DNA Double-strand Break Repair Research Articles

The RAD51 S181P mutation shortens lifespan of female mice

RAD51 is critical to the homologous recombination (HR) pathway that repairs DNA double strand breaks (DSBs) and protects replication forks (RFs). Previously, we showed that the S181P (SP) mutation in RAD51 causes defective RF maintenance but is proficient for DSB repair. Here we report that SP/SP female mice exhibit a shortened lifespan compared to +/+ females but not males. Histological analysis found that most mice in this study died from lymphoma, independent of genotype and sex. We propose that a potential cause for shortened lifespan in SP/SP females is due to the RF defect.

Endogenous base damage as a driver of genomic instability in homologous recombination-deficient cancers

Homologous recombination (HR) is a high-fidelity DNA double-strand break (DSB) repair pathway. Both familial and somatic loss of function mutation(s) in various HR genes predispose to a variety of cancer types, underscoring the importance of error-free repair of DSBs in human physiology. While environmental sources of DSBs have been known, more recent studies have begun to uncover the role of endogenous base damage in leading to these breaks. Base damage repair intermediates often consist of single-strand breaks, which if left unrepaired, can lead to DSBs as the replication fork encounters these lesions. This review summarizes various sources of endogenous base damage and how these lesions are repaired. We highlight how conversion of base repair intermediates, particularly those with 5′or 3′ blocked ends, to DSBs can be a predominant source of genomic instability in HR-deficient cancers. We also discuss how endogenous base damage and ensuing DSBs can be exploited to enhance the efficacy of Poly (ADP-ribose) polymerase inhibitors (PARPi), that are widely used in the clinics for the regimen of HR-deficient cancers.

Increased AID results in mutations at the CRLF2 locus implicated in Latin American ALL health disparities

Activation-induced cytidine deaminase (AID) is a B cell-specific mutator required for antibody diversification. However, it is also implicated in the etiology of several B cell malignancies. Evaluating the AID-induced mutation load in patients at-risk for certain blood cancers is critical in assessing disease severity and treatment options. We have developed a digital PCR (dPCR) assay that allows us to quantify mutations resulting from AID modification or DNA double-strand break (DSB) formation and repair at sites known to be prone to DSBs. Implementation of this assay shows that increased AID levels in immature B cells increase genome instability at loci linked to chromosomal translocation formation. This includes the CRLF2 locus that is often involved in translocations associated with a subtype of acute lymphoblastic leukemia (ALL) that disproportionately affects Hispanics, particularly those with Latin American ancestry. Using dPCR, we characterize the CRLF2 locus in B cell-derived genomic DNA from both Hispanic ALL patients and healthy Hispanic donors and found increased mutations in both, suggesting that vulnerability to DNA damage at CRLF2 may be driving this health disparity. Our ability to detect and quantify these mutations will potentiate future risk identification, early detection of cancers, and reduction of associated cancer health disparities.

Targeting PRMT5 enhances the radiosensitivity of tumor cells grown in vitro and in vivo

PRMT5 is a widely expressed arginine methyltransferase that regulates processes involved in tumor cell proliferation and survival. In the study described here, we investigated whether PRMT5 provides a target for tumor radiosensitization. Knockdown of PRMT5 using siRNA enhanced the radiosensitivity of a panel of cell lines corresponding to tumor types typically treated with radiotherapy. To extend these studies to an experimental therapeutic setting, the PRMT5 inhibitor LLY-283 was used. Exposure of the tumor cell lines to LLY-283 decreased PRMT5 activity and enhanced their radiosensitivity. This increase in radiosensitivity was accompanied by an inhibition of DNA double-strand break repair as determined by γH2AX foci and neutral comet analyses. For a normal fibroblast cell line, although LLY-283 reduced PRMT5 activity, it had no effect on their radiosensitivity. Transcriptome analysis of U251 cells showed that LLY-283 treatment reduced the expression of genes and altered the mRNA splicing pattern of genes involved in the DNA damage response. Subcutaneous xenografts were then used to evaluate the in vivo response to LLY-283 and radiation. Treatment of mice with LLY-283 decreased tumor PRMT5 activity and significantly enhanced the radiation-induced growth delay. These results suggest that PRMT5 is a tumor selective target for radiosensitization.

