MRN Complex Research Articles

Abstract 2082: Deciphering the nucleosome acidic patch binding at amino acid resolution

Abstract In all eukaryotic cells, nucleosomes provide basic organization of genomic DNA and serve as signaling hubs for gene expression, DNA replication and DNA damage repair. These genome-templated processes are tuned by chromatin enzymes that introduce chemical modifications to nucleosomes and control their positioning and occupancy. Mutations in genes encoding chromatin enzymes are highly correlated with numerous cancer types. A thorough exploration of how these chromatin enzymes act on their physiologic target, the nucleosome, is critical to understanding abnormal DNA methylation, histone posttranslational modifications, and chromatin composition, leading to cancer. In our recent nucleosome interactome proteomic screen, we demonstrated that the vast majority of nuclear proteins require a group of acidic residues on the nucleosome disk surface to interact with nucleosomes. This acidic patch and an adjacent region were recognized by over 50% and 18% of nuclear proteins, respectively, while almost half of the nucleosome disk surface contributed only minimally to nucleosome recognition. Overall, we paired about 300 proteins representing diverse nuclear functions including DNA methylation (DNMT1 and 3, Tet1), histone modification (HAT complexes, kinases), nucleosome remodeling (SWI/SNF), DNA repair (MRN complex), DNA replication (ORC complex), RNA processing, and cell cycle progression (APC/C) with specific nucleosome surface requirements. We have now repeated our screen at amino acid resolution using a library of fifteen nucleosomes containing single point mutations in and around the acid patch, including potential histone oncomutations identified to be overrepresented in cancers. Using our nucleosome library, we preformed pulldowns from human embryonic kidney 293 cell nuclear lysates to comprehensively evaluate the role of single histone residues in nucleosome recognition. Overall, this study dissects the primary nucleosome binding hot-spot at high resolution and provide a robust resource for further exploration of the nucleosome interactome in normal and cancer cell biology. Citation Format: Aleksandra Skrajna, Holly Simmons, Charlotte Smith, Jeanne-Marie E. Mcpherson, Dennis Goldfarb, Robert K. McGinty. Deciphering the nucleosome acidic patch binding at amino acid resolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2082.

Protein phosphatase 1 acts as a RIF1 effector to suppress DSB resection prior to Shieldin action.

SummaryDNA double-strand breaks (DSBs) are repaired mainly by non-homologous end joining (NHEJ) or homologous recombination (HR). RIF1 negatively regulates resection through the effector Shieldin, which associates with a short 3′ single-stranded DNA (ssDNA) overhang by the MRN (MRE11-RAD50-NBS1) complex, to prevent further resection and HR repair. In this study, we show that RIF1, but not Shieldin, inhibits the accumulation of CtIP at DSB sites immediately after damage, suggesting that RIF1 has another effector besides Shieldin. We find that protein phosphatase 1 (PP1), a known RIF1 effector in replication, localizes at damage sites dependent on RIF1, where it suppresses downstream CtIP accumulation and limits the resection by the MRN complex. PP1 therefore acts as a RIF1 effector distinct from Shieldin. Furthermore, PP1 deficiency in the context of Shieldin depletion elevates HR immediately after irradiation. We conclude that PP1 inhibits resection before the action of Shieldin to prevent precocious HR in the early phase of the damage response.

Abstract 119: Enteric glial cells promote chemoresistance in ATM-expressing cancer stem cells

