing for ligation, whereas HR requires 5 end resection followed by a search for homology. The main event that determines the mode of repair is the initiation of 5 resection because if resection starts, then NHEJ cannot occur. Nej1 is a canonical NHEJ factor that functions at the cross-roads of repair pathway choice and prior to its function in stimulating Dnl4 ligase. Nej1 competes with Dna2, inhibiting its recruitment to DSBs and thereby inhibiting resection. The highly conserved C-terminal region (CTR) of Nej1 (330- 338) is important for two events that drive NHEJ, stimulating ligation and inhibiting resection, but it is dispensable for end-bridging. By combining nej1 point mutants with nuclease-dead dna2-1, we find that Nej1-F335 is essential for end-joining whereas V338 promotes NHEJ indirectly through inhibiting Dna2-mediated resection.

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

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.

scholarly journals Replicated chromatin curtails 53BP1 recruitment in BRCA1-proficient and -deficient cells

2020 ◽  
Author(s):  
Jone Michelena ◽  
Stefania Pellegrino ◽  
Vincent Spegg ◽  
Matthias Altmeyer
Keyword(s):  
Single Cell ◽  
Repair Pathway ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Homology Directed Repair ◽  
Dna End Resection ◽  
Non Homologous End Joining ◽  
Cellular Phenotypes ◽  
Template Dna

AbstractDNA double-strand breaks can be repaired by two competing mechanisms, non-homologous end-joining (NHEJ) and homologous recombination (HR). Whether one or the other repair pathway is favored depends on the availability of an undamaged template DNA that allows for homology-directed repair. The tumor suppressor proteins 53BP1 and BRCA1 are considered antagonistic players in this repair pathway choice, as 53BP1 restrains DNA end resection, whereas BRCA1, together with its partner protein BARD1, displaces 53BP1 from damaged replicated chromatin and promotes HR. How cells switch from a 53BP1-dominated to a BRCA1-dominated response as they progress through the cell cycle is incompletely understood. Here we reveal, using high-throughput microscopy and applying single cell normalization to control for increased genome size as cells replicate their DNA, that 53BP1 recruitment to damaged replicated chromatin is inefficient in both BRCA1-proficient and BRCA1-deficient cells, in comparison to 53BP1 accumulation at damaged unreplicated chromatin. These findings substantiate a dual switch model from a 53BP1-dominated response in unreplicated chromatin to a BRCA1-BARD1-dominated response in replicated chromatin, in which replication-coupled dilution of 53BP1’s binding mark H4K20me2 functionally cooperates with BRCA1-BARD1-mediated suppression of 53BP1 binding. More generally, we suggest that appropriate normalization of single cell data, e.g. to DNA content, provides additional layers of information, which can be critical for quantifying and interpreting cellular phenotypes.

scholarly journals DNA-PKcs phosphorylation at the T2609 cluster alters the repair pathway choice during immunoglobulin class switch recombination

2020 ◽  
Vol 117 (37) ◽  
pp. 22953-22961 ◽  
Author(s):  
Jennifer L. Crowe ◽  
Xiaobin S. Wang ◽  
Zhengping Shao ◽  
Brian J. Lee ◽  
Verna M. Estes ◽  
...  
Keyword(s):  
Dna Repair ◽  
B Cells ◽  
Class Switch Recombination ◽  
Radiation Sensitivity ◽  
Repair Pathway ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Class Switch ◽  
Switch Regions ◽  
Pathway Choice

The DNA-dependent protein kinase (DNA-PK), which is composed of the KU heterodimer and the large catalytic subunit (DNA-PKcs), is a classical nonhomologous end-joining (cNHEJ) factor. Naïve B cells undergo class switch recombination (CSR) to generate antibodies with different isotypes by joining two DNA double-strand breaks at different switching regions via the cNHEJ pathway. DNA-PK and the cNHEJ pathway play important roles in the DNA repair phase of CSR. To initiate cNHEJ, KU binds to DNA ends and recruits and activates DNA-PK. Activated DNA-PK phosphorylates DNA-PKcs at the S2056 and T2609 clusters. Loss of T2609 cluster phosphorylation increases radiation sensitivity but whether T2609 phosphorylation has a role in physiological DNA repair remains elusive. Using the DNA-PKcs5A mouse model carrying alanine substitutions at the T2609 cluster, here we show that loss of T2609 phosphorylation of DNA-PKcs does not affect the CSR efficiency. Yet, the CSR junctions recovered from DNA-PKcs5A/5A B cells reveal increased chromosomal translocations, extensive use of distal switch regions (consistent with end resection), and preferential usage of microhomology—all signs of the alternative end-joining pathway. Thus, these results uncover a role of DNA-PKcs T2609 phosphorylation in promoting cNHEJ repair pathway choice during CSR.