M6A-mediated lnc-OXAR promotes oxaliplatin resistance by enhancing Ku70 stability in non-alcoholic steatohepatitis-related hepatocellular carcinoma

BackgroundThe escalating prevalence of metabolic diseases has led to a rapid increase in non-alcoholic steatohepatitis (NASH)-related hepatocellular carcinoma (NASH-HCC). While oxaliplatin (OXA)-based hepatic arterial infusion chemotherapy (HAIC) has shown promise in advanced-stage HCC patients, its efficacy in NASH-HCC remains uncertain. This study aims to assess the effectiveness of OXA-based HAIC and elucidate the mechanisms underlying OXA resistance in NASH-HCC.MethodsThe key lncRNAs were screened through RNA-seq analysis of NASH/non-NASH and OXA-sensitive/OXA-resistant (OXA-S/R) HCC tissues. The biological functions of the lnc-OXAR (OXA resistance–related lncRNA in NASH-HCC) in NASH-HCC were verified through a series of in vitro and in vivo experiments. The molecular mechanism of lnc-OXAR was elucidated by fluorescence in situ hybridization, immunoprecipitation-mass spectrometry (FISH), Immunoprecipitation-Mass Spectrometry (IP-MS), RNA pulldown, RNA immunoprecipitation (RIP), methylated RNA immunoprecipitation sequencing (MeRIP-Seq) and a dual-luciferase reporter assay.ResultsNASH-HCC exhibited reduced responsiveness to OXA-based HAIC compared to non-NASH HCC. We identified and validated a novel transcript namedlnc-OXAR, which played a crucial role in conferring OXA resistance to NASH-HCC. Inhibition of lnc-OXAR suppressed HCC cell growth and restored OXA sensitivity both in NASH-HCC mouse models and in vitro. Mechanistically, lnc-OXAR recruited Ku70 and cystatin A (CSTA), preventing Ku70 degradation and facilitating DNA double-strand break (DSB) repair, thereby promoting OXA resistance in NASH-HCC. Additionally, WTAP-mediated m6A modification enhanced the stability of lnc-OXAR in an IGF2BP2-dependent manner. Notably, silencing lnc-OXAR significantly enhanced the response to OXA in patient-derived xenograft (PDX) models derived from NASH-HCC.ConclusionsThe reduced responsiveness of NASH-HCC to OXA treatment can be attributed to the upregulation of lnc-OXAR. Our findings provide a rationale for stratifying HCC patients undergoing OXA-based HAIC based on etiology. Lnc-OXAR holds promise as a novel target for overcoming OXA resistance in NASH-HCC and improving prognosis.

Human translesion DNA polymerases ι and κ mediate tolerance to temozolomide in MGMT-deficient glioblastoma cells.

Glioblastoma (GBM) is a highly aggressive brain tumor associated with poor patient survival. The current standard treatment involves invasive surgery, radiotherapy, and chemotherapy employing temozolomide (TMZ). Resistance to TMZ is, however, a major challenge. Previous work from our group has identified candidate genes linked to TMZ resistance, including genes encoding translesion synthesis (TLS) DNA polymerases iota (Polɩ) and kappa (Polκ). These specialized enzymes are known for bypassing lesions and tolerating DNA damage. Here, we investigated the roles of Polɩ and Polκ in TMZ resistance, employing MGMT-deficient U251-MG glioblastoma cells, with knockout of either POLI or POLK genes encoding Polɩ and Polκ, respectively, and assess their viability and genotoxic stress responses upon subsequent TMZ treatment. Cells lacking either of these polymerases exhibited a significant decrease in viability following TMZ treatment compared to parental counterparts. The restoration of the missing polymerase led to a recovery of cell viability. Furthermore, knockout cells displayed increased cell cycle arrest, mainly in late S-phase, and lower levels of genotoxic stress after TMZ treatment, as assessed by a reduction of γH2AX foci and flow cytometry data. This implies that TMZ treatment does not trigger a significant H2AX phosphorylation response in the absence of these proteins. Interestingly, combining TMZ with Mirin (double-strand break repair pathway inhibitor) further reduced the cell viability and increased DNA damage and γH2AX positive cells in TLS KO cells, but not in parental cells. These findings underscore the crucial roles of Polɩ and Polκ in conferring TMZ resistance and the potential backup role of homologous recombination in the absence of these TLS polymerases. Targeting these TLS enzymes, along with double-strand break DNA repair inhibition, could, therefore, provide a promising strategy to enhance TMZ's effectiveness in treating GBM.

