Poly(ADP-ribose) binding and macroH2A mediate recruitment and functions of KDM5A at DNA lesions

The histone demethylase KDM5A erases histone H3 lysine 4 methylation, which is involved in transcription and DNA damage responses (DDRs). While DDR functions of KDM5A have been identified, how KDM5A recognizes DNA lesion sites within chromatin is unknown. Here, we identify two factors that act upstream of KDM5A to promote its association with DNA damage sites. We have identified a noncanonical poly(ADP-ribose) (PAR)–binding region unique to KDM5A. Loss of the PAR-binding region or treatment with PAR polymerase (PARP) inhibitors (PARPi’s) blocks KDM5A–PAR interactions and DNA repair functions of KDM5A. The histone variant macroH2A1.2 is also specifically required for KDM5A recruitment and function at DNA damage sites, including homology-directed repair of DNA double-strand breaks and repression of transcription at DNA breaks. Overall, this work reveals the importance of PAR binding and macroH2A1.2 in KDM5A recognition of DNA lesion sites that drive transcriptional and repair activities at DNA breaks within chromatin that are essential for maintaining genome integrity.

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Poly(ADP-ribose)-binding and macroH2A mediate recruitment and functions of KDM5A at DNA lesions

10.1101/2020.07.27.223131 ◽

2020 ◽

Author(s):

Ramhari Kumbhar ◽

Jullian Perren ◽

Fade Gong ◽

David Corujo ◽

Frank Medina ◽

...

Keyword(s):

Dna Damage ◽

Parp Inhibitors ◽

Dna Lesions ◽

Histone Demethylase ◽

Dna Double Strand Breaks ◽

Dna Breaks ◽

Binding Region ◽

Transcriptional Responses ◽

Dna Lesion ◽

Two Factors

AbstractThe histone demethylase KDM5A removes histone H3 lysine 4 methylation, which is involved in transcription and DNA damage responses (DDR). While DDR functions of KDM5A have been identified, how KDM5A recognizes DNA lesion sites within chromatin is unknown. Here, we identify two factors that act upstream of KDM5A to promote its association with DNA damage sites. We have identified a non-canonical poly(ADP-ribose), (PAR), binding region unique to KDM5A. Loss of the PAR-binding region or treatment with PAR polymerase (PARP) inhibitors (PARPi) blocks KDM5A-PAR interactions and DNA repair functions of KDM5A. The histone variant macroH2A1.2 is also specifically required for KDM5A recruitment and functions at DNA damage sites, including homology-directed repair of DNA double-strand breaks and repression of transcription at DNA breaks. Overall, this work reveals the importance of PAR-binding and macroH2A1.2 in KDM5A recognition of damage sites that drive transcriptional and repair activities at DNA breaks within chromatin that are essential for maintaining genome integrity.SummaryThe histone demethylase KDM5A demethylates H3K4 to promote repair and transcriptional responses at DNA breaks. We identified poly(ADP-ribose)-binding and macroH2A1.2 as modulators of KDM5A association with DNA damage sites, revealing how KDM5A engages DNA breaks within chromatin.

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Making Connections: Integrative Signaling Mechanisms Coordinate DNA Break Repair in Chromatin

Frontiers in Genetics ◽

10.3389/fgene.2021.747734 ◽

2021 ◽

Vol 12 ◽

Author(s):

Anthony Sanchez ◽

Doohyung Lee ◽

Dae In Kim ◽

Kyle M. Miller

Keyword(s):

