scholarly journals The enrichment of breakpoints in late-replicating chromatin provides novel insights into chromoanagenesis mechanisms

2020 ◽  
Author(s):  
Nicolas Chatron ◽  
Giuliana Giannuzzi ◽  
Pierre-Antoine Rollat-Farnier ◽  
Flavie Diguet ◽  
Eleonora Porcu ◽  
...  
Keyword(s):  
Selective Pressure ◽  
Chromosomal Rearrangements ◽  
End Joining ◽  
Premature Chromosome Condensation ◽  
Complex Chromosomal Rearrangement ◽  
Three Samples ◽  
Complex Chromosomal Rearrangements ◽  
Non Homologous End Joining ◽  
Reduced Mobility ◽  
Dna Ends

AbstractThe rise of pangenomic molecular assays allowed uncovering complex rearrangements named chromoanagenesis that were hypothesized to result from catastrophic shattering events. Constitutional cases have typically been reported individually preventing identification of common features and uncovering the mechanisms at play. We characterized 20 new chromoanagenesis and discovered yet undescribed features. While literature differentiates chromothripsis and its shattering event repaired through non-homologous end joining from chromoanasynthesis born to aberrant replicative processes, we identified shattered chromosomes repaired through a combination of mechanisms. In particular, three samples present with “rearrangement hubs” comprising a fragmented kilobase-long sequence threaded throughout the rearrangement.To assess the mechanisms at play, we merged our data with those of 20 published constitutional complex chromosomal rearrangement cases. We evaluated if the distribution of their 1032 combined breakpoints was distinctive using bootstrap simulations and found that breakpoints tend to keep away from haplosensitive genes suggesting selective pressure. We then compared their distribution with that of 13,310 and 468 breakpoints of cancer complex chromosomal rearrangements and constitutional simple rearrangement samples, respectively. Both complex rearrangement groups showed breakpoint enrichment in late replicating regions suggesting similar origins for constitutional and cancer cases. Simple rearrangement breakpoints but not complex ones were depleted from lamina-associated domains (LADs), possibly as a consequence of reduced mobility of DNA ends bound to lamina.The enrichment of breakpoints in late-replicating chromatin for both constitutional and cancer chromoanagenesis provides an orthogonal support to the premature chromosome condensation hypothesis that was put forward to explain chromoanagenesis.

scholarly journals Activation of DNA-PK by hairpinned DNA ends reveals a stepwise mechanism of kinase activation

2020 ◽  
Vol 48 (16) ◽  
pp. 9098-9108 ◽  
Author(s):  
Katheryn Meek
Keyword(s):  
Protein Kinase ◽  
End Joining ◽  
Dependent Protein Kinase ◽  
Kinase Activation ◽  
Stepwise Mechanism ◽  
Dependent Protein ◽  
Enzymatic Activation ◽  
Non Homologous End Joining ◽  
Dna Ends ◽  
Step Mechanism

Abstract As its name implies, the DNA dependent protein kinase (DNA-PK) requires DNA double-stranded ends for enzymatic activation. Here, I demonstrate that hairpinned DNA ends are ineffective for activating the kinase toward many of its well-studied substrates (p53, XRCC4, XLF, HSP90). However, hairpinned DNA ends robustly stimulate certain DNA-PK autophosphorylations. Specifically, autophosphorylation sites within the ABCDE cluster are robustly phosphorylated when DNA-PK is activated by hairpinned DNA ends. Of note, phosphorylation of the ABCDE sites is requisite for activation of the Artemis nuclease that associates with DNA-PK to mediate hairpin opening. This finding suggests a multi-step mechanism of kinase activation. Finally, I find that all non-homologous end joining (NHEJ) defective cells (whether deficient in components of the DNA-PK complex or components of the ligase complex) are similarly deficient in joining DNA double-stranded breaks (DSBs) with hairpinned termini.

scholarly journals Characterization of gamma irradiation-induced mutations in Arabidopsis mutants deficient in non-homologous end joining

2020 ◽  
Vol 61 (5) ◽  
pp. 639-647
Author(s):  
Yan Du ◽  
Yoshihiro Hase ◽  
Katsuya Satoh ◽  
Naoya Shikazono
Keyword(s):  
Gamma Rays ◽  
Wild Type ◽  
End Joining ◽  
Complex Type ◽  
Single Base ◽  
In The Wild ◽  
Non Homologous End Joining ◽  
Dna Ends ◽  
Wild Type Control