Abstract In colon cancer, the subset of cells fueling the clonal growth of the tumor have been shown to also possess enhanced chemoresistance abilities. These cells, known as cancer stem cells (CSC), are controlled by molecules and cells within the tumor microenvironment. In the latter reside enteric glial cells (EGC) that heavily regulate intestinal epithelial barrier function in a healthy colon. However, EGC influence on colon carcinogenesis remains poorly understood. Recent work from our lab shows that EGC promote CSC-driven tumorigenesis via the release of prostaglandin E2. Here, we investigate the impact of EGC on CSC chemoresistance. CD24Hi-CD44Hi CSC were FACS-isolated from human colon cancer primary tumors or cell lines and 3D-grown in the presence of murine or human EGC seeded on Transwell filters in vitro in the presence of the chemotherapeutic drug 5-Fluorouracil (5-FU) or injected subcutaneously with(out) EGC in vivo in 5-FU treated-immunodeficient mice. EGC impact was assessed on 5-FU-induced restriction of tumor formation and growth in vitro and in vivo. EGC-conditioned medium (CM) impact on 5-FU-induced apoptosis in CSC was measured by flow cytometry. EGC impact on expression of 48 chemoresistance-related genes in CSC was assessed by high throughput RT-qPCR. Impact of inhibition of ATM activity and ATM activation by the Mre11-Rad50-Nbs1 (MRN) complex in CSC was tested using 0.1µM KU-55933 and 12µM mirin. To understand the impact of 5-FU on EGC, levels of senescence markers p16, p19, p21 and H3K9me3 and β-gal activity were assessed. Proteomic analyses of EGC-CM were conducted by mass spectrometry. EGC promoted growth of CSC-derived tumors in the presence of 5-FU in vivo. EGC increased the number and size of tumor-organoids grown from CSC isolated from cell lines (HT29, HCT116, HCT15) and primary tumors in the presence of 5-FU in vitro. EGC-CM reduced apoptosis in 5-FU-treated CSC. Out of all genes tested, only ATM mRNA was significantly enriched in 5-FU-treated CSC cultured with EGC vs. alone. Co-culture studies using CSC derived from the ATM deficient SW1222 cell line or using the ATM inhibitor KU-55933 reduced EGC pro-chemoresistance effects. Furthermore, inhibition of ATM activation by the upstream DNA damage sensor MRN complex using mirin abolished EGC protective effects on CSC resistance to 5-FU. 5-FU-treated EGC showed increased levels of senescence markers and protein secretion, indicative of a senescence-associated secretory phenotype (SASP). Moreover, CM from 5-FU-treated EGC further reduced 5-FU-induced apoptosis in CSC as compared to CM from untreated EGC. Mass spectrometric analyses revealed that IGFBP7, a putative ATM activator, was >100 times more abundant in the CM of 5-FU-treated EGC vs. untreated EGC. Our data strongly indicate that EGC promote CSC chemoresistance via increased ATM signaling. Future studies will investigate the implication of IGFBP7 and downstream targets of ATM involved in DNA repair. Citation Format: Gregory Bacola, Simon Vales, Alice Prigent, Kelsie A. Dougherty, Deanna M. Peperno, Shaian Lashani, Bradley A. Wieland, Melissa Touvron, Lisa Oliver, François M. Vallette, Michel Neunlist, Laurianne Van Landeghem. Enteric glial cells promote chemoresistance in ATM-expressing cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 119.

Abstract 1972: A novel signaling pathway delays abscission in cytokinesis with chromatin bridges

Abstract Chromatin bridges are strands of missegregated chromatin connecting the anaphase poles or daughter nuclei and have been linked to tumourigenesis. In response to chromatin bridges in cytokinesis, cells delay abscission, the severing of the narrow cytoplasmic canal that connects the two daughter cells, to prevent chromatin breakage or tetraploiudization that are associated with genomic instability and cancer predisposition. In mammalian cells, this abscission delay is called “the abscission checkpoint” and is dependent on the localization and catalytic activity of the Chromosomal Passenger Complex (CPC) at the midbody. The CPC comprises the catalytic subunit Aurora B kinase, the scaffolding protein INCENP and the non-enzymatic subunits Survivin and Borealin; however, the molecular mechanisms that signal chromatin bridges to the CPC have not been previously identified. In the present study, we show that inhibition of the DNA damage kinases ATM or Chk2 impairs CPC-localization to the midbody and correlates with premature abscission and chromatin breakage in cytokinesis with trapped chromatin in human carcinoma cell lines. ATM phosphorylates Chk2-threonine 68 (T68) to activate Chk2 at the midbody. In turn, active Chk2 phosphorylates INCENP at the newly identified site serine 91 (S91) to promote CPC-localization to the midbody, to delay abscission and prevent chromatin breakage. Expression of siRNA-resistant phosphomimetic mutant INCENP-S91D or GFP:INCENP(FB) that is constitutively targeted to the Flemming body (FB), but not WT INCENP, rescues CPC-midbody-localization and prevents chromatin breakage in Chk2-deficient or ATM-deficient cells. In contrast, in the absence of the endogenous INCENP, the non-phosphorylatable mutant INCENP-S91A does not localize to the midbody and its expression promotes chromatin breakage. Furthermore, the Mre11-Rad50-Nbs1 (MRN) complex is required for ATM activation at the midbody. These results identify an MRN-ATM-Chk2-INCENP signaling pathway that prevents chromosome breakage in cytokinesis with chromatin bridges, by promoting CPC-midbody localization. Citation Format: Eleni Petsalaki, George Zachos. A novel signaling pathway delays abscission in cytokinesis with chromatin bridges [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1972.