Multifunctional Glycopeptide-Based Hydrogel via Dual-Modulation for the Prevention and Repair of Radiation-Induced Skin Injury.

The radiation-induced skin injury (RISI) remains a great challenge for clinical wound management and care after radiotherapy, as patients will suffer from the acute radiation injury and long-term chronic inflammatory damage during the treatment. The excessive ROS in the early acute stage and prolonged inflammatory response in the late healing process always hinder therapeutic efficiency. Herein, we developed an extracellular matrix (ECM)-mimetic multifunctional glycopeptide hydrogel (oCP@As) to promote and accelerate RISI repair via a dual-modulation strategy in different healing stages. The oCP@As hydrogel not only can form an ECM-like nanofiber structure through the Schiff base reaction but also exhibits ROS scavenging and DNA double-strand break repair abilities, which can effectively reduce the acute radiation damage. Meanwhile, the introduction of oxidized chondroitin sulfate, which is the ECM polysaccharide-like component, enables regulation of the inflammatory response by adsorption of inflammatory factors, accelerating the repair of chronic inflammatory injury. The animal experiments demonstrated that oCP@As can significantly weaken RISI symptoms, promote epidermal tissue regeneration and angiogenesis, and reduce pro-inflammatory cytokine expression. Therefore, this multifunctional glycopeptide hydrogel dressing can effectively attenuate RISI symptoms and promote RISI healing, showing great potential for clinical applications in radiotherapy protection and repair.

Meiotic double-strand break repair DNA synthesis tracts in Arabidopsis thaliana.

We report here the successful labelling of meiotic prophase I DNA synthesis in the flowering plant, Arabidopsis thaliana. Incorporation of the thymidine analogue, EdU, enables visualisation of the footprints of recombinational repair of programmed meiotic DNA double-strand breaks (DSB), with ~400 discrete, SPO11-dependent, EdU-labelled chromosomal foci clearly visible at pachytene and later stages of meiosis. This number equates well with previous estimations of 200-300 DNA double-strand breaks per meiosis in Arabidopsis, confirming the power of this approach to detect the repair of most or all SPO11-dependent meiotic DSB repair events. The chromosomal distribution of these DNA-synthesis foci accords with that of early recombination markers and MLH1, which marks Class I crossover sites. Approximately 10 inter-homologue cross-overs (CO) have been shown to occur in each Arabidopsis male meiosis and, athough very probably under-estimated, an equivalent number of inter-homologue gene conversions (GC) have been described. Thus, at least 90% of meiotic recombination events, and very probably more, have not previously been accessible for analysis. Visual examination of the patterns of the foci on the synapsed pachytene chromosomes corresponds well with expectations from the different mechanisms of meiotic recombination and notably, no evidence for long Break-Induced Replication DNA synthesis tracts was found. Labelling of meiotic prophase I, SPO11-dependent DNA synthesis holds great promise for further understanding of the molecular mechanisms of meiotic recombination, at the heart of reproduction and evolution of eukaryotes.

A Genomewide Evolution-Based CRISPR/Cas9 with Donor-Free (GEbCD) for Developing Robust and Productive Industrial Yeast.