Abstract To investigate the involvement of the non-homologous end joining (NHEJ) pathway in plant mutagenesis by ionizing radiation, we conducted a genome-wide characterization of the mutations induced by gamma rays in NHEJ-deficient Arabidopsis mutants (AtKu70−/− and AtLig4−/−). Although both mutants were more sensitive to gamma rays than the wild-type control, the AtKu70−/− mutant was slightly more sensitive than the AtLig4−/− mutant. Single-base substitutions (SBSs) were the predominant mutations in the wild-type control, whereas deletions (≥2 bp) and complex-type mutations [i.e. more than two SBSs or short insertion and deletions (InDels) separated by fewer than 10 bp] were frequently induced in the mutants. Single-base deletions were the most frequent deletions in the wild-type control, whereas the most common deletions in the mutants were 11–30 bp. The apparent microhomology at the rejoined sites of deletions peaked at 2 bp in the wild-type control, but was 3–4 bp in the mutants. This suggests the involvement of alternative end joining and single-strand annealing pathways involving increased microhomology for rejoining DNA ends. Complex-type mutations comprising short InDels were frequently detected in the mutants, but not in the wild-type control. Accordingly, NHEJ is more precise than the backup pathways, and is the main pathway for rejoining the broken DNA ends induced by ionizing radiation in plants.

scholarly journals Non-homologous End Joining Requires That the DNA-PK Complex Undergo an Autophosphorylation-dependent Rearrangement at DNA Ends

2004 ◽  
Vol 279 (38) ◽  
pp. 39408-39413 ◽  
Author(s):  
Yeturu V. R. Reddy ◽  
Qi Ding ◽  
Susan P. Lees-Miller ◽  
Katheryn Meek ◽  
Dale A. Ramsden
Keyword(s):  
End Joining ◽  
Non Homologous End Joining ◽  
Dna Ends

scholarly journals Structural mechanism of DNA-end synapsis in the non-homologous end joining pathway for repairing double-strand breaks: bridge over troubled ends

2019 ◽  
Vol 47 (6) ◽  
pp. 1609-1619 ◽  
Author(s):  
Qian Wu
Keyword(s):  
Single Molecule ◽  
Genome Instability ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Structural Mechanism ◽  
Strand Breaks ◽  
Single Molecule Studies ◽  
Non Homologous End Joining ◽  
Dna Ends

Non-homologous end joining (NHEJ) is a major repair pathway for DNA double-strand breaks (DSBs), which is the most toxic DNA damage in cells. Unrepaired DSBs can cause genome instability, tumorigenesis or cell death. DNA end synapsis is the first and probably the most important step of the NHEJ pathway, aiming to bring two broken DNA ends close together and provide structural stability for end processing and ligation. This process is mediated through a group of NHEJ proteins forming higher-order complexes, to recognise and bridge two DNA ends. Spatial and temporal understanding of the structural mechanism of DNA-end synapsis has been largely advanced through recent structural and single-molecule studies of NHEJ proteins. This review focuses on core NHEJ proteins that mediate DNA end synapsis through their unique structures and interaction properties, as well as how they play roles as anchor and linker proteins during the process of ‘bridge over troubled ends'.

scholarly journals Suppression of non-homologous end joining does not rescue DNA repair defects in Fanconi anemia patient cells

2017 ◽  
Author(s):  
Supawat Thongthip ◽  
Brooke A. Conti ◽  
Francis P. Lach ◽  
Agata Smogorzewska
Keyword(s):  
Fanconi Anemia ◽  
Chromosomal Abnormalities ◽  
Chromosomal Rearrangements ◽  
Direct Consequence ◽  
End Joining ◽  
Fanconi Anemia Patient ◽  
Interstrand Crosslink ◽  
Ligase Iv ◽  
Short Palindromic Repeat ◽  
Non Homologous End Joining

ABSTRACTSevere cellular sensitivity and aberrant chromosomal rearrangements in response to DNA interstrand crosslink (ICL) inducing agents are hallmarks of Fanconi anemia (FA) deficient cells. These phenotypes have previously been ascribed to inappropriate activity of non-homologous end joining (NHEJ) rather than a direct consequence of DNA ICL repair defects. Here we used chemical inhibitors, RNAi, and Clusterd Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas9 to inactivate various components of NHEJ in cells from FA patients. We show that suppression of DNA-PKcs, DNA Ligase IV and 53BP1 is not capable of rescuing ICL-induced proliferation defects and only 53BP1 knockout partially suppresses the chromosomal abnormalities of FA patient cells.

scholarly journals XLF acts as a flexible connector during non-homologous end joining

