scholarly journals Functions of the CSB Protein at Topoisomerase 2 Inhibitors-Induced DNA Lesions

2021 ◽  
Vol 9 ◽  
Author(s):  
Franciele Faccio Busatto ◽  
Sofiane Y. Mersaoui ◽  
Yilun Sun ◽  
Yves Pommier ◽  
Jean-Yves Masson ◽  
...  
Keyword(s):  
Dna Cleavage ◽  
Excision Repair ◽  
Dna Lesions ◽  
Double Strand Breaks ◽  
Strand Breaks ◽  
Nucleotide Excision ◽  
Different Types ◽  
Topoisomerase 2 ◽  
Nucleotide Excision Repair Pathway

Topoisomerase 2 (TOP2) inhibitors are drugs widely used in the treatment of different types of cancer. Processing of their induced-lesions create double-strand breaks (DSBs) in the DNA, which is the main toxic mechanism of topoisomerase inhibitors to kill cancer cells. It was established that the Nucleotide Excision Repair pathway respond to TOP2-induced lesions, mainly through the Cockayne Syndrome B (CSB) protein. In this paper, we further define the mechanism and type of lesions induced by TOP2 inhibitors when CSB is abrogated. In the absence of TOP2, but not during pharmacological inhibition, an increase in R-Loops was detected. We also observed that CSB knockdown provokes the accumulation of DSBs induced by TOP2 inhibitors. Consistent with a functional interplay, interaction between CSB and TOP2 occurred after TOP2 inhibition. This was corroborated with in vitro DNA cleavage assays where CSB stimulated the activity of TOP2. Altogether, our results show that TOP2 is stimulated by the CSB protein and prevents the accumulation of R-loops/DSBs linked to genomic instability.

scholarly journals XPD/ERCC2 mutations interfere in cellular responses to oxidative stress

Mutagenesis ◽  
2019 ◽  
Vol 34 (4) ◽  
pp. 341-354 ◽  
Author(s):  
Leticia K Lerner ◽  
Natália C Moreno ◽  
Clarissa R R Rocha ◽  
Veridiana Munford ◽  
Valquíria Santos ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Lines ◽  
Excision Repair ◽  
Dna Lesions ◽  
Repair Capacity ◽  
Strand Breaks ◽  
Nucleotide Excision ◽  
Dna Strand ◽  
Ultraviolet Damage ◽  
Host Cell Reactivation

Abstract Nucleotide excision repair (NER) is a conserved, flexible mechanism responsible for the removal of bulky, helix-distorting DNA lesions, like ultraviolet damage or cisplatin adducts, but its role in the repair of lesions generated by oxidative stress is still not clear. The helicase XPD/ERCC2, one of the two helicases of the transcription complex IIH, together with XPB, participates both in NER and in RNA pol II-driven transcription. In this work, we investigated the responses of distinct XPD-mutated cell lines to the oxidative stress generated by photoactivated methylene blue (MB) and KBrO3 treatments. The studied cells are derived from patients with XPD mutations but expressing different clinical phenotypes, including xeroderma pigmentosum (XP), XP and Cockayne syndrome (XP-D/CS) and trichothiodystrophy (TTD). We show by different approaches that all XPD-mutated cell lines tested were sensitive to oxidative stress, with those from TTD patients being the most sensitive. Host cell reactivation (HCR) assays showed that XP-D/CS and TTD cells have severely impaired repair capacity of oxidised lesions in plasmid DNA, and alkaline comet assays demonstrated the induction of significantly higher amounts of DNA strand breaks after treatment with photoactivated MB in these cells compared to wild-type cells. All XPD-mutated cells presented strong S/G2 arrest and persistent γ-H2AX staining after photoactivated MB treatment. Taken together, these results indicate that XPD participates in the repair of lesions induced by the redox process, and that XPD mutations lead to differences in the response to oxidatively induced damage.

scholarly journals Leaky severe combined immunodeficiency and aberrant DNA rearrangements due to a hypomorphic RAG1 mutation