Dna Damage ◽

Protein Interactions ◽

Dna Lesions ◽

Genome Integrity ◽

Dna Double Strand Breaks ◽

Dna Breaks ◽

Dsb Repair ◽

Damage Sensing ◽

Dna Lesion ◽

Break Site

DNA double-strand breaks (DSBs) are hazardous to genome integrity and can promote mutations and disease if not handled correctly. Cells respond to these dangers by engaging DNA damage response (DDR) pathways that are able to identify DNA breaks within chromatin leading ultimately to their repair. The recognition and repair of DSBs by the DDR is largely dependent on the ability of DNA damage sensing factors to bind to and interact with nucleic acids, nucleosomes and their modified forms to target these activities to the break site. These contacts orientate and localize factors to lesions within chromatin, allowing signaling and faithful repair of the break to occur. Coordinating these events requires the integration of several signaling and binding events. Studies are revealing an enormously complex array of interactions that contribute to DNA lesion recognition and repair including binding events on DNA, as well as RNA, RNA:DNA hybrids, nucleosomes, histone and non-histone protein post-translational modifications and protein-protein interactions. Here we examine several DDR pathways that highlight and provide prime examples of these emerging concepts. A combination of approaches including genetic, cellular, and structural biology have begun to reveal new insights into the molecular interactions that govern the DDR within chromatin. While many questions remain, a clearer picture has started to emerge for how DNA-templated processes including transcription, replication and DSB repair are coordinated. Multivalent interactions with several biomolecules serve as key signals to recruit and orientate proteins at DNA lesions, which is essential to integrate signaling events and coordinate the DDR within the milieu of the nucleus where competing genome functions take place. Genome architecture, chromatin structure and phase separation have emerged as additional vital regulatory mechanisms that also influence genome integrity pathways including DSB repair. Collectively, recent advancements in the field have not only provided a deeper understanding of these fundamental processes that maintain genome integrity and cellular homeostasis but have also started to identify new strategies to target deficiencies in these pathways that are prevalent in human diseases including cancer.

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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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Poly(ADP-ribosyl)ation mediates early phase histone eviction at DNA lesions

Nucleic Acids Research ◽

10.1093/nar/gkaa022 ◽

2020 ◽

Vol 48 (6) ◽

pp. 3001-3013 ◽

Cited By ~ 4

Author(s):

Guang Yang ◽

Yibin Chen ◽

Jiaxue Wu ◽

Shih-Hsun Chen ◽

Xiuhua Liu ◽

...

Keyword(s):

Dna Damage ◽

Molecular Mechanism ◽

Parp Inhibitor ◽

Repair Process ◽

Dna Lesions ◽

Histone Chaperones ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Dna Lesion ◽

Damage Repair

Abstract Nucleosomal histones are barriers to the DNA repair process particularly at DNA double-strand breaks (DSBs). However, the molecular mechanism by which these histone barriers are removed from the sites of DNA damage remains elusive. Here, we have generated a single specific inducible DSB in the cells and systematically examined the histone removal process at the DNA lesion. We found that histone removal occurred immediately following DNA damage and could extend up to a range of few kilobases from the lesion. To examine the molecular mechanism underlying DNA damage-induced histone removal, we screened histone modifications and found that histone ADP-ribosylation was associated with histone removal at DNA lesions. PARP inhibitor treatment suppressed the immediate histone eviction at DNA lesions. Moreover, we examined histone chaperones and found that the FACT complex recognized ADP-ribosylated histones and mediated the removal of histones in response to DNA damage. Taken together, our results reveal a pathway that regulates early histone barrier removal at DNA lesions. It may also explain the mechanism by which PARP inhibitor regulates early DNA damage repair.

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Clinical PARP inhibitors do not abrogate PARP1 exchange at DNA damage sites in vivo

Nucleic Acids Research ◽

10.1093/nar/gkaa718 ◽

2020 ◽

Vol 48 (17) ◽

pp. 9694-9709

Author(s):

Zhengping Shao ◽

Brian J Lee ◽

Élise Rouleau-Turcotte ◽

Marie-France Langelier ◽

Xiaohui Lin ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Live Cell Imaging ◽

