Complex interplay between FMRP and DHX9 during DNA replication stress.

Mutations in, or deficiency of, fragile X messenger ribonucleoprotein (FMRP) is responsible for the Fragile X syndrome (FXS), the most common cause for inherited intellectual disability. FMRP is a nucleocytoplasmic protein, primarily characterized as a translation repressor with poorly understood nuclear function(s). We recently reported that FXS patient cells lacking FMRP sustain higher level of DNA double-strand breaks (DSBs) than normal cells, specifically at sequences prone to forming R-loops, a phenotype further exacerbated by DNA replication stress. Moreover, expression of FMRP, and not an FMRPI304N mutant known to cause FXS, reduced R-loop-associated DSBs. We subsequently reported that recombinant FMRP directly binds R-loops, primarily through the carboxyl terminal intrinsically disordered region. Here, we show that FMRP directly interacts with an RNA helicase, DHX9. This interaction, which is mediated by the amino terminal structured domain of FMRP, is reduced with FMRPI304N. We also show that FMRP inhibits DHX9 helicase activity on RNA:DNA hybrids and the inhibition is also dependent on the amino terminus. Furthermore, the FMRPI304N mutation causes both FMRP and DHX9 to persist on the chromatin in replication stress. These results suggest an antagonistic relationship between FMRP and DHX9 at the chromatin, where their proper interaction leads to dissociation of both proteins from the fully resolved R-loop. We propose that the absence or the loss of function of FMRP leads to persistent presence of DHX9 or both proteins, respectively, on the unresolved R-loop, ultimately leading to DSBs. Our study sheds new light on our understanding of the genome functions of FMRP.

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.

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.

Time dependent response of daunorubicin on cytotoxicity, cell cycle and DNA repair in acute lymphoblastic leukaemia.

BACKGROUND: Daunorubicin is commonly used in the treatment of acute lymphoblastic leukaemia (ALL). The aim of this study was to explore the kinetics of double strand break (DSB) formation of three ALL cell lines following exposure to daunorubicin and to investigate the effects of daunorubicin on the cell cycle and the protein kinases involved in specific checkpoints following DNA damage and recovery periods. METHODS: Three ALL cell lines CCRF-CEM and MOLT-4 derived from T lymphocytes and SUP-B15 derived from B lymphocytes were examined following 4 h treatment with daunorubicin chemotherapy and 4, 12 and 24 h recovery periods. Cell viability was measured via MTT (3-(4,5-dimethylthiazol-2-yl)-2-5 diphenyltetrazolium bromide) assay, reactive oxygen species (ROS) production by flow cytometry, double stranded DNA breaks by detecting γH2AX levels while stages of the cell cycle were detected following propidium iodide staining and flow cytometry. Western blotting was used to detect specific proteins while RNA was extracted from all cell lines and converted to cDNA to sequence Ataxia-telangiectasia mutated (ATM). RESULTS: Daunorubicin induced different degrees of toxicity in all cell lines and consistently generated reactive oxygen species. Daunorubicin was more potent at inducing DSB in MOLT-4 and CCRF-CEM cell lines while SUP-B15 cells showed delays in DSB repair and significantly more resistance to daunorubicin compared to the other cell lines as measured by γH2AX assay. Daunorubicin also causes cell cycle arrest in all three cell lines at different checkpoints at different times. These effects were not due to mutations in ATM as sequencing revealed none in any of the three cell lines. However, p53 was phosphorylated at serine 15 only in CCRF-CEM and MOLT-4 but not in SUP-B15 cells. The lack of active p53 may be correlated to the increase of SOD2 in SUP-B15 cells. CONCLUSIONS: The delay in DSB repair and lower sensitivity to daunorubicin seen in the B lymphocyte derived SUP-B15 cells could be due to loss of function of p53 that may be correlated to increased expression of SOD2 and lower ROS production.

Repair of DNA double-strand breaks in plant meiosis: role of eukaryotic RecA recombinases and their modulators.

