Elp1 facilitates RAD51-mediated homologous recombination repair via translational regulation

Abstract Background RAD51-dependent homologous recombination (HR) is one of the most important pathways for repairing DNA double-strand breaks (DSBs), and its regulation is crucial to maintain genome integrity. Elp1 gene encodes IKAP/ELP1, a core subunit of the Elongator complex, which has been implicated in translational regulation. However, how ELP1 contributes to genome maintenance is unclear. Methods To investigate the function of Elp1, Elp1-deficient mouse embryonic fibroblasts (MEFs) were generated. Metaphase chromosome spreading, immunofluorescence, and comet assays were used to access chromosome abnormalities and DSB formation. Functional roles of Elp1 in MEFs were evaluated by cell viability, colony forming capacity, and apoptosis assays. HR-dependent DNA repair was assessed by reporter assay, immunofluorescence, and western blot. Polysome profiling was used to evaluate translational efficiency. Differentially expressed proteins and signaling pathways were identified using a label-free liquid chromatography–tandem mass spectrometry (LC–MS/MS) proteomics approach. Results Here, we report that Elp1 depletion enhanced genomic instability, manifested as chromosome breakage and genotoxic stress-induced genomic DNA fragmentation upon ionizing radiation (IR) exposure. Elp1-deficient cells were hypersensitive to DNA damage and exhibited impaired cell proliferation and defective HR repair. Moreover, Elp1 depletion reduced the formation of IR-induced RAD51 foci and decreased RAD51 protein levels. Polysome profiling analysis revealed that ELP1 regulated RAD51 expression by promoting its translation in response to DNA damage. Notably, the requirement for ELP1 in DSB repair could be partially rescued in Elp1-deficient cells by reintroducing RAD51, suggesting that Elp1-mediated HR-directed repair of DSBs is RAD51-dependent. Finally, using proteome analyses, we identified several proteins involved in cancer pathways and DNA damage responses as being differentially expressed upon Elp1 depletion. Conclusions Our study uncovered a molecular mechanism underlying Elp1-mediated regulation of HR activity and provides a novel link between translational regulation and genome stability.

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Genome Maintenance Mechanisms at the Chromatin Level

International Journal of Molecular Sciences ◽

10.3390/ijms221910384 ◽

2021 ◽

Vol 22 (19) ◽

pp. 10384

Author(s):

Hirotomo Takatsuka ◽

Atsushi Shibata ◽

Masaaki Umeda

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Genome Stability ◽

Genotoxic Stress ◽

Genome Integrity ◽

Easy Access ◽

Order Structure ◽

Chromatin Modifications ◽

Level Control ◽

Regulatory Systems

Genome integrity is constantly threatened by internal and external stressors, in both animals and plants. As plants are sessile, a variety of environment stressors can damage their DNA. In the nucleus, DNA twines around histone proteins to form the higher-order structure “chromatin”. Unraveling how chromatin transforms on sensing genotoxic stress is, thus, key to understanding plant strategies to cope with fluctuating environments. In recent years, accumulating evidence in plant research has suggested that chromatin plays a crucial role in protecting DNA from genotoxic stress in three ways: (1) changes in chromatin modifications around damaged sites enhance DNA repair by providing a scaffold and/or easy access to DNA repair machinery; (2) DNA damage triggers genome-wide alterations in chromatin modifications, globally modulating gene expression required for DNA damage response, such as stem cell death, cell-cycle arrest, and an early onset of endoreplication; and (3) condensed chromatin functions as a physical barrier against genotoxic stressors to protect DNA. In this review, we highlight the chromatin-level control of genome stability and compare the regulatory systems in plants and animals to find out unique mechanisms maintaining genome integrity under genotoxic stress.

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PARP1-dependent recruitment of the FBXL10-RNF68-RNF2 ubiquitin ligase to sites of DNA damage controls H2A.Z loading

eLife ◽

10.7554/elife.38771 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 17

Author(s):

Gergely Rona ◽

Domenico Roberti ◽

Yandong Yin ◽

Julia K Pagan ◽

Harrison Homer ◽

...