Developing more robust and productive industrial yeast is crucial for high-efficiency biomanufacturing. However, the challenges posed by the long time required and the low abundance of mutations generated through genomewide evolutionary engineering hinder the development and optimization of desired hosts for industrial applications. To address these issues, we present a novel solution called the Genomewide Evolution-based CRISPR/Cas with Donor-free (GEbCD) system, in which nonhomologous-end-joining (NHEJ) repair can accelerate the acquisition of highly abundant yeast mutants. Together with modified rad52 of the DNA double-strand break repair in Saccharomyces cerevisiae, a hypermutation host was obtained with a 400-fold enhanced mutation ability. Under multiple environmental stresses the system could rapidly generate millions of mutants in a few rounds of iterative evolution. Using high-throughput screening, an industrial S. cerevisiae SISc-Δrad52-G4-72 (G4-72) was obtained that is strongly robust and has higher productivity. G4-72 grew stably and produced ethanol efficiently in multiple-stress environments, e.g. high temperature and high osmosis. In a pilot-scale fermentation with G4-72, the fermentation temperature was elevated by 8 °C and ethanol production was increased by 6.9% under the multiple stresses posed by the industrial fermentation substrate. Overall, the GEbCD system presents a powerful tool to rapidly generate abundant mutants and desired hosts, and offers a novel strategy for optimizing microbial chassis with regard to demands posed in industrial applications.

The Central Role of Cytogenetics in Radiation Biology.

Radiation cytogenetics has a rich history seldom appreciated by those outside the field. Early radiobiology was dominated by physics and biophysical concepts that borrowed heavily from the study of radiation-induced chromosome aberrations. From such studies, quantitative relationships between biological effect and changes in absorbed dose, dose rate and ionization density were codified into key concepts of radiobiological theory that have persisted for nearly a century. This review aims to provide a historical perspective of some of these concepts, including evidence supporting the contention that chromosome aberrations underlie development of many, if not most, of the biological effects of concern for humans exposed to ionizing radiations including cancer induction, on the one hand, and tumor eradication on the other. The significance of discoveries originating from these studies has widened and extended far beyond their original scope. Chromosome structural rearrangements viewed in mitotic cells were first attributed to the production of breaks by the radiations during interphase, followed by the rejoining or mis-rejoining among ends of other nearby breaks. These relatively modest beginnings eventually led to the discovery and characterization of DNA repair of double-strand breaks by non-homologous end joining, whose importance to various biological processes is now widely appreciated. Two examples, among many, are V(D)J recombination and speciation. Rapid technological advancements in cytogenetics, the burgeoning fields of molecular radiobiology and third-generation sequencing served as a point of confluence between the old and new. As a result, the emergent field of "cytogenomics" now becomes uniquely positioned for the purpose of more fully understanding mechanisms underlying the biological effects of ionizing radiation exposure.

HLTF disrupts Cas9-DNA post-cleavage complexes to allow DNA break processing

The outcome of CRISPR-Cas-mediated genome modifications is dependent on DNA double-strand break (DSB) processing and repair pathway choice. Homology-directed repair (HDR) of protein-blocked DSBs requires DNA end resection that is initiated by the endonuclease activity of the MRE11 complex. Using reconstituted reactions, we show that Cas9 breaks are unexpectedly not directly resectable by the MRE11 complex. In contrast, breaks catalyzed by Cas12a are readily processed. Cas9, unlike Cas12a, bridges the broken ends, preventing DSB detection and processing by MRE11. We demonstrate that Cas9 must be dislocated after DNA cleavage to allow DNA end resection and repair. Using single molecule and bulk biochemical assays, we next find that the HLTF translocase directly removes Cas9 from broken ends, which allows DSB processing by DNA end resection or non-homologous end-joining machineries. Mechanistically, the activity of HLTF requires its HIRAN domain and the release of the 3′-end generated by the cleavage of the non-target DNA strand by the Cas9 RuvC domain. Consequently, HLTF removes the H840A but not the D10A Cas9 nickase. The removal of Cas9 H840A by HLTF explains the different cellular impact of the two Cas9 nickase variants in human cells, with potential implications for gene editing.

An E3 ubiquitin ligase localization screen uncovers DTX2 as a novel ADP-ribosylation-dependent regulator of DNA double-strand break repair