Homologous recombination during meiosis is crucial for the DNA double-strand breaks (DSBs) repair that promotes the balanced segregation of homologous chromosomes and enhances genetic variation. In most eukaryotes, two recombinases RAD51 and DMC1 form nucleoprotein filaments on single-stranded DNA generated at DSB sites and play a central role in the meiotic DSB repair and genome stability. These nucleoprotein filaments perform homology search and DNA strand exchange to initiate repair using homologous template-directed sequences located elsewhere in the genome. Multiple factors can regulate the assembly, stability, and disassembly of RAD51 and DMC1 nucleoprotein filaments. In this review, we summarize the current understanding of the meiotic functions of RAD51 and DMC1 and the role of their positive and negative modulators. We discuss the current models and regulators of homology searches and strand exchange conserved during plant meiosis. Manipulation of these repair factors during plant meiosis also holds a great potential to accelerate plant breeding for crop improvements and productivity.

SFN Enhanced the Radiosensitivity of Cervical Cancer Cells via Activating LATS2 and Blocking Rad51/MDC1 Recruitment to DNA Damage Site.

BACKGROUND: Sulforaphane (SFN) is one kind of phytochemical anticancer drug. It inhibits cancer cell proliferation and promotes cell apoptosis while the mechanism behind is still uncertain. We aimed to explore its downstream target and the radiotherapy sensitization mechanism in cervical cancer. METHODS: We treated established cervical cancer cells line (SiHa, HeLa, C33A) with SFN followed by irradiation, and explored its survival, apoptosis, and DNA damage repair in vitro and validated the radiosensitivity of SFN treatment in vivo. We conducted mRNA sequencing to identify differentially expressed mRNAs after SFN treatment. We further investigated SFN downstream target and its involvement in DNA damage repair under irradiation. RESULTS: We found that SFN inhibited the survival of cervical cancer cells under radiotherapy treatment in vitro and prolonged the survival period after radiotherapy in the mouse tumorigenic model. SFN increased the protein expression of LATS2 and promoted apoptosis of cervical cancer cells. Overexpressed LATS2 decreased the cellular survival rate of cervical cancer cells. Additionally, SFN treatment and LATS2 overexpression prevented MDC1 and Rad51 from accumulating in the nucleus in cervical cancer cells after being exposed to ionized radiation. LATS2 loss intervened with SFN-alleviated RAD51 and MDC1 nucleus accumulation and resumed the repairment of DNA damage. CONCLUSION: We identified SFN as cervical cancer cells radiotherapy sensitizer and LATS2 served as a downstream target of SFN treatment. SFN treatment resulted in the inhibition of the homologous recombination (HR) pathway, and LATS2 has an indispensable contribution to this SFN-facilitated radiotherapy sensitization.

RNase H1 Hybrid-Binding Domain-Based Tools for Cellular Biology Studies of DNA-RNA Hybrids in Mammalian Cells.

R loops are abundant noncanonical DNA-RNA hybrid structures that can occur during DNA-based processes, such as transcription, replication and DNA damage, and can lead both to physiologically favorable and pathological outcomes. With an increasing body of work feeding the field of R loop biology, our understanding of the processes in which R loops intervene and the consequences of meddling with R loop formation and dissolution has greatly increased but it has also led to new questions and sometimes opposing possibilities. Proper detection of these structures is a crucial factor to advance our knowledge about R loops and factors associated with their formation and removal. Here, we describe the use of fluorescently tagged HBD, the hybrid-binding domain of RNase H1, as a tool for analyzing DNA-RNA hybrids in different contexts using live-cell microscopy and immunofluorescence experiments.

BMN673 Is a PARP Inhibitor with Unique Radiosensitizing Properties: Mechanisms and Potential in Radiation Therapy.