Keyword(s):

Dna Damage ◽

Homologous Recombination ◽

Ubiquitin Ligase ◽

Transcriptional Repression ◽

Genotoxic Stress ◽

Strand Break ◽

Homologous Recombination Repair ◽

Dependent Manner ◽

Ubiquitin Ligase Complex ◽

Recombination Repair

The mammalian FBXL10-RNF68-RNF2 ubiquitin ligase complex (FRRUC) mono-ubiquitylates H2A at Lys119 to repress transcription in unstressed cells. We found that the FRRUC is rapidly and transiently recruited to sites of DNA damage in a PARP1- and TIMELESS-dependent manner to promote mono-ubiquitylation of H2A at Lys119, a local decrease of H2A levels, and an increase of H2A.Z incorporation. Both the FRRUC and H2A.Z promote transcriptional repression, double strand break signaling, and homologous recombination repair (HRR). All these events require both the presence and activity of the FRRUC. Moreover, the FRRUC and its activity are required for the proper recruitment of BMI1-RNF2 and MEL18-RNF2, two other ubiquitin ligases that mono-ubiquitylate Lys119 in H2A upon genotoxic stress. Notably, whereas H2A.Z is not required for H2A mono-ubiquitylation, impairment of the latter results in the inhibition of H2A.Z incorporation. We propose that the recruitment of the FRRUC represents an early and critical regulatory step in HRR.

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PARP Inhibitor Olaparib Causes No Potentiation of the Bleomycin Effect in VERO Cells, Even in the Presence of Pooled ATM, DNA-PK, and LigIV Inhibitors

International Journal of Molecular Sciences ◽

10.3390/ijms21218288 ◽

2020 ◽

Vol 21 (21) ◽

pp. 8288

Author(s):

Valentina Perini ◽

Michelle Schacke ◽

Pablo Liddle ◽

Salomé Vilchez-Larrea ◽

Deborah J. Keszenman ◽

...

Keyword(s):

Dna Damage ◽

Homologous Recombination ◽

Synthetic Lethality ◽

Excision Repair ◽

Parp Inhibitor ◽

Genotoxic Stress ◽

Vero Cells ◽

Limiting Factor ◽

Parp Activation ◽

Alternative Hypotheses

Poly(ADP-ribosyl)polymerase (PARP) synthesizes poly(ADP-ribose) (PAR), which is anchored to proteins. PAR facilitates multiprotein complexes’ assembly. Nuclear PAR affects chromatin’s structure and functions, including transcriptional regulation. In response to stress, particularly genotoxic stress, PARP activation facilitates DNA damage repair. The PARP inhibitor Olaparib (OLA) displays synthetic lethality with mutated homologous recombination proteins (BRCA-1/2), base excision repair proteins (XRCC1, Polβ), and canonical nonhomologous end joining (LigIV). However, the limits of synthetic lethality are not clear. On one hand, it is unknown whether any limiting factor of homologous recombination can be a synthetic PARP lethality partner. On the other hand, some BRCA-mutated patients are not responsive to OLA for still unknown reasons. In an effort to help delineate the boundaries of synthetic lethality, we have induced DNA damage in VERO cells with the radiomimetic chemotherapeutic agent bleomycin (BLEO). A VERO subpopulation was resistant to BLEO, BLEO + OLA, and BLEO + OLA + ATM inhibitor KU55933 + DNA-PK inhibitor KU-0060648 + LigIV inhibitor SCR7 pyrazine. Regarding the mechanism(s) behind the resistance and lack of synthetic lethality, some hypotheses have been discarded and alternative hypotheses are suggested.

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WIP1 Promotes Homologous Recombination and Modulates Sensitivity to PARP Inhibitors

Cells ◽

10.3390/cells8101258 ◽

2019 ◽

Vol 8 (10) ◽

pp. 1258 ◽

Cited By ~ 5

Author(s):

Kamila Burdova ◽

Radka Storchova ◽

Matous Palek ◽

Libor Macurek

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Homologous Recombination ◽

Cancer Cells ◽

Genotoxic Stress ◽

Parp Inhibitors ◽

Cell Cycle Checkpoint ◽

Dna Lesion ◽

Cycle Checkpoint ◽

G2 Cells

Genotoxic stress triggers a combined action of DNA repair and cell cycle checkpoint pathways. Protein phosphatase 2C delta (referred to as WIP1) is involved in timely inactivation of DNA damage response by suppressing function of p53 and other targets at chromatin. Here we show that WIP1 promotes DNA repair through homologous recombination. Loss or inhibition of WIP1 delayed disappearance of the ionizing radiation-induced 53BP1 foci in S/G2 cells and promoted cell death. We identify breast cancer associated protein 1 (BRCA1) as interactor and substrate of WIP1 and demonstrate that WIP1 activity is needed for correct dynamics of BRCA1 recruitment to chromatin flanking the DNA lesion. In addition, WIP1 dephosphorylates 53BP1 at Threonine 543 that was previously implicated in mediating interaction with RIF1. Finally, we report that inhibition of WIP1 allowed accumulation of DNA damage in S/G2 cells and increased sensitivity of cancer cells to a poly-(ADP-ribose) polymerase inhibitor olaparib. We propose that inhibition of WIP1 may increase sensitivity of BRCA1-proficient cancer cells to olaparib.

