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20 pages, 3046 KiB

Open AccessArticle

Low CDK Activity and Enhanced Degradation by APC/C^CDH1 Abolishes CtIP Activity and Alt-EJ in Quiescent Cells

by Fanghua Li, Emil Mladenov, Yanjie Sun, Aashish Soni, Martin Stuschke, Beate Timmermann and George Iliakis

Cells 2023, 12(11), 1530; https://doi.org/10.3390/cells12111530 - 1 Jun 2023

Cited by 6 | Viewed by 1737

Abstract

Alt-EJ is an error-prone DNA double-strand break (DSBs) repair pathway coming to the fore when first-line repair pathways, c-NHEJ and HR, are defective or fail. It is thought to benefit from DNA end-resection—a process whereby 3′ single-stranded DNA-tails are generated—initiated by the CtIP/MRE11-RAD50-NBS1 [...] Read more.

Alt-EJ is an error-prone DNA double-strand break (DSBs) repair pathway coming to the fore when first-line repair pathways, c-NHEJ and HR, are defective or fail. It is thought to benefit from DNA end-resection—a process whereby 3′ single-stranded DNA-tails are generated—initiated by the CtIP/MRE11-RAD50-NBS1 (MRN) complex and extended by EXO1 or the BLM/DNA2 complex. The connection between alt-EJ and resection remains incompletely characterized. Alt-EJ depends on the cell cycle phase, is at maximum in G₂-phase, substantially reduced in G₁-phase and almost undetectable in quiescent, G₀-phase cells. The mechanism underpinning this regulation remains uncharacterized. Here, we compare alt-EJ in G₁- and G₀-phase cells exposed to ionizing radiation (IR) and identify CtIP-dependent resection as the key regulator. Low levels of CtIP in G₁-phase cells allow modest resection and alt-EJ, as compared to G₂-phase cells. Strikingly, CtIP is undetectable in G₀-phase cells owing to APC/C-mediated degradation. The suppression of CtIP degradation with bortezomib or CDH1-depletion rescues CtIP and alt-EJ in G₀-phase cells. CtIP activation in G₀-phase cells also requires CDK-dependent phosphorylation by any available CDK but is restricted to CDK4/6 at the early stages of the normal cell cycle. We suggest that suppression of mutagenic alt-EJ in G₀-phase is a mechanism by which cells of higher eukaryotes maintain genomic stability in a large fraction of non-cycling cells in their organisms. Full article

(This article belongs to the Special Issue Double-Strand DNA Break Repair and Human Disease II)

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20 pages, 2949 KiB

Open AccessArticle

The p38/MK2 Pathway Functions as Chk1-Backup Downstream of ATM/ATR in G₂-Checkpoint Activation in Cells Exposed to Ionizing Radiation

by Daxian Luo, Emil Mladenov, Aashish Soni, Martin Stuschke and George Iliakis

Cells 2023, 12(10), 1387; https://doi.org/10.3390/cells12101387 - 14 May 2023

Viewed by 1921

Abstract

We have recently reported that in G₂-phase cells (but not S-phase cells) sustaining low loads of DNA double-strand break (DSBs), ATM and ATR regulate the G₂-checkpoint epistatically, with ATR at the output-node, interfacing with the cell cycle through Chk1. [...] Read more.

We have recently reported that in G₂-phase cells (but not S-phase cells) sustaining low loads of DNA double-strand break (DSBs), ATM and ATR regulate the G₂-checkpoint epistatically, with ATR at the output-node, interfacing with the cell cycle through Chk1. However, although inhibition of ATR nearly completely abrogated the checkpoint, inhibition of Chk1 using UCN-01 generated only partial responses. This suggested that additional kinases downstream of ATR were involved in the transmission of the signal to the cell cycle engine. Additionally, the broad spectrum of kinases inhibited by UCN-01 pointed to uncertainties in the interpretation that warranted further investigations. Here, we show that more specific Chk1 inhibitors exert an even weaker effect on G₂-checkpoint, as compared to ATR inhibitors and UCN-01, and identify the MAPK p38α and its downstream target MK2 as checkpoint effectors operating as backup to Chk1. These observations further expand the spectrum of p38/MK2 signaling to G₂-checkpoint activation, extend similar studies in cells exposed to other DNA damaging agents and consolidate a role of p38/MK2 as a backup kinase module, adding to similar backup functions exerted in p53 deficient cells. The results extend the spectrum of actionable strategies and targets in current efforts to enhance the radiosensitivity in tumor cells. Full article

(This article belongs to the Special Issue Double-Strand DNA Break Repair and Human Disease II)

