DNA double-strand breaks and ATM activation by transcription-blocking DNA lesions

Background: Charged-particle radiotherapy is an emerging treatment modality for radioresistant tumors. The enhanced effectiveness of high-energy particles (such as heavy ions) has been related to the spatial clustering of DNA lesions due to highly localized energy deposition. Here, DNA damage patterns induced by single and multiple carbon ions were analyzed in the nuclear chromatin environment by different high-resolution microscopy approaches. Material and Methods: Using the heavy-ion microbeam SNAKE, fibroblast monolayers were irradiated with defined numbers of carbon ions (1/10/100 ions per pulse, ipp) focused to micrometer-sized stripes or spots. Radiation-induced lesions were visualized as DNA damage foci (γH2AX, 53BP1) by conventional fluorescence and stimulated emission depletion (STED) microscopy. At micro- and nanoscale level, DNA double-strand breaks (DSBs) were visualized within their chromatin context by labeling the Ku heterodimer. Single and clustered pKu70-labeled DSBs were quantified in euchromatic and heterochromatic regions at 0.1 h, 5 h and 24 h post-IR by transmission electron microscopy (TEM). Results: Increasing numbers of carbon ions per beam spot enhanced spatial clustering of DNA lesions and increased damage complexity with two or more DSBs in close proximity. This effect was detectable in euchromatin, but was much more pronounced in heterochromatin. Analyzing the dynamics of damage processing, our findings indicate that euchromatic DSBs were processed efficiently and repaired in a timely manner. In heterochromatin, by contrast, the number of clustered DSBs continuously increased further over the first hours following IR exposure, indicating the challenging task for the cell to process highly clustered DSBs appropriately. Conclusion: Increasing numbers of carbon ions applied to sub-nuclear chromatin regions enhanced the spatial clustering of DSBs and increased damage complexity, this being more pronounced in heterochromatic regions. Inefficient processing of clustered DSBs may explain the enhanced therapeutic efficacy of particle-based radiotherapy in cancer treatment.

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Recent advances in the nucleolar responses to DNA double-strand breaks

Nucleic Acids Research ◽

10.1093/nar/gkaa713 ◽

2020 ◽

Vol 48 (17) ◽

pp. 9449-9461

Author(s):

Lea Milling Korsholm ◽

Zita Gál ◽

Blanca Nieto ◽

Oliver Quevedo ◽

Stavroula Boukoura ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Dna Damage Response ◽

Ribosomal Dna ◽

Repetitive Sequences ◽

Dna Lesions ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Damage Response

Abstract DNA damage poses a serious threat to human health and cells therefore continuously monitor and repair DNA lesions across the genome. Ribosomal DNA is a genomic domain that represents a particular challenge due to repetitive sequences, high transcriptional activity and its localization in the nucleolus, where the accessibility of DNA repair factors is limited. Recent discoveries have significantly extended our understanding of how cells respond to DNA double-strand breaks (DSBs) in the nucleolus, and new kinases and multiple down-stream targets have been identified. Restructuring of the nucleolus can occur as a consequence of DSBs and new data point to an active regulation of this process, challenging previous views. Furthermore, new insights into coordination of cell cycle phases and ribosomal DNA repair argue against existing concepts. In addition, the importance of nucleolar-DNA damage response (n-DDR) mechanisms for maintenance of genome stability and the potential of such factors as anti-cancer targets is becoming apparent. This review will provide a detailed discussion of recent findings and their implications for our understanding of the n-DDR. The n-DDR shares features with the DNA damage response (DDR) elsewhere in the genome but is also emerging as an independent response unique to ribosomal DNA and the nucleolus.

