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

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

doi:10.3390/cells11132099

Cited by 6 publications

(12 citation statements)

References 73 publications

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“…RPA70 intensity in the S phase is measured by gating S-phase cells as shown in Figure 6 A. Irradiated CHO cells show an increase in RPA70 signal when compared to 0 Gy controls, indicating IR-induced DNA end resection ( Figure 6 B upper panel). DNA-PKcs inhibition ( Figure 6 B second panel from top) has no effect on resection, as reported earlier [ 26 , 27 , 38 ]. Inhibition of ATM ( Figure 6 B third panel from top) or ATR individually ( Figure 6 B fourth panel from top) partly suppresses resection.…”

Section: Resultssupporting

confidence: 84%

“…Suppression of resection through ATMi or ATRi individually, but particularly in combination, reflects well the observed impact of these kinases on the intra-S-phase checkpoint. In addition, the DNA-PKcs mutant, V3, shows clearly elevated resection in S phase when compared to wild-type CHO cells (Figure 6C), which is in line with similar results obtained in G 2 -phase irradiated cells [26,27,38].…”

Section: The Degree Of Dna End-resection At Dsbs In the S-phase Corre...supporting

confidence: 88%

“…The possibility that the delayed intra-S-phase checkpoint recovery in DNA-PKcs–deficient cells is due to compromised DSB repair through a defect in c-NHEJ is unlikely, as XRCC4 and LIG4 mutants, despite the potentiated checkpoint, exhibited checkpoint recovery when compared to their corresponding DNA-PK deficient counterparts ( Figure 3 E,F and Figure S1 ). In addition, recent studies from our group show that only DNA-PKcs mutation causes hyperresection upon irradiation in G 2 -phase cells, while cells with defects in other c-NHEJ factors show normal resection levels, comparable to control cells [ 26 , 38 ]. It has been shown that DNA-PKcs may crosstalk with ATM and ATR [ 53 , 54 , 55 , 56 , 57 ], suggesting a non-canonical, c-NHEJ–independent, function.…”

Section: Discussionmentioning

confidence: 92%

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ATR Contributes More Than ATM in Intra-S-Phase Checkpoint Activation after IR, and DNA-PKcs Facilitates Recovery: Evidence for Modular Integration of ATM/ATR/DNA-PKcs Functions

Soni

Duan

Stuschke

et al. 2022

IJMS

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The intra-S-phase checkpoint was among the first reported cell cycle checkpoints in mammalian cells. It transiently slows down the rate of DNA replication after DNA damage to facilitate repair and thus prevents genomic instability. The ionizing radiation (IR)-induced intra-S-phase checkpoint in mammalian cells is thought to be mainly dependent upon the kinase activity of ATM. Defects in the intra-S-phase checkpoint result in radio-resistant DNA synthesis (RDS), which promotes genomic instability. ATM belongs to the PI3K kinase family along with ATR and DNA-PKcs. ATR has been shown to be the key kinase for intra-S-phase checkpoint signaling in yeast and has also been implicated in this checkpoint in higher eukaryotes. Recently, contributions of DNA-PKcs to IR-induced G2-checkpoint could also be established. Whether and how ATR and DNA-PKcs are involved in the IR-induced intra-S-phase checkpoint in mammalian cells is incompletely characterized. Here, we investigated the contributions of ATM, ATR, and DNA-PKcs to intra-S-phase checkpoint activation after exposure to IR of human and hamster cells. The results suggest that the activities of both ATM and ATR are essential for efficient intra-S-phase checkpoint activation. Indeed, in a wild-type genetic background, ATR inhibition generates stronger checkpoint defects than ATM inhibition. Similar to G2 checkpoint, DNA-PKcs contributes to the recovery from the intra-S-phase checkpoint. DNA-PKcs–deficient cells show persistent, mainly ATR-dependent intra-S-phase checkpoints. A correlation between the degree of DSB end resection and the strength of the intra-S-phase checkpoint is observed, which again compares well to the G2 checkpoint response. We conclude that the organization of the intra-S-phase checkpoint has a similar mechanistic organization to that of the G2 checkpoint in cells irradiated in the G2 phase.

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Section: Resultssupporting

confidence: 84%

Section: The Degree Of Dna End-resection At Dsbs In the S-phase Corre...supporting

confidence: 88%

Section: Discussionmentioning

confidence: 92%

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ATR Contributes More Than ATM in Intra-S-Phase Checkpoint Activation after IR, and DNA-PKcs Facilitates Recovery: Evidence for Modular Integration of ATM/ATR/DNA-PKcs Functions

Soni

Duan

Stuschke

et al. 2022

IJMS

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“…A commonly accepted model is that, prior to DNA ligation, DNA-PKcs must be displaced from DNA ends by autophosphorylation to free them up for ligation [70]. However, new data suggest that DNA-PKcs could remain bound to DNA ends and help coordinate the overall repair reaction including the transfer of DNA ends for processing by HR [71].…”

Section: Classical Non-homologous End Joining (C-nhej)mentioning

confidence: 99%

New Facets of DNA Double Strand Break Repair: Radiation Dose as Key Determinant of HR versus c-NHEJ Engagement