Parp Inhibitors ◽

Dna Lesions ◽

Dna Breaks ◽

Downstream Effector ◽

Mutation Analyses ◽

Molecular Nature

Abstract DNA breaks recruit and activate PARP1/2, which deposit poly-ADP-ribose (PAR) to recruit XRCC1-Ligase3 and other repair factors to promote DNA repair. Clinical PARP inhibitors (PARPi) extend the lifetime of damage-induced PARP1/2 foci, referred to as ‘trapping’. To understand the molecular nature of ‘trapping’ in cells, we employed quantitative live-cell imaging and fluorescence recovery after photo-bleaching. Unexpectedly, we found that PARP1 exchanges rapidly at DNA damage sites even in the presence of clinical PARPi, suggesting the persistent foci are not caused by physical stalling. Loss of Xrcc1, a major downstream effector of PAR, also caused persistent PARP1 foci without affecting PARP1 exchange. Thus, we propose that the persistent PARP1 foci are formed by different PARP1 molecules that are continuously recruited to and exchanging at DNA lesions due to attenuated XRCC1-LIG3 recruitment and delayed DNA repair. Moreover, mutation analyses of the NAD+ interacting residues of PARP1 showed that PARP1 can be physically trapped at DNA damage sites, and identified H862 as a potential regulator for PARP1 exchange. PARP1-H862D, but not PARylation-deficient PARP1-E988K, formed stable PARP1 foci upon activation. Together, these findings uncovered the nature of persistent PARP1 foci and identified NAD+ interacting residues involved in the PARP1 exchange.

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DNA damage and osmotic regulation in the kidney

AJP Renal Physiology ◽

10.1152/ajprenal.00041.2005 ◽

2005 ◽

Vol 289 (1) ◽

pp. F2-F7 ◽

Cited By ~ 16

Author(s):

Natalia I. Dmitrieva ◽

Maurice B. Burg ◽

Joan D. Ferraris

Keyword(s):

Dna Damage ◽

Cultured Cells ◽

Osmotic Regulation ◽

Dna Double Strand Breaks ◽

Dna Breaks ◽

Atm Kinase ◽

Strand Breaks ◽

Cycle Arrest ◽

And Function

Renal medullary cells normally are exposed to extraordinarily high interstitial NaCl concentration as part of the urinary concentrating mechanism, yet they survive and function. Acute elevation of NaCl to a moderate level causes transient cell cycle arrest in culture. Higher levels of NaCl, within the range found in the inner medulla, cause apoptosis. Recently, it was surprising to discover that even moderately high levels of NaCl cause DNA double-strand breaks. The DNA breaks persist in cultured cells that are proliferating rapidly after adaptation to high NaCl, and DNA breaks normally are present in the renal inner medulla in vivo. High NaCl inhibits repair of broken DNA both in culture and in vivo, but the DNA is rapidly repaired if the level of NaCl is reduced. The inhibition of DNA repair is associated with suppressed activity of some DNA damage-response proteins like Mre11, Chk1, and H2AX but not that of others, like GADD45, p53, ataxia telangiectasia-mutated kinase (ATM), and Ku86. In this review, we consider possible mechanisms by which the renal cells escape the known dangerous consequences of persistent DNA damage. Furthermore, we consider that the persistent DNA damage may be a sensor of hypertonicity that activates ATM kinase to provide a signal that contributes to protective osmotic regulation.

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DNA damage interactions on both nanometer and micrometer scale determine overall cellular damage

Scientific Reports ◽

10.1038/s41598-018-34323-9 ◽

2018 ◽

Vol 8 (1) ◽

Cited By ~ 16

Author(s):

Thomas Friedrich ◽

Katarina Ilicic ◽

Christoph Greubel ◽

Stefanie Girst ◽

Judith Reindl ◽

...

Keyword(s):

Dna Damage ◽

Cellular Level ◽

Dna Lesions ◽

Double Strand Breaks ◽

Cellular Damage ◽

Biophysical Model ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Dna Lesion ◽

Micrometer Scale

Abstract DNA double strand breaks (DSB) play a pivotal role for cellular damage, which is a hazard encountered in toxicology and radiation protection, but also exploited e.g. in eradicating tumors in radiation therapy. It is still debated whether and in how far clustering of such DNA lesions leads to an enhanced severity of induced damage. Here we investigate - using focused spots of ionizing radiation as damaging agent - the spatial extension of DNA lesion patterns causing cell inactivation. We find that clustering of DNA damage on both the nm and µm scale leads to enhanced inactivation compared to more homogeneous lesion distributions. A biophysical model interprets these observations in terms of enhanced DSB production and DSB interaction, respectively. We decompose the overall effects quantitatively into contributions from these lesion formation processes, concluding that both processes coexist and need to be considered for determining the resulting damage on the cellular level.