DNA double-strand breaks (DSBs) elicit an elaborate response to signal damage and trigger repair via two major pathways: non-homologous end-joining (NHEJ), which functions throughout the interphase, and homologous recombination (HR), restricted to S/G2 phases. The DNA damage response (DDR) relies, on post-translational modifications of nuclear factors to coordinate the mending of breaks. Ubiquitylation of histones and chromatin-associated factors regulates DSB repair and numerous E3 ubiquitin ligases are involved in this process. Despite significant progress, our understanding of ubiquitin-mediated DDR regulation remains incomplete.Here, we have performed a localization screen to identify RING/U-box E3 ligases involved in genome maintenance. Our approach uncovered 7 novel E3 ligases that are recruited to microirradiation stripes, suggesting potential roles in DNA damage signaling and repair. Amongst these factors, the DELTEX family E3 ligase DTX2 is rapidly mobilized to lesions in a poly ADP-ribosylation-dependent manner. DTX2 is recruited and retained at DSBs via its WWE and DTC domains. In cells, both domains are required for optimal binding to mono and poly ADP-ribosylated proteins with WWEs playing a prominent role in this process. Supporting its involvement in DSB repair, DTX2 depletion decreases HR efficiency and moderately enhances NHEJ. Furthermore, DTX2 depletion impeded BRCA1 foci formation and increased 53BP1 accumulation at DSBs, suggesting a fine-tuning role for this E3 ligase in repair pathway choice. Finally, DTX2 depletion sensitized cancer cells to X-rays and PARP inhibition and these susceptibilities could be rescued by DTX2 re-expression. Altogether, our work identifies DTX2 as a novel ADP-ribosylation-dependent regulator of HR-mediated DSB repair.

Nuclear F-actin assembly on damaged chromatin is regulated by DYRK1A and Spir1 phosphorylation.

Nuclear actin-based movements support DNA double-strand break (DSB) repair. However, molecular determinants that promote filamentous actin (F-actin) formation on the damaged chromatin remain undefined. Here we describe the DYRK1A kinase as a nuclear activity that promotes local F-actin assembly to support DSB mobility and repair, accomplished in part by its targeting of actin nucleator spire homolog 1 (Spir1). Indeed, perturbing DYRK1A-dependent phosphorylation of S482 mis-regulated Spir1 accumulation at damaged-modified chromatin, and led to compromised DSB-associated actin polymerization and attenuated DNA repair. Our findings uncover a role of the DYRK1A-Spir1 axis in nuclear actin dynamics during early DSB responses, and highlight the intricate details of nuclear cytoskeletal network in DSB repair and genome stability maintenance.

Ubiquitin-induced RNF168 condensation promotes DNA double-strand break repair

Rapid accumulation of repair factors at DNA double-strand breaks (DSBs) is essential for DSB repair. Several factors involved in DSB repair have been found undergoing liquid-liquid phase separation (LLPS) at DSB sites to facilitate DNA repair. RNF168, a RING-type E3 ubiquitin ligase, catalyzes H2A.X ubiquitination for recruiting DNA repair factors. Yet, whether RNF168 undergoes LLPS at DSB sites remains unclear. Here, we identified K63-linked polyubiquitin-triggered RNF168 condensation which further promoted RNF168-mediated DSB repair. RNF168 formed liquid-like condensates upon irradiation in the nucleus while purified RNF168 protein also condensed in vitro. An intrinsically disordered region containing amino acids 460-550 was identified as the essential domain for RNF168 condensation. Interestingly, LLPS of RNF168 was significantly enhanced by K63-linked polyubiquitin chains, and LLPS largely enhanced the RNF168-mediated H2A.X ubiquitination, suggesting a positive feedback loop to facilitate RNF168 rapid accumulation and its catalytic activity. Functionally, LLPS deficiency of RNF168 resulted in delayed recruitment of 53BP1 and BRCA1 and subsequent impairment in DSB repair. Taken together, our finding demonstrates the pivotal effect of LLPS in RNF168-mediated DSB repair.

Profiling Tel1 Signaling Reveals a Non-Canonical Motif Targeting DNA Repair and Telomere Control Machineries.