BMN673 is a relatively new PARP inhibitor (PARPi) that exhibits superior efficacy in vitro compared to olaparib and other clinically relevant PARPi. BMN673, similar to most clinical PARPi, inhibits the catalytic activities of PARP-1 and PARP-2 and shows impressive anticancer potential as monotherapy in several pre-clinical and clinical studies. Tumor resistance to PARPi poses a significant challenge in the clinic. Thus, combining PARPi with other treatment modalities, such as radiotherapy (RT), is being actively pursued to overcome such resistance. However, the modest to intermediate radiosensitization exerted by olaparib, rucaparib, and veliparib, limits the rationale and the scope of such combinations. The recently reported strong radiosensitizing potential of BMN673 forecasts a paradigm shift on this front. Evidence accumulates that BMN673 may radiosensitize via unique mechanisms causing profound shifts in the balance among DNA double-strand break (DSB) repair pathways. According to one of the emerging models, BMN673 strongly inhibits classical non-homologous end-joining (c-NHEJ) and increases reciprocally and profoundly DSB end-resection, enhancing error-prone DSB processing that robustly potentiates cell killing. In this review, we outline and summarize the work that helped to formulate this model of BMN673 action on DSB repair, analyze the causes of radiosensitization and discuss its potential as a radiosensitizer in the clinic. Finally, we highlight strategies for combining BMN673 with other inhibitors of DNA damage response for further improvements.

Proficiency in homologous recombination repair is prerequisite for activation of G2-checkpoint at low radiation doses.

Pathways of repair of DNA double strand breaks (DSBs) cooperate with DNA damage cell cycle checkpoints to safeguard genomic stability when cells are exposed to ionizing radiation (IR). It is widely accepted that checkpoints facilitate the function of DSB repair pathways. Whether DSB repair proficiency feeds back into checkpoint activation is less well investigated. Here, we study activation of the G2-checkpoint in cells deficient in homologous recombination repair (HRR) after exposure to low IR doses (∼1 Gy) in the G2-phase. We report that in the absence of functional HRR, activation of the G2-checkpoint is severely impaired. This response is specific for HRR, as cells deficient in classical non-homologous end joining (c-NHEJ) develop a similar or stronger G2-checkpoint than wild-type (WT) cells. Inhibition of ATM or ATR leaves largely unaffected residual G2-checkpoint in HRR-deficient cells, suggesting that the G2-checkpoint engagement of ATM/ATR is coupled to HRR. HRR-deficient cells show in G2-phase reduced DSB-end-resection, as compared to WT-cells or c-NHEJ mutants, confirming the reported link between resection and G2-checkpoint activation. Strikingly, at higher IR doses (≥4 Gy) HRR-deficient cells irradiated in G2-phase activate a weak but readily detectable ATM/ATR-dependent G2-checkpoint, whereas HRR-deficient cells irradiated in S-phase develop a stronger G2-checkpoint than WT-cells. We conclude that HRR and the ATM/ATR-dependent G2-checkpoint are closely intertwined in cells exposed to low IR-doses in G2-phase, where HRR dominates; they uncouple as HRR becomes suppressed at higher IR doses. Notably, this coupling is specific for cells irradiated in G2-phase, and cells irradiated in S-phase utilize a different mechanistic setup.

Analysis of chromatid-break-repair detects a homologous recombination to non-homologous end-joining switch with increasing load of DNA double-strand breaks.

We recently reported that when low doses of ionizing radiation induce low numbers of DNA double-strand breaks (DSBs) in G2-phase cells, about 50 % of them are repaired by homologous recombination (HR) and the remaining by classical non-homologous end-joining (c-NHEJ). However, with increasing DSB-load, the contribution of HR drops to undetectable (at ∼10 Gy) as c-NHEJ dominates. It remains unknown whether the approximately equal shunting of DSBs between HR and c-NHEJ at low radiation doses and the predominant shunting to c-NHEJ at high doses, applies to every DSB, or whether the individual characteristics of each DSB generate processing preferences. When G2-phase cells are irradiated, only about 10 % of the induced DSBs break the chromatids. This breakage allows analysis of the processing of this specific subset of DSBs using cytogenetic methods. Notably, at low radiation doses, these DSBs are almost exclusively processed by HR, suggesting that chromatin characteristics awaiting characterization underpin chromatid breakage and determine the preferential engagement of HR. Strikingly, we also discovered that with increasing radiation dose, a pathway switch to c-NHEJ occurs in the processing of this subset of DSBs. Here, we confirm and substantially extend our initial observations using additional methodologies. Wild-type cells, as well as HR and c-NHEJ mutants, are exposed to a broad spectrum of radiation doses and their response analyzed specifically in G2 phase. Our results further consolidate the observation that at doses <2 Gy, HR is the main option in the processing of the subset of DSBs generating chromatid breaks and that a pathway switch at doses between 4-6 Gy allows the progressive engagement of c-NHEJ. PARP1 inhibition, irrespective of radiation dose, leaves chromatid break repair unaffected suggesting that the contribution of alternative end-joining is undetectable under these experimental conditions.