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The role of ubiquitin-dependent segregase p97 (VCP or Cdc48) in chromatin dynamics after DNA double strand breaks

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2016.0282 ◽

2017 ◽

Vol 372 (1731) ◽

pp. 20160282 ◽

Cited By ~ 19

Author(s):

Ignacio Torrecilla ◽

Judith Oehler ◽

Kristijan Ramadan

Keyword(s):

Dna Repair ◽

Current Knowledge ◽

Dna Lesions ◽

Chromatin Remodelling ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Dsb Repair ◽

Chromatin Dynamics ◽

Strand Breaks ◽

Signalling Molecules

DNA double strand breaks (DSBs) are the most cytotoxic DNA lesions and, if not repaired, lead to chromosomal rearrangement, genomic instability and cell death. Cells have evolved a complex network of DNA repair and signalling molecules which promptly detect and repair DSBs, commonly known as the DNA damage response (DDR). The DDR is orchestrated by various post-translational modifications such as phosphorylation, methylation, ubiquitination or SUMOylation. As DSBs are located in complex chromatin structures, the repair of DSBs is engineered at two levels: (i) at sites of broken DNA and (ii) at chromatin structures that surround DNA lesions. Thus, DNA repair and chromatin remodelling machineries must work together to efficiently repair DSBs. Here, we summarize the current knowledge of the ubiquitin-dependent molecular unfoldase/segregase p97 (VCP in vertebrates and Cdc48 in worms and lower eukaryotes) in DSB repair. We identify p97 as an essential factor that regulates DSB repair. p97-dependent extraction of ubiquitinated substrates mediates spatio-temporal protein turnover at and around the sites of DSBs, thus orchestrating chromatin remodelling and DSB repair. As p97 is a druggable target, p97 inhibition in the context of DDR has great potential for cancer therapy, as shown for other DDR components such as PARP, ATR and CHK1. This article is part of the themed issue ‘Chromatin modifiers and remodellers in DNA repair and signalling’.

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Repair of Directly and Indirectly UV-Induced DNA Lesions and of DNA Double-Strand Breaks in Cells from Skin Cancer-Prone Patients with the Disorders Dysplastic Nevus Syndrome or Basal Cell Nevus Syndrome

Recent Results in Cancer Research - Risk and Progression Factors in Carcinogenesis ◽

10.1007/978-3-642-60393-8_24 ◽

1997 ◽

pp. 337-351 ◽

Cited By ~ 5

Author(s):

T. M. Rünger ◽

B. Epe ◽

K. Möller ◽

B. Dekant ◽

D. Hellfritsch

Keyword(s):

Skin Cancer ◽

Basal Cell ◽

Dna Lesions ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Basal Cell Nevus Syndrome ◽

Strand Breaks ◽

Dysplastic Nevus ◽

Dysplastic Nevus Syndrome ◽

In Cells

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

10.1101/2020.04.27.064105 ◽

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.

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DNA Lesions: DNA Double Strand Breaks

Encyclopedia of Metalloproteins ◽

10.1007/978-1-4614-1533-6_100443 ◽

2013 ◽

pp. 757-757

Keyword(s):

Dna Lesions ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Strand Breaks

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Reading chromatin signatures after DNA double-strand breaks

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2016.0280 ◽

2017 ◽

Vol 372 (1731) ◽

pp. 20160280 ◽

Cited By ~ 26

Author(s):

Marcus D. Wilson ◽

Daniel Durocher

Keyword(s):

Dna Lesions ◽

Double Strand Breaks ◽

Cellular Viability ◽

Dna Double Strand Breaks ◽

Dsb Repair ◽

Post Translational Modifications ◽

Strand Breaks ◽

Cellular Processes ◽

Landing Platform ◽

Biochemical Studies

DNA double-strand breaks (DSBs) are DNA lesions that must be accurately repaired in order to preserve genomic integrity and cellular viability. The response to DSBs reshapes the local chromatin environment and is largely orchestrated by the deposition, removal and detection of a complex set of chromatin-associated post-translational modifications. In particular, the nucleosome acts as a central signalling hub and landing platform in this process by organizing the recruitment of repair and signalling factors, while at the same time coordinating repair with other DNA-based cellular processes. While current research has provided a descriptive overview of which histone marks affect DSB repair, we are only beginning to understand how these marks are interpreted to foster an efficient DSB response. Here we review how the modified chromatin surrounding DSBs is read, with a focus on the insights gleaned from structural and biochemical studies. This article is part of the themed issue ‘Chromatin modifiers and remodellers in DNA repair and signalling’.