2020 ◽  
Author(s):  
Sean M. Carney ◽  
Andrew T. Moreno ◽  
Sadie C. Piatt ◽  
Metztli Cisneros-Aguirre ◽  
Felicia Wednesday Lopezcolorado ◽  
...  
Keyword(s):  
Single Molecule ◽  
Critical Factor ◽  
Tail Length ◽  
Binding Motif ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Synaptic Complex ◽  
Intrinsically Disordered ◽  
Non Homologous End Joining ◽  
Dna Ends

AbstractNon-homologous end joining (NHEJ) is the predominant pathway that repairs DNA double strand breaks in vertebrates. During NHEJ DNA ends are held together by a multi-protein synaptic complex until they are ligated. Here we investigate the role of the intrinsically disordered C-terminal tail of XLF, a critical factor in end synapsis. We demonstrate that the XLF tail along with the Ku binding motif (KBM) at the extreme C-terminus are required for end joining. While the underlying sequence of the tail can be varied, a minimal tail length is required for NHEJ. Single-molecule FRET experiments that observe end synapsis in real-time show that this defect is due to a failure to closely align DNA ends. Our data supports a model in which a single C-terminal tail tethers XLF to Ku while allowing XLF to form interactions with XRCC4 that enable synaptic complex formation.

scholarly journals Two-Stage Synapsis of DNA Ends during Non-homologous End Joining

2016 ◽  
Vol 61 (6) ◽  
pp. 850-858 ◽  
Author(s):  
Thomas G.W. Graham ◽  
Johannes C. Walter ◽  
Joseph J. Loparo
Keyword(s):  
End Joining ◽  
Two Stage ◽  
Non Homologous End Joining ◽  
Dna Ends

scholarly journals Combining Nanopore and Illumina Sequencing Permits Detailed Analysis of Insertion Mutations and Structural Variations Produced by PEG-Mediated Transformation in Ostreococcus tauri

2021 ◽  
Author(s):  
Julie Thomy ◽  
Frederic Sanchez ◽  
Marta Gut ◽  
Fernando Cruz ◽  
Tyler Alioto ◽  
...  
Keyword(s):  
Insertional Mutagenesis ◽  
Chromosomal Rearrangements ◽  
Molecular Techniques ◽  
End Joining ◽  
Structural Variations ◽  
Vector Integration ◽  
Ostreococcus Tauri ◽  
Unicellular Green Alga ◽  
Genomic Studies ◽  
Non Homologous End Joining

Ostreococcus tauri is a simple unicellular green alga representing an ecologically important group of phytoplankton in oceans worldwide. Modern molecular techniques must be developed in order to understand the mechanisms that permit adaptation of microalgae to their environment. We present for the first time in O. tauri a detailed characterization of individual genomic integration events of foreign DNA of plasmid origin after PEG-mediated transformation. Vector integration appears to be random, occurring mainly at a single locus, and thus confirming the utility of this technique for insertional mutagenesis. While the mechanism of double-stranded DNA repair in the O. tauri model remains to be elucidated, we clearly demonstrate by genome resequencing that the integration of the vector leads to frequent structural variations (deletions/insertions and duplications) and some chromosomal rearrangements in the genome at the insertion loci, and often within the vector sequence itself. From these observations, we speculate that a non-homologous end joining-like mechanism is required during random insertion events, as described in plants and other freshwater algal models. PEG-mediated transformation is therefore a promising molecular biology tool, not only for functional genomic studies, but also for biotechnological research in ecologically important marine algae.

scholarly journals To Join or Not to Join: Decision Points Along the Pathway to Double-Strand Break Repair vs. Chromosome End Protection

2021 ◽  
Vol 9 ◽  
Author(s):  
Stephanie M. Ackerson ◽  
Carlan Romney ◽  
P. Logan Schuck ◽  
Jason A. Stewart
Keyword(s):  
Genome Stability ◽  
Strand Break ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Decision Points ◽  
Chromosome Fusions ◽  
Non Homologous End Joining ◽  
Dna Ends

The regulation of DNA double-strand breaks (DSBs) and telomeres are diametrically opposed in the cell. DSBs are considered one of the most deleterious forms of DNA damage and must be quickly recognized and repaired. Telomeres, on the other hand, are specialized, stable DNA ends that must be protected from recognition as DSBs to inhibit unwanted chromosome fusions. Decisions to join DNA ends, or not, are therefore critical to genome stability. Yet, the processing of telomeres and DSBs share many commonalities. Accordingly, key decision points are used to shift DNA ends toward DSB repair vs. end protection. Additionally, DSBs can be repaired by two major pathways, namely homologous recombination (HR) and non-homologous end joining (NHEJ). The choice of which repair pathway is employed is also dictated by a series of decision points that shift the break toward HR or NHEJ. In this review, we will focus on these decision points and the mechanisms that dictate end protection vs. DSB repair and DSB repair choice.

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