Blood ◽  
2009 ◽  
Vol 113 (13) ◽  
pp. 2965-2975 ◽  
Author(s):  
William Giblin ◽  
Monalisa Chatterji ◽  
Gerwin Westfield ◽  
Tehmina Masud ◽  
Brian Theisen ◽  
...  
Keyword(s):  
Immune System ◽  
Dna Cleavage ◽  
Severe Combined Immunodeficiency ◽  
Double Strand Breaks ◽  
Mutant Form ◽  
Dna Rearrangements ◽  
Strand Breaks ◽  
Immune System Dysfunction

Abstract The RAG1/2 endonuclease initiates programmed DNA rearrangements in progenitor lymphocytes by generating double-strand breaks at specific recombination signal sequences. This process, known as V(D)J recombination, assembles the vastly diverse antigen receptor genes from numerous V, D, and J coding segments. In vitro biochemical and cellular transfection studies suggest that RAG1/2 may also play postcleavage roles by forming complexes with the recombining ends to facilitate DNA end processing and ligation. In the current study, we examine the in vivo consequences of a mutant form of RAG1, RAG1-S723C, that is proficient for DNA cleavage, yet exhibits defects in postcleavage complex formation and end joining in vitro. We generated a knockin mouse model harboring the RAG1-S723C hypomorphic mutation and examined the immune system in this fully in vivo setting. RAG1-S723C homozygous mice exhibit impaired lymphocyte development and decreased V(D)J rearrangements. Distinct from RAG nullizygosity, the RAG1-S723C hypomorph results in aberrant DNA double-strand breaks within rearranging loci. RAG1-S723C also predisposes to thymic lymphomas associated with chromosomal translocations in a p53 mutant background, and heterozygosity for the mutant allele accelerates age-associated immune system dysfunction. Thus, our study provides in vivo evidence that implicates aberrant RAG1/2 activity in lymphoid tumor development and premature immunosenescence.

scholarly journals A Domesticated piggyBac Transposase Plays Key Roles in Heterochromatin Dynamics and DNA Cleavage during Programmed DNA Deletion in Tetrahymena thermophila

2010 ◽  
Vol 21 (10) ◽  
pp. 1753-1762 ◽  
Author(s):  
Chao-Yin Cheng ◽  
Alexander Vogt ◽  
Kazufumi Mochizuki ◽  
Meng-Chao Yao
Keyword(s):  
Dna Cleavage ◽  
Double Strand Breaks ◽  
Cellular Functions ◽  
Transposase Gene ◽  
Strand Breaks ◽  
Dna Deletion ◽  
Final Assembly ◽  
Dna Elimination ◽  
Cleavage Step

Transposons comprise large fractions of eukaryotic genomes and provide genetic reservoirs for the evolution of new cellular functions. We identified TPB2, a homolog of the piggyBac transposase gene that is required for programmed DNA deletion in Tetrahymena. TPB2 was expressed exclusively during the time of DNA excision, and its encoded protein Tpb2p was localized in DNA elimination heterochromatin structures. Notably, silencing of TPB2 by RNAi disrupts the final assembly of these heterochromatin structures and prevents DNA deletion to occur. In vitro studies revealed that Tpb2p is an endonuclease that produces double-strand breaks with four-base 5′ protruding ends, similar to the ends generated during DNA deletion. These findings suggest that Tpb2p plays a key role in the assembly of specialized DNA elimination chromatin architectures and is likely responsible for the DNA cleavage step of programmed DNA deletion.

scholarly journals Transcription-coupled DNA Double-Strand Breaks Are Mediated via the Nucleotide Excision Repair and the Mre11-Rad50-Nbs1 Complex

2008 ◽  
Vol 19 (9) ◽  
pp. 3969-3981 ◽  
Author(s):  
Josée Guirouilh-Barbat ◽  
Christophe Redon ◽  
Yves Pommier
Keyword(s):  
Nucleotide Excision Repair ◽  
Excision Repair ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Cellular Effects ◽  
Neutral Comet Assay ◽  
Nucleotide Excision ◽  
Full Activation ◽  
Transcription And Replication