The stability of the genome relies on Phosphatidyl Inositol 3-Kinase-related Kinases (PIKKs) that sense DNA damage and trigger elaborate downstream signaling responses. In S. cerevisiae, the Tel1 kinase (ortholog of human ATM) is activated at DNA double strand breaks (DSBs) and short telomeres. Despite the well-established roles of Tel1 in the control of telomere maintenance, suppression of chromosomal rearrangements, activation of cell cycle checkpoints, and repair of DSBs, the substrates through which Tel1 controls these processes remain incompletely understood. Here we performed an in depth phosphoproteomic screen for Tel1-dependent phosphorylation events. To achieve maximal coverage of the phosphoproteome, we developed a scaled-up approach that accommodates large amounts of protein extracts and chromatographic fractions. Compared to previous reports, we expanded the number of detected Tel1-dependent phosphorylation events by over 10-fold. Surprisingly, in addition to the identification of phosphorylation sites featuring the canonical motif for Tel1 phosphorylation (S/T-Q), the results revealed a novel motif (D/E-S/T) highly prevalent and enriched in the set of Tel1-dependent events. This motif is unique to Tel1 signaling and not shared with the Mec1 kinase, providing clues to how Tel1 plays specialized roles in DNA repair and telomere length control. Overall, these findings define a Tel1-signaling network targeting numerous proteins involved in DNA repair, chromatin regulation, and telomere maintenance that represents a framework for dissecting the molecular mechanisms of Tel1 action.

Non-homologous end joining shapes the genomic rearrangement landscape of chromothripsis from mitotic errors

Mitotic errors generate micronuclei entrapping mis-segregated chromosomes, which are susceptible to catastrophic fragmentation through chromothripsis. The reassembly of fragmented chromosomes by error-prone DNA double-strand break (DSB) repair generates diverse genomic rearrangements associated with human diseases. How specific repair pathways recognize and process these lesions remains poorly understood. Here we use CRISPR/Cas9 to systematically inactivate distinct DSB repair pathways and interrogate the rearrangement landscape of fragmented chromosomes. Deletion of canonical non-homologous end joining (NHEJ) components substantially reduces complex rearrangements and shifts the rearrangement landscape toward simple alterations without the characteristic patterns of chromothripsis. Following reincorporation into the nucleus, fragmented chromosomes localize within sub-nuclear micronuclei bodies (MN bodies) and undergo ligation by NHEJ within a single cell cycle. In the absence of NHEJ, chromosome fragments are rarely engaged by alternative end-joining or recombination-based mechanisms, resulting in delayed repair kinetics, persistent 53BP1-labeled MN bodies, and cell cycle arrest. Thus, we provide evidence supporting NHEJ as the exclusive DSB repair pathway generating complex rearrangements from mitotic errors.

Progerin Can Induce DNA Damage in the Absence of Global Changes in Replication or Cell Proliferation.

Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare genetic condition characterized by features of accelerated aging, and individuals with HGPS seldom live beyond their mid-teens. The syndrome is commonly caused by a point mutation in the LMNA gene which codes for lamin A and its splice variant lamin C, components of the nuclear lamina. The mutation causing HGPS leads to production of a truncated, farnesylated form of lamin A referred to as "progerin." Progerin is also expressed at low levels in healthy individuals and appears to play a role in normal aging. HGPS is associated with an accumulation of genomic DNA double-strand breaks (DSBs) and alterations in the nature of DSB repair. The source of DSBs in HGPS is often attributed to stalling and subsequent collapse of replication forks in conjunction with faulty recruitment of repair factors to damage sites. In this work, we used a model system involving immortalized human cell lines to investigate progerin-induced genomic damage. Using an immunofluorescence approach to visualize phosphorylated histone H2AX foci which mark sites of genomic damage, we report that cells engineered to express progerin displayed a significant elevation of endogenous damage in the absence of any change in the cell cycle profile or doubling time of cells. Genomic damage was enhanced and persistent in progerin-expressing cells treated with hydroxyurea. Overexpression of wild-type lamin A did not elicit the outcomes associated with progerin expression. Our results show that DNA damage caused by progerin can occur independently from global changes in replication or cell proliferation.

ARID1A is involved in DNA double-strand break repair in gastric cancer.