Abstract LB218: MRE11A expression regulated by UBQLN4 is related to cisplatin-resistance and survival in patients with esophageal squamous cell carcinoma

Abstract Platinum-based combination regimens are the standard of care to treat esophageal squamous cell carcinoma (ESCC) in both pre- and post-operative settings. Thus, resistance to cisplatin-based chemotherapies leads to worse survival outcomes in patients with ESCC. Therefore, there is an urgent need to understand aberrant mechanism driving cisplatin-resistance in ESCC. UBQLN4 is a proteasomal shuttle factor that interacts with ubiquitinated proteins and leads to proteasome-mediated protein degradation. We have recently demonstrated that UBQLN4 is highly expressed in solid aggressive tumors. However, the role of UBQLN4 in driving cisplatin-resistance in ESCC remains unknown. We hypothesize that UBQLN4 interacts with ubiquitinated-MRE11A, an essential component of the MRN complex and the DNA damage repairing pathway and thus, lead to cisplatin-resistance in ESCC tumors. In this study we demonstrated that MRE11A knockdown in ESCC cell lines reduced DNA damage and increased cisplatin-resistance, while MRE11A overexpression caused the opposite effects. MRE11A and UBQLN4 negatively correlated in TE-4, TE-8, and TE-10 cell lines. In established cisplatin-resistant ESCC cell lines (TE-8 and TE-10), MRE11A expression was significantly lower compared to their respective parental cell lines (p<0.01). UBQLN4 overexpression was associated with cisplatin-resistance in ESCC cell lines. Mechanistically, UBQLN4 bound to ubiquitinated MRE11A, enhanced MRE11A proteasomal degradation, and promoted cisplatin-resistance. Immunohistochemistry analysis was conducted in total 120 ESCC patients' specimens. In endoscopic core biopsies taken from 61 ESCC patients who underwent cisplatin-based neoadjuvant chemotherapy, low MRE11A and high UBQLN4 protein levels were associated with reduced pathological response to neoadjuvant chemotherapy (p<0.001 and p<0.001, respectively). Analysis of surgically-resected ESCC tissues from 59 patients who underwent upfront surgery and the following cisplatin-based adjuvant chemotherapy revealed that low MRE11A and high UBLQN4 expression were independent factors to predict shorter overall survival in multivariable analysis (p=0.02, HR=3.74, 95% C.I., 1.19-11.76; p=0.01, HR=5.11, 95% C.I., 1.45-18.03, respectively). In summary, UBQLN4 targets ubiquitinated-MRE11A to the proteasome for degradation during DNA damage induced by cisplatin in ESCC cell lines; thus, regulating MRE11A protein levels. MRE11A and UBQLN4 may serve as predictors for treatment response to cisplatin regimens and prognosis in ESCC patients. Citation Format: Tomohiro Murakami, Yoshiaki Shoji, Tomohiko Nishi, Shu-Ching Chang, Ron D. Jachimowicz, Yosef Shiloh, Hiroya Takeuchi, Yuko Kitagawa, Dave S. Hoon, Matias A. Bustos. MRE11A expression regulated by UBQLN4 is related to cisplatin-resistance and survival in patients with esophageal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB218.

Inhibition of MRN activity by a telomere protein motif

The MRN complex (MRX in Saccharomyces cerevisiae, made of Mre11, Rad50 and Nbs1/Xrs2) initiates double-stranded DNA break repair and activates the Tel1/ATM kinase in the DNA damage response. Telomeres counter both outcomes at chromosome ends, partly by keeping MRN-ATM in check. We show that MRX is disabled by telomeric protein Rif2 through an N-terminal motif (MIN, MRN/X-inhibitory motif). MIN executes suppression of Tel1, DNA end-resection and non-homologous end joining by binding the Rad50 N-terminal region. Our data suggest that MIN promotes a transition within MRX that is not conductive for endonuclease activity, DNA-end tethering or Tel1 kinase activation, highlighting an Achilles’ heel in MRN, which we propose is also exploited by the RIF2 paralog ORC4 (Origin Recognition Complex 4) in Kluyveromyces lactis and the Schizosaccharomyces pombe telomeric factor Taz1, which is evolutionarily unrelated to Orc4/Rif2. This raises the possibility that analogous mechanisms might be deployed in other eukaryotes as well.