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Telomeric dysfunction triggers an unstable growth arrest leading to irreparable genomic lesions and entry into cellular senescence

10.1101/442533 ◽

2018 ◽

Author(s):

Sabrina Ghadaouia ◽

Marc Alexandre Olivier ◽

Aurélie Martinez ◽

Nicolas Malaquin ◽

Guillaume B Cardin ◽

...

Keyword(s):

Dna Damage ◽

Replicative Senescence ◽

Secondary Growth ◽

Growth Arrest ◽

Dna Lesions ◽

Genome Replication ◽

Dna Double Strand Breaks ◽

Dna Breaks ◽

Telomere Attrition ◽

Genome Damage

ABSTRACTReplicative senescence is the permanent growth arrest caused by gradual telomere attrition occurring at each round of genome replication. Critically shortened telomeres lose their protective shelterin complex and t-loop structure revealing uncapped chromosome ends that are recognized as DNA double-strand breaks causing a p53-dependent DNA damage response (DDR) towards proliferation arrest. Because telomeres are heterogeneous in length within a single cell, the number of short telomeres necessary for senescence onset remains ill defined. Using controlled Tin2-mediated shelterin inactivation, we show that telomere uncapping is not sufficient to trigger senescence. While uncapping generates expected telomeric DNA damage detection, the associated weak DDR allows a rapid bypass of the primary growth arrest and re-entry into the cell cycle despite dysfunctional telomeres. During the ensuing mitosis, fused telomeres lead to additional DNA breaks and to genomic instability including chromosomes bridges or micronuclei, which sustain a secondary entry into stable growth arrest. The loss of p53 prevented both primary and secondary growth arrest, leading to amplified genomic instablility. Our results support a new multistep model for entry into telomere-mediated replicative senescence in normal cells, which is not directly induced by telomere uncapping, but rather by an amplification of DNA lesions caused by telomere fusions that leads to permanent irreparable genome damage.

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Focused Ion Microbeam Irradiation Induces Clustering of DNA Double-Strand Breaks in Heterochromatin Visualized by Nanoscale-Resolution Electron Microscopy

International Journal of Molecular Sciences ◽

10.3390/ijms22147638 ◽

2021 ◽

Vol 22 (14) ◽

pp. 7638

Author(s):

Yvonne Lorat ◽

Judith Reindl ◽

Anna Isermann ◽

Christian Rübe ◽

Anna A. Friedl ◽

...

Keyword(s):

Electron Microscopy ◽

Dna Damage ◽

Spatial Clustering ◽

Dna Lesions ◽

Double Strand Breaks ◽

Stimulated Emission ◽

Nuclear Chromatin ◽

Carbon Ions ◽

Dna Double Strand Breaks ◽

Strand Breaks

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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The Role of RNA in DNA Breaks, Repair and Chromosomal Rearrangements

Biomolecules ◽

10.3390/biom11040550 ◽

2021 ◽

Vol 11 (4) ◽

pp. 550

Author(s):

Matvey Mikhailovich Murashko ◽

Ekaterina Mikhailovna Stasevich ◽

Anton Markovich Schwartz ◽

Dmitriy Vladimirovich Kuprash ◽

Aksinya Nicolaevna Uvarova ◽

...

Keyword(s):

Chromosomal Rearrangements ◽

Gene Mutations ◽

Nonhomologous End Joining ◽

Published Data ◽

Dna Double Strand Breaks ◽

Dna Breaks ◽

End Joining ◽

Homology Directed Repair ◽

Chromosome Aberration Frequency

Incorrect reparation of DNA double-strand breaks (DSB) leading to chromosomal rearrangements is one of oncogenesis’s primary causes. Recently published data elucidate the key role of various types of RNA in DSB formation, recognition and repair. With growing interest in RNA biology, increasing RNAs are classified as crucial at the different stages of the main pathways of DSB repair in eukaryotic cells: nonhomologous end joining (NHEJ) and homology-directed repair (HDR). Gene mutations or variation in expression levels of such RNAs can lead to local DNA repair defects, increasing the chromosome aberration frequency. Moreover, it was demonstrated that some RNAs could stimulate long-range chromosomal rearrangements. In this review, we discuss recent evidence demonstrating the role of various RNAs in DSB formation and repair. We also consider how RNA may mediate certain chromosomal rearrangements in a sequence-specific manner.

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