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Molecular flexibility of DNA as a key determinant of RAD51 recruitment

10.1101/392795 ◽

2018 ◽

Author(s):

Federico Paoletti ◽

Afaf El-Sagheer ◽

Jun Allard ◽

Tom Brown ◽

Omer Dushek ◽

...

Keyword(s):

Dna Damage ◽

Surface Plasmon Resonance ◽

Homologous Recombination ◽

Surface Plasmon ◽

Plasmon Resonance ◽

Genome Stability ◽

General Framework ◽

Binding Kinetics ◽

Double Stranded Dna ◽

Conceptual Question

AbstractThe timely activation of homologous recombination is essential for the maintenance of genome stability, in which the RAD51 recombinase plays a central role. Biochemically, human RAD51 polymerises faster on single-stranded DNA (ssDNA) compared to double-stranded DNA (dsDNA), raising a key conceptual question: how does it discriminate between them? In this study, we tackled this problem by systematically assessing RAD51 binding kinetics on ssDNA and dsDNA differing in length and flexibility using surface plasmon resonance. By fitting detailed polymerisation models informed by our experimental datasets, we show that RAD51 is a mechano-sensor that exhibits a larger polymerisation rate constant on flexible ssDNA compared to rigid ssDNA or dsDNA. This model presents a new general framework suggesting that the flexibility of DNA, which may increase locally as a result of DNA damage, plays an important role in rapidly recruiting repair factors that multimerise at sites of DNA damage.

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Mtor-Independent Translation in Early Hematopoietic Development

Blood ◽

10.1182/blood-2018-99-120191 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3827-3827

Author(s):

Christina Spevak ◽

Harold Elias ◽

Lavanya Kannan ◽

Gaelle Martin ◽

Shanmugapriya Selvaraj ◽

...

Keyword(s):