scholarly journals RB Regulates DNA Double Strand Break Repair Pathway Choice by Mediating CtIP Dependent End Resection

2020 ◽  
Vol 21 (23) ◽  
pp. 9176
Author(s):  
Yuning Jiang ◽  
Jason C. Yam ◽  
Clement C. Tham ◽  
Chi Pui Pang ◽  
Wai Kit Chu
Keyword(s):  
Genome Instability ◽  
Synthetic Lethality ◽  
Strand Break ◽  
Suppressor Gene ◽  
Repair Pathway ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Dsb Repair ◽  
Pathway Choice ◽  
End Resection

Inactivation of the retinoblastoma tumor suppressor gene (RB1) leads to genome instability, and can be detected in retinoblastoma and other cancers. One damaging effect is causing DNA double strand breaks (DSB), which, however, can be repaired by homologous recombination (HR), classical non-homologous end joining (C-NHEJ), and micro-homology mediated end joining (MMEJ). We aimed to study the mechanistic roles of RB in regulating multiple DSB repair pathways. Here we show that HR and C-NHEJ are decreased, but MMEJ is elevated in RB-depleted cells. After inducing DSB by camptothecin, RB co-localizes with CtIP, which regulates DSB end resection. RB depletion leads to less RPA and native BrdU foci, which implies less end resection. In RB-depleted cells, less CtIP foci, and a lack of phosphorylation on CtIP Thr847, are observed. According to the synthetic lethality principle, based on the altered DSB repair pathway choice, after inducing DSBs by camptothecin, RB depleted cells are more sensitive to co-treatment with camptothecin and MMEJ blocker poly-ADP ribose polymerase 1 (PARP1) inhibitor. We propose a model whereby RB can regulate DSB repair pathway choice by mediating the CtIP dependent DNA end resection. The use of PARP1 inhibitor could potentially improve treatment outcomes for RB-deficient cancers.

scholarly journals Covalent linkage of the DNA repair template to the CRISPR/Cas9 complex enhances homology-directed repair

2017 ◽  
Author(s):  
Natasa Savic ◽  
Femke Ringnalda ◽  
Katja Bargsten ◽  
Yizhou Li ◽  
Christian Berk ◽  
...  
Keyword(s):  
Dna Repair ◽  
Mammalian Cells ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Homology Directed Repair ◽  
Genetic Modifications ◽  
Repair Template ◽  
Single Base Pair ◽  
Non Homologous End Joining

AbstractThe CRISPR/Cas9 targeted nuclease technology allows the insertion of genetic modifications with single base-pair precision. The preference of mammalian cells to repair Cas9-induced DNA double-strand breaks via non-homologous end joining (NHEJ) rather than via homology-directed repair (HDR) however leads to relatively low rates of correctly edited loci. Here we demonstrate that covalently linking the DNA repair template to Cas9 increases the ratio of HDR over NHEJ up to 23-fold, and therefore provides advantages for clinical applications where high-fidelity repair is needed.

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 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 Gene targeting facilitated by engineered sequence-specific nucleases and its potential for applications in crop improvement

2021 ◽  
Author(s):  
Daisuke Miki ◽  
Rui Wang ◽  
Jing Li ◽  
Dali Kong ◽  
Lei Zhang ◽  
...  
Keyword(s):  
Gene Targeting ◽  
Agronomic Traits ◽  
Higher Plants ◽  
Crop Improvement ◽  
Strand Break ◽  
End Joining ◽  
Homology Directed Repair ◽  
Target Dna ◽  
Targeted Gene Editing ◽  
Non Homologous End Joining

Abstract Humans are currently facing the problem of how to ensure that there is enough food to feed all of the world’s population. Ensuring that the food supply is sufficient will likely require the modification of crop genomes to improve their agronomic traits. The development of engineered sequence-specific nucleases (SSNs) paved the way for targeted gene editing in organisms, including plants. SSNs generate a double-strand break (DSB) at the target DNA site in a sequence-specific manner. These DSBs are predominantly repaired via error-prone non-homologous end joining (NHEJ), and are only rarely repaired via error-free homology-directed repair (HDR) if an appropriate donor template is provided. Gene targeting (GT), i.e., the integration or replacement of a particular sequence, can be achieved with combinations of SSNs and repair donor templates. Although its efficiency is extremely low, GT has been achieved in some higher plants. Here, we provide an overview of SSN-facilitated GT in higher plants and discuss the potential of GT as a powerful tool for generating crop plants with desirable features.

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