Chromatin-associated MRN complex protects highly transcribing genes from genomic instability.

MRN-MDC1 plays a central role in the DNA damage response (DDR) and repair. Using proteomics of isolated chromatin fragments, we identified DDR factors, such as MDC1, among those highly associating with a genomic locus upon transcriptional activation. Purification of MDC1 in the absence of exogenous DNA damage revealed interactions with factors involved in gene expression and RNA processing, in addition to DDR factors. ChIP-seq showed that MRN subunits, MRE11 and NBS1, colocalized throughout the genome, notably at TSSs and bodies of actively transcribing genes, which was dependent on the RNAPII transcriptional complex rather than transcription per se. Depletion of MRN increased RNAPII abundance at MRE11/NBS1-bound genes. Prolonged MRE11 or NBS1 depletion induced single-nucleotide polymorphisms across actively transcribing MRN target genes. These data suggest that association of MRN with the transcriptional machinery constitutively scans active genes for transcription-induced DNA damage to preserve the integrity of the coding genome.

Structure of the ATP‐free Mre11‐Rad50 DNA damage repair complex bound to DNA substrates

DNA double strand breaks, where both strands of the double-stranded DNA are broken, are among the most harmful forms of DNA damage because an intact template is not present for repair. The Mre11-Rad50–Nbs1 (Xrs2 in yeast) complex plays a central and critical role in the detection and repair of DSBs and is conserved in all kingdoms of life, as Mre11Rad50 (MR) in prokaryotes and as MRN/X in eukaryotes. Although there are some structural models available for MR complex bound to DNA substrates, there are limitations in these structures. Therefore, our goal is to examine the MR complex bound to different DNA substrates using an integrated structural biology approach. Using uniformly deuterated, 13 CH -methyl labeled samples, we measured distance restraints for the ~120 kDa P. furiosus MR complex bound to spin-labeled double-stranded or single-stranded DNA substrates 3 via paramagnetic relaxation enhancements (PREs). Additional distance and global restraints were measured from Luminescence Resonance Energy Transfer (LRET) and Small Angle X-ray Scatter (SAXS) experiments. These data then served as restraints for calculating structural models using multi-body docking implemented in HADDOCK. Our models revealed novel modes of DNA binding for the ATP-free MR complex and provide the first depiction of a single-stranded DNA-bound complex. We tested our models through biochemical activity assays to determine the effect of disruption of key interactions, via mutation, in our models.

P97/VCP inhibition causes excessive MRE11-dependent DNA end resection promoting cell killing after ionizing radiation.

SummaryThe ATPase p97 is a central component of the ubiquitin-proteasome degradation system. p97 uses its ATPase activity and co-factors to extract ubiquitinated substrates from different cellular locations, including DNA lesions, thereby regulating DNA repair pathway choice. Here, we find that p97 physically and functionally interacts with the MRE11-RAD50-NBS1 (MRN) complex on chromatin and that inactivation of p97 blocks the disassembly of the MRN complex from the sites of DNA damage upon ionizing radiation (IR). The inhibition of p97 function results in excessive 5′-DNA end resection mediated by MRE11 that leads to defective DNA repair and radiosensitivity. In addition, p97 inhibition by the specific small-molecule inhibitor CB-5083 increases tumor cell killing following IR both in vitro and in vivo. Mechanistically, this is mediated via increased MRE11 nuclease accumulation. This suggests that p97 inhibitors might be exploited to improve outcomes for radiotherapy patients.

Phosphopeptide interactions of the Nbs1 N-terminal FHA-BRCT1/2 domains

Human Nbs1, a component of the MRN complex involved in DNA double strand break repair, contains a concatenated N-terminal FHA-BRCT1/2 sequence that supports interaction with multiple phosphopeptide binding partners. MDC1 binding localizes Nbs1 to the damage site, while binding of CDK-phosphorylated CtIP activates additional ATM-dependent CtIP phosphorylation, modulating substrate-dependent resection. We have investigated the phosphopeptide binding characteristics of Nbs1 BRCT1/2 based on a molecular modeling approach that revealed structural homology with the tandem TopBP1 BRCT7/8 domains. Relevance of the model was substantiated by the ability of TopBP1-binding FANCJ phosphopeptide to interact with hsNbsBRCT1/2, albeit with lower affinity. The modeled BRCT1/2 is characterized by low pSer/pThr selectivity, preference for a cationic residue at the + 2 position, and an inter-domain binding cleft selective for hydrophobic residues at the + 3/ + 4 positions. These features provide insight into the basis for interaction of SDT motifs with the BRCT1/2 domains and allowed identification of CtIP pSer347- and pThr847-containing phosphopeptides as high and lower affinity ligands, respectively. Among other binding partners considered, rodent XRCC1 contains an SDT sequence in the second linker consistent with high-affinity Nbs1 binding, while human XRCC1 lacks this motif, but contains other phosphorylated sequences that exhibit low-affinity binding.