The canine RAD51 mutation leads to the attenuation of interaction with PALB2.

RAD51 forms a complex with BRCA2 and plays a central role in the DNA damage response pathway that is associated with homologous recombination. The structures of RAD51 and its homologues are highly conserved from prokaryotes to higher eukaryotes. Although a large number of BRCA2 mutations have been reported, there are only a few reports on the mutations of RAD51, which have been shown in humans and dogs. However, several mutations of canine RAD51 were identified from mammary gland tumour tissues in a recent study. Some of these mutations seem to have an influence on the homo-oligomerization or interaction with "Partner and localizer of BRCA2" (PALB2). In this study, we cloned the canine PALB2 homologue and investigated the effect on its interaction with the RAD51 mutants to evaluate the alteration in the function of RAD51 mutants. The A209S and T225S mutants of RAD51 show an attenuation of the interaction between RAD51 and PALB2. These results indicate that the canine RAD51 mutations can potentially alter the homologous recombination pathways in response to DNA damage in dogs.

A cell-by-cell Monte Carlo simulation for assessing radiation-induced DNA double strand breaks.

This paper presents a cell-by-cell Monte Carlo simulation study that combines charged particle track structure data with an interphase cell nucleus model to quantify DNA double strand breaks (DSBs), spatial distribution of DSBs in a cell nucleus, and resulting potentially lethal or mutagenic events (PLMEs) between DSBs in close proximity. Cell nucleus is simulated according to the chromosome territory-interchromatin compartment (CT-IC) model in that chromatin content is unevenly distributed in chromatin domains (CDs) and IC with a chromatin compaction ratio of 22:1. A particle track structure coordinate (PTSC) library was first generated for each particle type, energy, and dose based on a large number of particle track data obtained by running the Monte Carlo track structure code Geant4-DNA. To assess the DNA DSBs of a cell for a specific particle type, energy, and dose, the corresponding PTSC was selected and "map overlaid" onto 960 unique cell nucleus data sets containing chromatin fiber (CF) locations. Clustering algorithm DBSCAN was next used to identify the clustered energy deposition events occurring inside the CF. These events were then converted to DNA DSBs using a probabilistic approach. The locations of the DSBs thus obtained were, in turn, used to calculate PLMEs within the cell nucleus that can result from DSB proximity and complexity. The results obtained from this simulation study are correctly correlated to the experimental data of DSB yield and the RBE-LET relationships for various types of charged particles and of various energies. The results show agreement with other published radiobiological models.

The yield of DNA double strand breaks determined after exclusion of those forming from heat-labile lesions predicts tumor cell radiosensitivity to killing.

BACKGROUND AND PURPOSE: The radiosensitivity to killing of tumor cells and in-field normal tissue are key determinants of radiotherapy response. In vitro radiosensitivity of tumor- and normal-tissue-derived cells often predicts radiation response, but high determination cost in time and resources compromise utility as routine response-predictor. Efforts to use induction or repair of DNA double-strand-breaks (DSBs) as surrogate-predictors of cell radiosensitivity to killing have met with limited success. Here, we re-visit this issue encouraged by our recent observations that ionizing radiation (IR) induces not only promptly-forming DSBs (prDSBs), but also DSBs developing after irradiation from the conversion to breaks of thermally-labile sugar-lesions (tlDSBs). MATERIALS AND METHODS: We employ pulsed-field gel-electrophoresis and flow-cytometry protocols to measure total DSBs (tDSB=prDSB+tlDSBs) and prDSBs, as well as γH2AX and parameters of chromatin structure. RESULTS: We report a fully unexpected and in many ways unprecedented correlation between yield of prDSBs and radiosensitivity to killing in a battery of ten tumor cell lines that is not matched by yields of tDSBs or γH2AX, and cannot be explained by simple parameters of chromatin structure. CONCLUSIONS: We propose the introduction of prDSBs-yield as a novel and powerful surrogate-predictor of cell radiosensitivity to killing with potential for clinical application.