The cellular activity of Yondelis (trabectedin, Ecteinascidin 743, Et743) is known to depend on transcription-coupled nucleotide excision repair (TCR). However, the subsequent cellular effects of Et743 are not fully understood. Here we show that Et743 induces both transcription- and replication-coupled DNA double-strand breaks (DSBs) that are detectible by neutral COMET assay and as γ-H2AX foci that colocalize with 53BP1, Mre11, Ser1981-pATM, and Thr68-pChk2. The transcription coupled-DSBs (TC-DSBs) induced by Et743 depended both on TCR and Mre11-Rad50-Nbs1 (MRN) and were associated with DNA-PK–dependent γ-H2AX foci. In contrast to DNA-PK, ATM phosphorylated H2AX both in NER-proficient and -deficient cells, but its full activation was dependent on H2AX as well as DNA-PK, suggesting a positive feedback loop: DNA-PK-γ-H2AX-ATM. Knocking-out H2AX or inactivating DNA-PK reduced Et743's antiproliferative activity, whereas ATM and MRN tended to act as survival factors. Our results highlight the interplays between ATM and DNA-PK and their impacts on H2AX phosphorylation and cell survival. They also suggest that γ-H2AX may serve as a biomarker in patients treated with Et743 and that molecular profiling of tumors for TCR, MRN, ATM, and DNA-PK might be useful to anticipate tumor response to Et743 treatment.

scholarly journals PARP1 promotes nucleotide excision repair through DDB2 stabilization and recruitment of ALC1

2012 ◽  
Vol 199 (2) ◽  
pp. 235-249 ◽  
Author(s):  
Alex Pines ◽  
Mischa G. Vrouwe ◽  
Jurgen A. Marteijn ◽  
Dimitris Typas ◽  
Martijn S. Luijsterburg ◽  
...  
Keyword(s):  
Nucleotide Excision Repair ◽  
Chromatin Remodeling ◽  
Excision Repair ◽  
Dna Lesions ◽  
Subsequent Removal ◽  
Wd40 Repeat ◽  
Nucleotide Excision ◽  
Efficient Recognition

The WD40-repeat protein DDB2 is essential for efficient recognition and subsequent removal of ultraviolet (UV)-induced DNA lesions by nucleotide excision repair (NER). However, how DDB2 promotes NER in chromatin is poorly understood. Here, we identify poly(ADP-ribose) polymerase 1 (PARP1) as a novel DDB2-associated factor. We demonstrate that DDB2 facilitated poly(ADP-ribosyl)ation of UV-damaged chromatin through the activity of PARP1, resulting in the recruitment of the chromatin-remodeling enzyme ALC1. Depletion of ALC1 rendered cells sensitive to UV and impaired repair of UV-induced DNA lesions. Additionally, DDB2 itself was targeted by poly(ADP-ribosyl)ation, resulting in increased protein stability and a prolonged chromatin retention time. Our in vitro and in vivo data support a model in which poly(ADP-ribosyl)ation of DDB2 suppresses DDB2 ubiquitylation and outline a molecular mechanism for PARP1-mediated regulation of NER through DDB2 stabilization and recruitment of the chromatin remodeler ALC1.

scholarly journals Interactome analysis and docking sites prediction of (AtCHR8, AtCUL4 and AtERCC1/UVR7) proteins in Arabidopsis Thaliana

2020 ◽  
Vol 2 (1) ◽  
pp. 52-68
Author(s):  
Mohamed Ragab Abdel Gawwad ◽  
Ali Taha Ozdemir
Keyword(s):  
Dna Damage ◽  
Arabidopsis Thaliana ◽  
Protein Interactions ◽  
Excision Repair ◽  
Dna Lesions ◽  
Damage Recognition ◽  
Nucleotide Excision ◽  
Nucleotide Excision Repair Pathway ◽  
Docking Sites ◽  
Dna Damage Recognition