Defects in DNA damage repair can cause genetic mutations, which in turn can cause different types of cancers. Chromatin remodeling complexes, which help repair damaged DNA, can cause the chromatin structure to change as a result of DNA damage. ARID1A may play a role in the process of DNA damage repair, and arid1a may be related to the occurrence and development of gastric cancer (GC). This study aimed to investigate the mechanism of ARID1A regulating the DNA damage repair of gastric adenocarcinoma cell lines AGS and SGC-7901 and its effect on migration, proliferation and apoptosis. The expression of ARID1A plasmid was detected by Western blot and real-time polymerase chain reaction (PCR). The effect of etoposide (ETO) on the survival rate of AGS and SGC-7901 gastric adenocarcinoma cell lines was detected by MTT assay. The DNA double-strand break model was established by ETO and then passed through the comet assay and immunofluorescence co-localization to observe DNA damage; western blot method was used to detect the effect of ARID1A on the expression of related proteins in DNA damage repair pathway in gastric adenocarcinoma cells; scratch test and colony formation experiments were used to observe ARID1A migration and proliferation of gastric adenocarcinoma cells. The flow cytometry was used to detect the effect of ARID1A on apoptosis of gastric adenocarcinoma cells. The expression of mRNA and protein was increased after transfection of ARID1A plasmid. ETO was confirmed by MTT assay to inhibit cell survival in a dose-dependent manner. After the DNA double-strand break model was established by ETO, the expression levels of phospho-ataxia telangiectasia mutated (p-ATM) protein increased in the overexpressed ARID1A group. Meanwhile, the overexpressed ARID1A group had a shortened tail moment, and γ-H2AX and ARID1A co-localized in the DNA damage site of the nucleus. The over-expressed ARID1A group had weaker wound healing ability, reduced number of clone formation, and increased apoptosis rate. ARID1A may repair DNA double-strand breaks caused by ETO by p-ATM pathway; ARID1A can inhibit the migration and proliferation of gastric adenocarcinoma cells and promote apoptosis. Our findings indicate that ARID1A could serve as a therapeutic target and biomarker for GC patients.

In-depth mapping of DNA-PKcs signaling uncovers noncanonical features of its kinase specificity

DNA-PKcs is a DNA damage sensor kinase with established roles in DNA double-strand break repair via nonhomologous end joining. Recent studies have revealed additional roles of DNA-PKcs in the regulation of transcription, translation, and DNA replication. However, the substrates through which DNA-PKcs regulates these processes remain largely undefined. Here, we utilized quantitative phosphoproteomics to generate a high coverage map of DNA-PKcs signaling in response to ionizing radiation and mapped its interplay with the ATM kinase. Beyond the detection of the canonical S/T-Q phosphorylation motif, we uncovered a noncanonical mode of DNA-PKcs signaling targeting S/T-ψ-D/E motifs. Sequence and structural analyses of the DNA-PKcs substrate recognition pocket revealed unique features compared to closely related phosphatidylinositol 3-kinase-related kinases that may explain its broader substrate preference. These findings expand the repertoire of DNA-PKcs and ATM substrates while establishing a novel preferential phosphorylation motif for DNA-PKcs.

Cumulative Dose from Recurrent CT Scans: Exploring the DNA Damage Response in Human Non-Transformed Cells.

Recurrent computed tomography (CT) examination has become a common diagnostic procedure for several diseases and injuries. Though each singular CT scan exposes individuals at low doses of low linear energy transfer (LET) radiation, the cumulative dose received from recurrent CT scans poses an increasing concern for potential health risks. Here, we evaluated the biological effects of recurrent CT scans on the DNA damage response (DDR) in human fibroblasts and retinal pigment epithelial cells maintained in culture for five months and subjected to four CT scans, one every four weeks. DDR kinetics and eventual accumulation of persistent-radiation-induced foci (P-RIF) were assessed by combined immunofluorescence for γH2AX and 53BP1, i.e., γH2AX/53BP1 foci. We found that CT scan repetitions significantly increased both the number and size of γH2AX/53BP1 foci. In particular, after the third CT scan, we observed the appearance of giant foci that might result from the overlapping of individual small foci and that do not associate with irreversible growth arrest, as shown by DNA replication in the foci-carrying cells. Whether these giant foci represent coalescence of unrepaired DNA damage as reported following single exposition to high doses of high LET radiation is still unclear. However, morphologically, these giant foci resemble the recently described compartmentalization of damaged DNA that should facilitate the repair of DNA double-strand breaks but also increase the risk of chromosomal translocations. Overall, these results indicate that for a correct evaluation of the damage following recurrent CT examinations, it is necessary to consider the size and composition of the foci in addition to their number.