Protein Expression ◽

Translational Regulation ◽

Differentially Expressed ◽

Rna Seq ◽

Total Rna ◽

Polysome Profiling ◽

Versus Protein ◽

Mtor Protein ◽

Mtor Signalling ◽

Independent Translation

Abstract Mechanisms of translational regulation are poorly understood in hematopoietic stem cells (HSCs) and committed progenitors (MP). In order to investigate the impact of translational regulation on early mouse hematopoietic development, we characterized the translatome using a combination of polysome profiling, RNA-sequencing (RNA-seq) of polysome-associated and total cellular mRNA, and whole proteome evaluation. Comparison of RNA-seq data from HSC-enriched LSK (Lin-Sca-1+c-Kit+) and MP (Lin-Sca-1-c-Kit+) cells showed more differentially expressed mRNAs in polysomal RNA than total RNA (412 vs 280 mRNAs, respectively) among ~15,000 mRNAs analyzed. In addition, polysomal mRNAs were enriched for a unique set of functional pathways (e.g. inflammatory response, apoptosis and p53) compared to total RNA in both LSK and MP cells. Interestingly, although LSK cells showed ~20% lower global translation than MPs as demonstrated by polysome profiling (Figure 1A), they exhibited significantly higher translational efficiency (TE = polysome RNA abundance/total RNA abundance) for mRNAs that support HSC maintenance (e.g. glycolysis, fatty acid metabolism, oxidative phosphorylation, mTOR signalling). Integration of proteomic and RNA-seq data demonstrated that 605 out of 784 (77.2%) differentially expressed genes (DEGs) between LSK and MP cells identified based on total RNA-seq data (Groups I & III; Fig 1B) also showed a corresponding change in protein expression, while remaining 179 DEGs (22.8%; Groups II & IV; Fig. 1B) showed an anti-correlation. Remarkably, in the latter group, expression of 129 proteins (72.1% of all differentially expressed proteins in LSKs vs MPs) correlated with their TEs (Figure 1C). While gene set enrichment analysis of published HSC regulators showed an enrichment in LSKs in total RNA-seq data, such an enrichment was not observed when evaluating mRNAs with differential TEs. However, mRNAs with high TE confirmed a surprising enrichment in mTOR-responsive genes independent of their total RNA expression in LSKs, but not in MP cells (Figure 1D). To investigate the biochemical basis of this observation, we performed western blot analysis of LSK and MP cells and observed decreased mTOR protein expression and signaling in purified MP cells, despite their higher global levels of translation. In addition, despite abundant expression of mTOR protein in LSK cells, 4E-BP1, a known target of mTOR, was only phosphorylated at the priming residues Thr-37/46 but not at the downstream Ser-65, a residue that initiates cap-dependent translation. In contrast, MP cells phosphorylated Ser-65, consistent with its increased translation despite the absence of mTOR signalling. mTOR inhibition with Torin-1 did not alter 4E-BP Ser-65 phosphorylation or translation in MPs ex vivo. The presence of mTOR-independent translation in MPs was corroborated by in vivo rapamycin treatment studies, which induced increased colony formation by LSKs, but not MPs. Decreased mTOR activity in MPs was due to degradation of mTOR protein mediated by the proteasome since mTOR protein expression was restored following treatment with the proteasome inhibitors bortezomib and MG132, as well as deletion of the E3 ubiquitin ligase, c-Cbl. Indeed, LSKs and MPs exhibit differential dependencies on mTOR signaling for translation, as mTOR protein is post-translationally downregulated in MPs by a previously undescribed mechanism for mTOR proteosomal degradation mediated by c-Cbl. These findings establish the presence of developmental stage-specific mechanisms of translational regulation in early hematopoiesis. Figure legend. (A) Representative polysome profiles from LSK (Lin-Sca-1+c-Kit+) and MP (Lin-Sca-1-c-Kit+) cells. Polysome/subpolysome ratios (poly/subpoly) were calculated by dividing total RNA abundance from polysomes (fractions 5-10) by subpolysomes (fractions 1-4) (n=3, p < 0.05). (B) Comparison of total RNA versus protein expression in LSK versus MP cells. Four groups of mRNAs were identified based on comparisons of their total mRNA and protein expression. Percentage of total analyzed mRNAs is indicated in each quadrant. (C) Comparison of TEs versus protein expression in LSK/MP cells (D) Enrichment plots comparing total RNA and TE in LSK and MP cells, using previously validated mTOR gene sets. Disclosures No relevant conflicts of interest to declare.

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BRCA2 Regulates Homologous Recombination in Response to DNA Damage: Implications for Genome Stability and Carcinogenesis

Cancer Research ◽

10.1158/0008-5472.can-04-3071 ◽

2005 ◽

Vol 65 (10) ◽

pp. 4117-4125 ◽

Cited By ~ 25

Author(s):

Christine Abaji ◽

Isabelle Cousineau ◽

Abdellah Belmaaza

Keyword(s):

Dna Damage ◽

Homologous Recombination ◽

Genome Stability

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Activated IGF-1R inhibits hyperglycemia-induced DNA damage and promotes DNA repair by homologous recombination

AJP Renal Physiology ◽

10.1152/ajprenal.00094.2005 ◽

2005 ◽

Vol 289 (5) ◽

pp. F1144-F1152 ◽

Cited By ~ 36

Author(s):

Shuo Yang ◽

Janaki Chintapalli ◽

Lakshmi Sodagum ◽

Stuart Baskin ◽

Ashwani Malhotra ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Homologous Recombination ◽