The UV irradiation is a major DNA damaging factor in plants. Arabidopsis thaliana uses various repair pathways for these kinds of DNA lesions. One of them is the nucleotide excision repair pathway. The AtCUL4, ERCC1/UVR7 and CHR8 are vital proteins for nucleotide excision pathway and mutations in these proteins cause flaws in the repair mechanism. Two of these proteins play crucial role during DNA damage recognition and the other is involved in the excision of damaged bases. During NER processes, Arabidopsis uses different sets of proteins during the DNA damage recognition for transcriptionally active and genomic DNA. In order to get better insight into these proteins, we used bioinformatics tools to predict, analyze, and validate 3D structures of ERCC1/UVR7, AtCUL4 and CHR8. We also predicted the subcellular and sub-nuclear localization of proteins. Subsequently, we predicted the docking sites for each individual proteins and searched for interacting residues which mediate the protein-protein interactions. 

scholarly journals A versatile system to introduce clusters of genomic double-strand breaks in large cell populations

2020 ◽  
Author(s):  
Thorsten Kolb ◽  
Umar Khalid ◽  
Milena Simović ◽  
Manasi Ratnaparkhe ◽  
John Wong ◽  
...  
Keyword(s):  
Primary Cultures ◽  
Large Cell ◽  
Time Lapse ◽  
Genome Rearrangements ◽  
Dna Lesions ◽  
Double Strand Breaks ◽  
Brdu Incorporation ◽  
Dna Double Strand Breaks ◽  
Strand Breaks

ABSTRACTIn vitro assays for clustered DNA lesions will facilitate the analysis of the mechanisms underlying complex genome rearrangements such as chromothripsis, including the recruitment of repair factors to sites of DNA double-strand breaks. We present a novel method generating localized DNA double-strand breaks using UV-irradiation with photomasks. The size of the damage foci and the spacing between lesions are fully adjustable, making the assay suitable for different cell types and targeted areas. We validated this set-up with genomically stable epithelial cells, normal fibroblasts, pluripotent stem cells and patient-derived primary cultures. Our method does not require a specialized device such as a laser, making it accessible to a broad range of users. Sensitization by BrdU incorporation is not required, which enables analyzing the DNA damage response in post-mitotic cells. Irradiated cells can be cultivated further, followed by time-lapse imaging or used for downstream biochemical analyses, thanks to the high-throughput of the system. Importantly, we showed genome rearrangements in the irradiated cells, providing a proof of principle for the induction of structural variants by localized DNA lesions.

scholarly journals Two human homologs of Rad23 are functionally interchangeable in complex formation and stimulation of XPC repair activity.

1997 ◽  
Vol 17 (12) ◽  
pp. 6924-6931 ◽  
Author(s):  
K Sugasawa ◽  
J M Ng ◽  
C Masutani ◽  
T Maekawa ◽  
A Uchida ◽  
...  
Keyword(s):  
Excision Repair ◽  
Early Stage ◽  
Dna Lesions ◽  
Repair Protein ◽  
Nucleotide Excision ◽  
Repair Activity ◽  
In Vitro Repair ◽  
Stimulation Of ◽  
Active Genes

XPC-hHR23B protein complex is specifically involved in nucleotide excision repair (NER) of DNA lesions on transcriptionally inactive sequences as well as the nontranscribed strand of active genes. Here we demonstrate that not only highly purified recombinant hHR23B (rhHR23B) but also a second human homolog of the Saccharomyces cerevisiae Rad23 repair protein, hHR23A, stimulates the in vitro repair activity of recombinant human XPC (rhXPC), revealing functional redundancy between these human Rad23 homologs. Coprecipitation experiments with His-tagged rhHR23 as well as sedimentation velocity analysis showed that both rhHR23 proteins in vitro reconstitute a physical complex with rhXPC. Both complexes were more active than free rhXPC, indicating that complex assembly is required for the stimulation. rhHR23B was shown to stimulate an early stage of NER at or prior to incision. Furthermore, both rhHR23 proteins function in a defined NER system reconstituted with purified proteins, indicating direct involvement of hHR23 proteins in the DNA repair reaction via interaction with XPC.

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