A Role for RAGE in DNA Double Strand Breaks (DSBs) Detected in Pathological Placentas and Trophoblast Cells.

Impaired DNA damage responses are associated with several diseases, including pregnancy complications. Recent research identified an ATM-kinase dependent function for the nuclear isoform of the receptor for advanced glycation end-products (RAGE) during double strand break (DSB)-repair. RAGE contributes to end-resectioning of broken DNA sites by binding with the MRE11-Rad50-Nbs1 (MRN) complex. Placental research is limited regarding the impact of genomic instability and the mechanism for potential repair. We tested the hypothesis regarding the involvement of RAGE during the repair of placental DNA-DSBs. We first identified that the pregnancy complications of PE and preterm labor (PTL) experience loss of genomic integrity and an in vitro trophoblast cell model was used to characterize trophoblast DSBs. Colocalized immunofluorescence of γ-H2AX and RAGE support the potential involvement of RAGE in cellular responses to DNA-DSBs. Immunoblotting for both molecules in PE and PTL placenta samples and in trophoblast cells validated a connection. Co-immunoprecipitation studies revealed interactions between RAGE and pATM and MRE11 during DNA-DSBs. Reduced cellular invasion confirmed the role of genomic instability in trophoblastic function. Collectively, these experiments identified genomic instability in pregnancy complications, the impact of defective DNA on trophoblast function, and a possible RAGE-mediated mechanism during DNA-DSB repair.

Drosophila telomere capping protein HOAP interacts with DSB sensor proteins Mre11 and Nbs.

In eukaryotes, specific DNA-protein structures called telomeres exist at linear chromosome ends. Telomere stability is maintained by a specific capping protein complex. This capping complex is essential for the inhibition of the DNA damage response (DDR) at telomeres and contributes to genome integrity. In Drosophila, the central factors of telomere capping complex are HOAP and HipHop. Furthermore, a DDR protein complex Mre11-Rad50-Nbs (MRN) is known to be important for the telomere association of HOAP and HipHop. However, whether MRN interacts with HOAP and HipHop, and the telomere recognition mechanisms of HOAP and HipHop are poorly understood. Here, we show that Nbs interacts with Mre11 and transports the Mre11-Rad50 complex from the cytoplasm to the nucleus. In addition, we report that HOAP interacts with both Mre11 and Nbs. The N-terminal region of HOAP is essential for its co-localization with HipHop. Finally, we reveal that Nbs interacts with the N-terminal region of HOAP.

DROSHA is recruited to DNA damage sites by the MRN complex to promote non-homologous end joining.

ABSTRACTThe DNA damage response (DDR) is the signaling cascade that recognizes DNA double-strand breaks (DSBs) and promotes their resolution via the DNA repair pathways of non-homologous end joining (NHEJ) or homologous recombination (HR). We and others have shown that DDR activation requires DROSHA; however, whether DROSHA exerts its functions by associating with damage sites, what controls its recruitment, and how DROSHA influences DNA repair remains poorly understood. Here, we show that DROSHA associates with DSBs independently of transcription. Neither H2AX, nor ATM or DNA-PK kinase activities are required for recruitment of DROSHA to break sites. Rather, DROSHA interacts with RAD50, and inhibition of the MRN complex by mirin treatment abolishes this interaction. MRN complex inactivation by RAD50 knockdown or mirin treatment prevents DROSHA recruitment to DSBs and, as a consequence, also prevents 53BP1 (also known as TP53BP1) recruitment. During DNA repair, DROSHA inactivation reduces NHEJ and boosts HR frequency. Indeed, DROSHA knockdown also increases the association of downstream HR factors such as RAD51 to DNA ends. Overall, our results demonstrate that DROSHA is recruited at DSBs by the MRN complex and directs DNA repair towards NHEJ.

Regulation of MRE11A by UBQLN4 leads to cisplatin resistance in patients with esophageal squamous cell carcinoma.