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19 pages, 5735 KiB

Open AccessArticle

TRIP13 Participates in Immediate-Early Sensing of DNA Strand Breaks and ATM Signaling Amplification through MRE11

by Hyeongsun Jeong, Minwoo Wie, In-Joon Baek, Gyuwon Sohn, Si-Hyeon Um, Seon-Gyeong Lee, Yuri Seo, Jaesun Ra, Eun A Lee, Shinseog Kim, Byung Gyu Kim, Rajashree A. Deshpande, Tanya T. Paull, Joo Seok Han, Taejoon Kwon and Kyungjae Myung

Cells 2022, 11(24), 4095; https://doi.org/10.3390/cells11244095 - 16 Dec 2022

Cited by 5 | Viewed by 2737

Abstract

Thyroid hormone receptor-interacting protein 13 (TRIP13) participates in various regulatory steps related to the cell cycle, such as the mitotic spindle assembly checkpoint and meiotic recombination, possibly by interacting with members of the HORMA domain protein family. Recently, it was reported that TRIP13 [...] Read more.

Thyroid hormone receptor-interacting protein 13 (TRIP13) participates in various regulatory steps related to the cell cycle, such as the mitotic spindle assembly checkpoint and meiotic recombination, possibly by interacting with members of the HORMA domain protein family. Recently, it was reported that TRIP13 could regulate the choice of the DNA repair pathway, i.e., homologous recombination (HR) or nonhomologous end-joining (NHEJ). However, TRIP13 is recruited to DNA damage sites within a few seconds after damage and may therefore have another function in DNA repair other than regulation of the pathway choice. Furthermore, the depletion of TRIP13 inhibited both HR and NHEJ, suggesting that TRIP13 plays other roles besides regulation of choice between HR and NHEJ. To explore the unidentified functions of TRIP13 in the DNA damage response, we investigated its genome-wide interaction partners in the context of DNA damage using quantitative proteomics with proximity labeling. We identified MRE11 as a novel interacting partner of TRIP13. TRIP13 controlled the recruitment of MDC1 to DNA damage sites by regulating the interaction between MDC1 and the MRN complex. Consistently, TRIP13 was involved in ATM signaling amplification. Our study provides new insight into the function of TRIP13 in immediate-early DNA damage sensing and ATM signaling activation. Full article

(This article belongs to the Special Issue Double-Strand DNA Break Repair and Human Disease II)

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20 pages, 3733 KiB

Open AccessArticle

Smurf1 Suppression Enhances Temozolomide Chemosensitivity in Glioblastoma by Facilitating PTEN Nuclear Translocation

by Lei Dong, Yang Li, Liqun Liu, Xinyi Meng, Shengzhen Li, Da Han, Zhenyu Xiao and Qin Xia

Cells 2022, 11(20), 3302; https://doi.org/10.3390/cells11203302 - 20 Oct 2022

Cited by 4 | Viewed by 2216 | Correction

Abstract

The tumor suppressor PTEN mainly inhibits the PI3K/Akt pathway in the cytoplasm and maintains DNA stability in the nucleus. The status of PTEN remains therapeutic effectiveness for chemoresistance of the DNA alkylating agent temozolomide (TMZ) in glioblastoma (GB). However, the underlying mechanisms of [...] Read more.

The tumor suppressor PTEN mainly inhibits the PI3K/Akt pathway in the cytoplasm and maintains DNA stability in the nucleus. The status of PTEN remains therapeutic effectiveness for chemoresistance of the DNA alkylating agent temozolomide (TMZ) in glioblastoma (GB). However, the underlying mechanisms of PTEN’s interconnected role in the cytoplasm and nucleus in TMZ resistance are still unclear. In this study, we report that TMZ-induced PTEN nuclear import depends on PTEN ubiquitylation modification by Smurf1. The Smurf1 suppression decreases the TMZ-induced PTEN nuclear translocation and enhances the DNA damage. In addition, Smurf1 degrades cytoplasmic PTEN K289E (the nuclear-import-deficient PTEN mutant) to activate the PI3K/Akt pathway under TMZ treatment. Altogether, Smurf1 interconnectedly promotes PTEN nuclear function (DNA repair) and cytoplasmic function (activation of PI3K/Akt pathway) to resist TMZ. These results provide a proof-of-concept demonstration for a potential strategy to overcome the TMZ resistance in PTEN wild-type GB patients by targeting Smurf1. Full article

(This article belongs to the Special Issue Double-Strand DNA Break Repair and Human Disease II)

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19 pages, 3229 KiB

Open AccessArticle

The Ubiquitin Ligase RNF138 Cooperates with CtIP to Stimulate Resection of Complex DNA Double-Strand Breaks in Human G1-Phase Cells

by Nicole B. Averbeck, Carina Barent, Burkhard Jakob, Tatyana Syzonenko, Marco Durante and Gisela Taucher-Scholz

Cells 2022, 11(16), 2561; https://doi.org/10.3390/cells11162561 - 17 Aug 2022

Cited by 3 | Viewed by 2073

Abstract

DNA double-strand breaks (DSBs) represent the molecular origin of ionizing-radiation inflicted biological effects. An increase in the ionization density causes more complex, clustered DSBs that can be processed by resection also in G1 phase, where repair of resected DSBs is considered erroneous and [...] Read more.