Mladenov,

Mladenova,

Stuschke

et al. 2023

IJMS

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Radiation therapy is an essential component of present-day cancer management, utilizing ionizing radiation (IR) of different modalities to mitigate cancer progression. IR functions by generating ionizations in cells that induce a plethora of DNA lesions. The most detrimental among them are the DNA double strand breaks (DSBs). In the course of evolution, cells of higher eukaryotes have evolved four major DSB repair pathways: classical non-homologous end joining (c-NHEJ), homologous recombination (HR), alternative end-joining (alt-EJ), and single strand annealing (SSA). These mechanistically distinct repair pathways have different cell cycle- and homology-dependencies but, surprisingly, they operate with widely different fidelity and kinetics and therefore contribute unequally to cell survival and genome maintenance. It is therefore reasonable to anticipate tight regulation and coordination in the engagement of these DSB repair pathway to achieve the maximum possible genomic stability. Here, we provide a state-of-the-art review of the accumulated knowledge on the molecular mechanisms underpinning these repair pathways, with emphasis on c-NHEJ and HR. We discuss factors and processes that have recently come to the fore. We outline mechanisms steering DSB repair pathway choice throughout the cell cycle, and highlight the critical role of DNA end resection in this process. Most importantly, however, we point out the strong preference for HR at low DSB loads, and thus low IR doses, for cells irradiated in the G2-phase of the cell cycle. We further explore the molecular underpinnings of transitions from high fidelity to low fidelity error-prone repair pathways and analyze the coordination and consequences of this transition on cell viability and genomic stability. Finally, we elaborate on how these advances may help in the development of improved cancer treatment protocols in radiation therapy.

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“…Another form of homology directed repair is the single-strand annealing (SSA), which is initiated when long stretches of ssDNA, generated by DNA end-resection, reveal homologies within the same DNA molecule. SSA is mostly active during the S- and G 2 -phase of the cell cycle, where DNA end-resection is at its maximum [ 37 ]. RAD52, an essential molecule in this process, catalyzes annealing between these homologous repeats, which causes the deletion of the intervening DNA segment during the subsequent repair steps [ 38 , 39 , 40 ].…”

Section: Introductionmentioning

confidence: 99%

Increased Gene Targeting in Hyper-Recombinogenic LymphoBlastoid Cell Lines Leaves Unchanged DSB Processing by Homologous Recombination

Mladenov

Paul-Konietzko

Mladenova

et al. 2022

IJMS

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In the cells of higher eukaryotes, sophisticated mechanisms have evolved to repair DNA double-strand breaks (DSBs). Classical nonhomologous end joining (c-NHEJ), homologous recombination (HR), alternative end joining (alt-EJ) and single-strand annealing (SSA) exploit distinct principles to repair DSBs throughout the cell cycle, resulting in repair outcomes of different fidelity. In addition to their functions in DSB repair, the same repair pathways determine how cells integrate foreign DNA or rearrange their genetic information. As a consequence, random integration of DNA fragments is dominant in somatic cells of higher eukaryotes and suppresses integration events at homologous genomic locations, leading to very low gene-targeting efficiencies. However, this response is not universal, and embryonic stem cells display increased targeting efficiency. Additionally, lymphoblastic chicken and human cell lines DT40 and NALM6 show up to a 1000-fold increased gene-targeting efficiency that is successfully harnessed to generate knockouts for a large number of genes. We inquired whether the increased gene-targeting efficiency of DT40 and NALM6 cells is linked to increased rates of HR-mediated DSB repair after exposure to ionizing radiation (IR). We analyzed IR-induced γ-H2AX foci as a marker for the total number of DSBs induced in a cell and RAD51 foci as a marker for the fraction of those DSBs undergoing repair by HR. We also evaluated RPA accretion on chromatin as evidence for ongoing DNA end resection, an important initial step for all pathways of DSB repair except c-NHEJ. We finally employed the DR-GFP reporter assay to evaluate DSB repair by HR in DT40 cells. Collectively, the results obtained, unexpectedly show that DT40 and NALM6 cells utilized HR for DSB repair at levels very similar to those of other somatic cells. These observations uncouple gene-targeting efficiency from HR contribution to DSB repair and suggest the function of additional mechanisms increasing gene-targeting efficiency. Indeed, our results show that analysis of the contribution of HR to DSB repair may not be used as a proxy for gene-targeting efficiency.

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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

Cited by 6 publications

References 73 publications

ATR Contributes More Than ATM in Intra-S-Phase Checkpoint Activation after IR, and DNA-PKcs Facilitates Recovery: Evidence for Modular Integration of ATM/ATR/DNA-PKcs Functions

ATR Contributes More Than ATM in Intra-S-Phase Checkpoint Activation after IR, and DNA-PKcs Facilitates Recovery: Evidence for Modular Integration of ATM/ATR/DNA-PKcs Functions

New Facets of DNA Double Strand Break Repair: Radiation Dose as Key Determinant of HR versus c-NHEJ Engagement

Increased Gene Targeting in Hyper-Recombinogenic LymphoBlastoid Cell Lines Leaves Unchanged DSB Processing by Homologous Recombination

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