Genomic Dna ◽

Mesangial Cells ◽

Reactive Oxygen ◽

Genotoxic Stress ◽

Ros Generation ◽

Oxygen Intermediates ◽

Strand Breaks

The IGF-1R is a genetic determinant of oxidative stress and longevity. Hyperglycemia induces an exponential increase in the production of a key danger signal, reactive oxygen intermediates, which target genomic DNA. Here, we report for the first time that ligand activation of the IGF-1R prevents hyperglycemia-induced genotoxic stress and enhances DNA repair, maintaining genomic integrity and cell viability. We performed single gel electrophoresis (comet assay) to evaluate DNA damage in serum-starved SV40 murine mesangial cells (MMC) and normal human mesangial cells (NHMC), maintained at high ambient glucose concentration. Hyperglycemia inflicted an impressive array of DNA damage in the form of single-strand breaks (SSBs) and double-strand breaks (DSBs). The inclusion of IGF-1 to culture media of MMC and NHMC prevented hyperglycemia-induced DNA damage. To determine whether DNA damage was mediated by reactive oxygen species (ROS), ROS generation was evaluated, in the presence of IGF-1, or the free radical scavenger n-acetyl-cysteine (NAC). IGF-1 and NAC inhibited hyperglycemic-induced ROS production and hyperglycemia-induced DNA damage. We next asked whether IGF-1 promotes the repair of DSB under hyperglycemic conditions, by homologous recombination (HRR) or nonhomologous end joining (NHEJ). Repair of DSB by NHEJ and HRR was operative in MMC maintained under hyperglycemic conditions. IGF-1 increased HRR by nearly twofold, whereas IGF-1 did not affect DNA repair by NHEJ. IGF-1R enhancement of HRR correlated with the translocation of Rad51 to foci of DNA damage. Inhibition of Rad51 expression by short interfering RNA experiments markedly decreased percentage of MMC positive for Rad51 nuclear foci and increased hyperglycemic DNA damage. We conclude that the activated IGF-1R rescues mesangial cells from hyperglycemia-induced danger signals that target genomic DNA by suppressing ROS and enhancing DNA repair by HRR.

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A novel motif of Rad51 serves as an interaction hub for recombination auxiliary factors

eLife ◽

10.7554/elife.64131 ◽

2021 ◽

Vol 10 ◽

Author(s):

Negar Afshar ◽

Bilge Argunhan ◽

Maierdan Palihati ◽

Goki Taniguchi ◽

Hideo Tsubouchi ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Structural Analysis ◽

Homologous Recombination ◽

Genome Stability ◽

Single Motif ◽

Evolutionarily Conserved ◽

Recombinational Dna Repair

Homologous recombination (HR) is essential for maintaining genome stability. Although Rad51 is the key protein that drives HR, multiple auxiliary factors interact with Rad51 to potentiate its activity. Here, we present an interdisciplinary characterization of the interactions between Rad51 and these factors. Through structural analysis, we identified an evolutionarily conserved acidic patch of Rad51. The neutralization of this patch completely abolished recombinational DNA repair due to defects in the recruitment of Rad51 to DNA damage sites. This acidic patch was found to be important for the interaction with Rad55-Rad57 and essential for the interaction with Rad52. Furthermore, biochemical reconstitutions demonstrated that neutralization of this acidic patch also impaired the interaction with Rad54, indicating that a single motif is important for the interaction with multiple auxiliary factors. We propose that this patch is a fundamental motif that facilitates interactions with auxiliary factors and is therefore essential for recombinational DNA repair.

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Imaging the response to DNA damage in heterochromatin domains reveals core principles of heterochromatin maintenance

10.1101/818914 ◽

2019 ◽

Cited By ~ 1

Author(s):

Anna Fortuny ◽

Audrey Chansard ◽

Pierre Caron ◽

Odile Chevallier ◽

Olivier Leroy ◽

...

Keyword(s):

Dna Damage ◽

Cell Fate ◽

Histone Modifications ◽

Mammalian Cells ◽

Genome Stability ◽

Molecular Mechanisms ◽

Transcriptional Silencing ◽

Genotoxic Stress ◽

Higher Order ◽

Uv Damage

ABSTRACTHeterochromatin is a critical chromatin compartment, whose integrity governs genome stability and cell fate transitions. How heterochromatin features, including higher-order chromatin folding and histone modifications associated with transcriptional silencing, are maintained following a genotoxic stress challenge is unknown. Here, we establish a system for targeting UV damage to pericentric heterochromatin in mammalian cells and for tracking the heterochromatin response to UV in real time. We uncover profound heterochromatin compaction changes during repair, orchestrated by the UV damage sensor DDB2, which stimulates linker histone displacement from chromatin. Despite massive heterochromatin unfolding, heterochromatin-specific histone modifications and transcriptional silencing are maintained. We unveil a central role for the methyltransferase SETDB1 in the maintenance of heterochromatic histone marks after UV, SETDB1 coordinating histone methylation with new histone deposition in damaged heterochromatin, thus protecting cells from genome instability. Our data shed light on fundamental molecular mechanisms safeguarding higher-order chromatin integrity following DNA damage.

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