DNA double-strand breaks (DSBs) represent the molecular origin of ionizing-radiation inflicted biological effects. An increase in the ionization density causes more complex, clustered DSBs that can be processed by resection also in G1 phase, where repair of resected DSBs is considered erroneous and may contribute to the increased biological effectiveness of heavy ions in radiotherapy. To investigate the resection regulation of complex DSBs, we exposed G1 cells depleted for different candidate factors to heavy ions or α-particle radiation. Immunofluorescence microscopy was used to monitor the resection marker RPA, the DSB marker γH2AX and the cell-cycle markers CENP-F and geminin. The Fucci system allowed to select G1 cells, cell survival was measured by clonogenic assay. We show that in G1 phase the ubiquitin ligase RNF138 functions in resection regulation. RNF138 ubiquitinates the resection factor CtIP in a radiation-dependent manner to allow its DSB recruitment in G1 cells. At complex DSBs, RNF138′s participation becomes more relevant, consistent with the observation that also resection is more frequent at these DSBs. Furthermore, deficiency of RNF138 affects both DSB repair and cell survival upon induction of complex DSBs. We conclude that RNF138 is a regulator of resection that is influenced by DSB complexity and can affect the quality of DSB repair in G1 cells. Full article

(This article belongs to the Special Issue Double-Strand DNA Break Repair and Human Disease II)

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21 pages, 4083 KiB

Open AccessArticle

Increased Resection at DSBs in G₂-Phase Is a Unique Phenotype Associated with DNA-PKcs Defects That Is Not Shared by Other Factors of c-NHEJ

by Huaping Xiao, Fanghua Li, Emil Mladenov, Aashish Soni, Veronika Mladenova, Bing Pan, Rositsa Dueva, Martin Stuschke, Beate Timmermann and George Iliakis

Cells 2022, 11(13), 2099; https://doi.org/10.3390/cells11132099 - 2 Jul 2022

Cited by 5 | Viewed by 2445

Abstract

The load of DNA double-strand breaks (DSBs) induced in the genome of higher eukaryotes by different doses of ionizing radiation (IR) is a key determinant of DSB repair pathway choice, with homologous recombination (HR) and ATR substantially gaining ground at doses below 0.5 [...] Read more.

The load of DNA double-strand breaks (DSBs) induced in the genome of higher eukaryotes by different doses of ionizing radiation (IR) is a key determinant of DSB repair pathway choice, with homologous recombination (HR) and ATR substantially gaining ground at doses below 0.5 Gy. Increased resection and HR engagement with decreasing DSB-load generate a conundrum in a classical non-homologous end-joining (c-NHEJ)-dominated cell and suggest a mechanism adaptively facilitating resection. We report that ablation of DNA-PKcs causes hyper-resection, implicating DNA-PK in the underpinning mechanism. However, hyper-resection in DNA-PKcs-deficient cells can also be an indirect consequence of their c-NHEJ defect. Here, we report that all tested DNA-PKcs mutants show hyper-resection, while mutants with defects in all other factors of c-NHEJ fail to do so. This result rules out the model of c-NHEJ versus HR competition and the passive shift from c-NHEJ to HR as the causes of the increased resection and suggests the integration of DNA-PKcs into resection regulation. We develop a model, compatible with the results of others, which integrates DNA-PKcs into resection regulation and HR for a subset of DSBs. For these DSBs, we propose that the kinase remains at the break site, rather than the commonly assumed autophosphorylation-mediated removal from DNA ends. Full article

(This article belongs to the Special Issue Double-Strand DNA Break Repair and Human Disease II)

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15 pages, 3988 KiB

Open AccessArticle

CNOT6: A Novel Regulator of DNA Mismatch Repair

by Peng Song, Shaojun Liu, Dekang Liu, Guido Keijzers, Daniela Bakula, Shunlei Duan, Niels de Wind, Zilu Ye, Sergey Y. Vakhrushev, Morten Scheibye-Knudsen and Lene Juel Rasmussen

Cells 2022, 11(3), 521; https://doi.org/10.3390/cells11030521 - 2 Feb 2022

Cited by 7 | Viewed by 3263

Abstract

DNA mismatch repair (MMR) is a highly conserved pathway that corrects both base–base mispairs and insertion-deletion loops (IDLs) generated during DNA replication. Defects in MMR have been linked to carcinogenesis and drug resistance. However, the regulation of MMR is poorly understood. Interestingly, CNOT6 [...] Read more.