The contribution of thermally labile sugar lesions to DNA double-strand break formation in cells grown in the presence of BrdU.

PURPOSE: Radiosensitization by bromodeoxyuridine (BrdU) is commonly attributed to an increase in the yield of double-strand breaks (DSB) in the DNA and an associated decrease in the reparability of these lesions. Radiation chemistry provides a mechanism for the increased yield of DSB through the generation, after bromine loss, of a highly reactive uracilyl radical that attacks the sugar moiety of the nucleotide to produce a single-strand break (SSB). The effects underpinning DSB repair inhibition remain, in contrast, incompletely characterized. A possible source of reduced reparability is a change in the nature or complexity of the DSB in BrdU-substituted DNA. Recent studies show that DSB-complexity or DSB-nature may also be affected by the presence within the cluster of thermally labile sugar lesions (TLSL) that break the DNA backbone only if they chemically evolve to SSB, a process thought to occur within the first hour post-irradiation. Since BrdU radiosensitization might be associated with increased yields and reduced reparability of DSB, we investigated whether BrdU underpins these effects by shifting the balance in the generation of TLSL. METHODS AND MATERIALS: We employed asymmetric-field-inversion gel electrophoresis (AFIGE), a pulsed-field gel electrophoresis (PFGE) method to quantitate DSB in a battery of five cells lines grown in the presence of different concentrations of BrdU. We measured specifically the yields of promptly forming DSB (prDSB) using low temperature lysis protocols, and the yields of total DSB (tDSB = prDSB + tlDSB; tlDSB form after evolution to SSB of TLSL) using high temperature lysis protocols. RESULTS: We report that incorporation of BrdU generates similar increases in the formation of tlDSB and prDSB, but variations are noted among the different cell lines tested. CONCLUSIONS: The similar increase in the yields of tlDSB and prDSB in BrdU substituted DNA showed that shifts in the yields of these forms of lesions could not be invoked to explain BrdU radiosensitization.

Cytogenetic evidence that DNA topoisomerase II is not involved in radiation induced chromsome-type aberrations.

ICRF-187 (Cardioxane™, Chiron) is a catalytic inhibitor of DNA topoisomerase II (Topo II), proposed to act by blocking Topo II-mediated DNA cleavage without stabilizing DNA-Topo II-"cleavable complexes". In this study ICRF-187 was used to evaluate the potential involvement of DNA topoisomerase II in the formation of the radiation-induced chromosome-type aberrations in the G0 phase of the cell cycle in human lymphocytes from three healthy male donors. This is based on many evidences that DNA topoisomerases are involved in DNA recombination, mainly of illegitimate type (non-homologous) both in vitro and in vivo. The results obtained clearly indicated that ICRF-187 did not induce per se any chromosomal damage. When challenged with the non-catalytic Topo II poison VP-16 (etoposide), which acts by stabilizing the "cleavable complex" generating "protein concealed" DSB's and thus chromosomal aberrations, it completely abolished the significant induction of chromosome-type aberrations and formation of dicentric chromosomes. This indicates that ICRF-187 acts effectively as catalytic inhibitor of Topo II. On the other hand, when X-ray treatments were challenged with ICRF-187 using experimental conditions as for VP-16 treatments, no modification of the incidence of chromosome-type aberrations and dicentric chromosomes was observed. On this basis, we conclude that Topo II is not involved in the formation of X-ray-induced chromosome-type aberrations and dicentric chromosomes in human lymphocytes in the G0 phase of the cell cycle.