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Additive Effects of SbcCD and PolX Deficiencies in the In Vivo Repair of DNA Double-Strand Breaks in Deinococcus radiodurans

Journal of Bacteriology ◽

10.1128/jb.00452-07 ◽

2007 ◽

Vol 189 (13) ◽

pp. 4784-4790 ◽

Cited By ~ 36

Author(s):

Esma Bentchikou ◽

Pascale Servant ◽

Geneviève Coste ◽

Suzanne Sommer

Keyword(s):

Double Mutant ◽

Deinococcus Radiodurans ◽

Dna Lesions ◽

Double Strand Breaks ◽

Repair System ◽

Dna Double Strand Breaks ◽

End Joining ◽

Γ Irradiation ◽

Strand Breaks

ABSTRACT Orthologs of proteins SbcD (Mre11) and SbcC (Rad50) exist in all kingdoms of life and are involved in a wide variety of DNA repair and maintenance functions, including homologous recombination and nonhomologous end joining. Here, we have inactivated the sbcC and/or sbcD genes of Deinococcus radiodurans, a highly radioresistant bacterium able to mend hundreds of radiation-induced DNA double-strand breaks (DSB). Mutants devoid of the SbcC and/or SbcD proteins displayed reduced survival and presented a delay in kinetics of DSB repair and cell division following γ-irradiation. It has been recently reported that D. radiodurans DNA polymerase X (PolX) possesses a structure-modulated 3′-to-5′ exonuclease activity reminiscent of specific nuclease activities displayed by the SbcCD complex from Escherichia coli. We constructed a double mutant devoid of SbcCD and PolX proteins. The double-mutant ΔsbcCD ΔpolX Dr (where Dr indicates D. radiodurans) bacteria are much more sensitive to γ-irradiation than the single mutants, suggesting that the deinococcal SbcCD and PolX proteins may play important complementary roles in processing damaged DNA ends. We propose that they are part of a backup repair system acting to rescue cells containing DNA lesions that are excessively numerous or difficult to repair.

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Coexposure to benzo[a]pyrene plus UVA induced DNA double strand breaks: visualization of Ku assembly in the nucleus having DNA lesions

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2004.07.174 ◽

2004 ◽

Vol 322 (2) ◽

pp. 631-636 ◽

Cited By ~ 20

Author(s):

Tatsushi Toyooka ◽

Yuko Ibuki ◽

Manabu Koike ◽

Norio Ohashi ◽

Sentaro Takahashi ◽

...

Keyword(s):

Dna Lesions ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Strand Breaks

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DNA Double-Strand Breaks: A Double-Edged Sword for Trypanosomatids

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.669041 ◽

2021 ◽

Vol 9 ◽

Author(s):

Marcelo Santos da Silva

Keyword(s):

Antigenic Variation ◽

Gene Copy Number ◽

Dna Lesions ◽

Double Strand Breaks ◽

Gene Copy ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Genomic Changes ◽

Gene Copy Number Variation ◽

Number Variation

For nearly all eukaryotic cells, stochastic DNA double-strand breaks (DSBs) are one of the most deleterious types of DNA lesions. DSB processing and repair can cause sequence deletions, loss of heterozygosity, and chromosome rearrangements resulting in cell death or carcinogenesis. However, trypanosomatids (single-celled eukaryotes parasites) do not seem to follow this premise strictly. Several studies have shown that trypanosomatids depend on DSBs to perform several events of paramount importance during their life cycle. For Trypanosoma brucei, DSBs formation is associated with host immune evasion via antigenic variation. In Trypanosoma cruzi, DSBs play a crucial role in the genetic exchange, a mechanism that is still little explored but appear to be of fundamental importance for generating variability. In Leishmania spp., DSBs are necessary to generate genomic changes by gene copy number variation (CNVs), events that are essential for these organisms to overcome inhospitable conditions. As DSB repair in trypanosomatids is primarily conducted via homologous recombination (HR), most of the events associated with DSBs are HR-dependent. This review will discuss the latest findings on how trypanosomatids balance the benefits and inexorable challenges caused by DSBs.

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