DNA mismatch repair (MMR) is a highly conserved pathway that corrects both base–base mispairs and insertion-deletion loops (IDLs) generated during DNA replication. Defects in MMR have been linked to carcinogenesis and drug resistance. However, the regulation of MMR is poorly understood. Interestingly, CNOT6 is one of four deadenylase subunits in the conserved CCR4-NOT complex and it targets poly(A) tails of mRNAs for degradation. CNOT6 is overexpressed in acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML) and androgen-independent prostate cancer cells, which suggests that an altered expression of CNOT6 may play a role in tumorigenesis. Here, we report that a depletion of CNOT6 sensitizes human U2OS cells to N-methyl-N′nitro-N-nitrosoguanidine (MNNG) and leads to enhanced apoptosis. We also demonstrate that the depletion of CNOT6 upregulates MMR and decreases the mutation frequency in MMR-proficient cells. Furthermore, the depletion of CNOT6 increases the stability of mRNA transcripts from MMR genes, leading to the increased expression of MMR proteins. Our work provides insight into a novel CNOT6-dependent mechanism for regulating MMR. Full article

(This article belongs to the Special Issue Double-Strand DNA Break Repair and Human Disease II)

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23 pages, 3344 KiB

Open AccessArticle

Shift in G₁-Checkpoint from ATM-Alone to a Cooperative ATM Plus ATR Regulation with Increasing Dose of Radiation

by Fanghua Li, Emil Mladenov, Rositsa Dueva, Martin Stuschke, Beate Timmermann and George Iliakis

Cells 2022, 11(1), 63; https://doi.org/10.3390/cells11010063 - 27 Dec 2021

Cited by 8 | Viewed by 4086

Abstract

The current view of the involvement of PI3-kinases in checkpoint responses after DNA damage is that ATM is the key regulator of G₁-, S- or G₂-phase checkpoints, that ATR is only partly involved in the regulation of S- and [...] Read more.

The current view of the involvement of PI3-kinases in checkpoint responses after DNA damage is that ATM is the key regulator of G₁-, S- or G₂-phase checkpoints, that ATR is only partly involved in the regulation of S- and G₂-phase checkpoints and that DNA-PKcs is not involved in checkpoint regulation. However, further analysis of the contributions of these kinases to checkpoint responses in cells exposed to ionizing radiation (IR) recently uncovered striking integrations and interplays among ATM, ATR and DNA-PKcs that adapt not only to the phase of the cell cycle in which cells are irradiated, but also to the load of DNA double-strand breaks (DSBs), presumably to optimize their processing. Specifically, we found that low IR doses in G₂-phase cells activate a G₂-checkpoint that is regulated by epistatically coupled ATM and ATR. Thus, inhibition of either kinase suppresses almost fully its activation. At high IR doses, the epistatic ATM/ATR coupling relaxes, yielding to a cooperative regulation. Thus, single-kinase inhibition suppresses partly, and only combined inhibition suppresses fully G₂-checkpoint activation. Interestingly, DNA-PKcs integrates with ATM/ATR in G₂-checkpoint control, but functions in its recovery in a dose-independent manner. Strikingly, irradiation during S-phase activates, independently of dose, an exclusively ATR-dependent G₂ checkpoint. Here, ATM couples with DNA-PKcs to regulate checkpoint recovery. In the present work, we extend these studies and investigate organization and functions of these PI3-kinases in the activation of the G₁ checkpoint in cells irradiated either in the G₀ or G₁ phase. We report that ATM is the sole regulator of the G₁ checkpoint after exposure to low IR doses. At high IR doses, ATM remains dominant, but contributions from ATR also become detectable and are associated with limited ATM/ATR-dependent end resection at DSBs. Under these conditions, only combined ATM + ATR inhibition fully abrogates checkpoint and resection. Contributions of DNA-PKcs and CHK2 to the regulation of the G₁ checkpoint are not obvious in these experiments and may be masked by the endpoint employed for checkpoint analysis and perturbations in normal progression through the cell cycle of cells exposed to DNA-PKcs inhibitors. The results broaden our understanding of organization throughout the cell cycle and adaptation with increasing IR dose of the ATM/ATR/DNA-PKcs module to regulate checkpoint responses. They emphasize notable similarities and distinct differences between G₁-, G₂- and S-phase checkpoint regulation that may guide DSB processing decisions. Full article

(This article belongs to the Special Issue Double-Strand DNA Break Repair and Human Disease II)

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