Resistance to standard cisplatin‐based chemotherapies leads to worse survival outcomes for patients with esophageal squamous cell carcinoma (ESCC). Therefore, there is an urgent need to understand the aberrant mechanisms driving resistance in ESCC tumors. We hypothesized that ubiquilin‐4 (UBQLN4), a protein that targets ubiquitinated proteins to the proteasome, regulates the expression of Meiotic Recombination 11 Homolog A (MRE11A), a critical component of the MRN complex and DNA damage repair pathways. Initially, immunohistochemistry analysis was conducted in specimens from patients with ESCC (n = 120). In endoscopic core ESCC biopsies taken from 61 patients who underwent neoadjuvant chemotherapy (NAC) (5‐fluorouracil and cisplatin), low MRE11A and high UBQLN4 protein levels were associated with reduced pathological response to NAC (P < 0.001 and P < 0.001, respectively). Multivariable analysis of surgically resected ESCC tissues from 59 patients revealed low MRE11A and high UBLQN4 expression as independent factors that can predict shorter overall survival [P = 0.01, hazard ratio (HR) = 5.11, 95% confidence interval (CI), 1.45–18.03; P = 0.02, HR = 3.74, 95% CI, 1.19–11.76, respectively]. Suppression of MRE11A expression was associated with cisplatin resistance in ESCC cell lines. Additionally, MRE11A was found to be ubiquitinated after cisplatin treatment. We observed an amplification of UBQLN4 gene copy numbers and an increase in UBQLN4 protein levels in ESCC tissues. Binding of UBQLN4 to ubiquitinated‐MRE11A increased MRE11A degradation, thereby regulating MRE11A protein levels following DNA damage and promoting cisplatin resistance. In summary, MRE11A and UBQLN4 protein levels can serve as predictors for NAC response and as prognostic markers in ESCC patients.

MRNIP is essential for meiotic progression and spermatogenesis in mice

Meiotic homologous recombination (HR) initiates with the programmed generation of DNA double-strand breaks (DSBs), which result in the exchange of genetic information and genome diversity. This process requires the tight cooperation of the MRE11-RAD50-NBS1 (MRN) complex to promote DSB formation and DNA end resection. However, the mechanism regulating MRN complex remains to be explored. In the present study, we report that MRN-interacting protein, MRNIP, is a novel factor for HR and is crucial for the expression of the MRN complex and loading of recombinases DMC1/RAD51. Knockout of Mrnip in mice led to aberrant synapsis, impaired HR, and male subfertility. In conclusion, MRNIP is a novel HR factor that probably promotes meiotic progression through the MRN complex.

FK228 sensitizes radioresistant small cell lung cancer cells to radiation

BackgroundConcurrent thoracic radiation plus chemotherapy is the mainstay of first-line treatment for limited-stage small cell lung cancer (LS-SCLC). Despite initial high responsiveness to combined chemo- and radiotherapy, SCLC almost invariably relapses and develops resistance within one year, leading to poor prognosis in patients with LS-SCLC. Developing new chemical agents that increase ionizing radiation’s cytotoxicity against SCLC is urgently needed.ResultsDual histone deacetylase (HDAC) and PI3K inhibitor FK228 not only displayed potent anticancer activity, but also enhanced the therapeutic effects of radiotherapy in SCLC cells. Mechanistically, radioresistant SCLC cells exhibit a lower level of histone H3K9 acetylation and a higher expression level of the MRE11-RAD50-NBS1 (MRN) complex and show more efficient and redundant DNA damage repair capacities than radiosensitive SCLC cells. FK228 pretreatment resulted in marked induction of H3k9 acetylation, attenuated homologous recombination (HR) repair competency and impaired non-homologous end joining (NHEJ) repair efficacy, leading to the accumulation of radiation-induced DNA damage and radiosensitization.ConclusionThe study uncovered that FK228 sensitized human radioresistant SCLC cells to radiation mainly through induction of chromatin decondensation and suppression of DNA damage signaling and repair. Our study provides a rational basis for a further clinical study to test the potential of FK228 as a radiosensitizing agent to increase the radiation-induced tumor cell kill in LS-SCLC patients.

RNA polymerase III is required for the repair of DNA double-strand breaks by homologous recombination

End resection in homologous recombination (HR) and HR-mediated repair of DNA double-strand breaks (DSBs) removes several kilobases from 5' strands of DSBs, but 3' strands are exempted from degradation. The mechanism by which the 3' overhangs are protected has not been determined. Here, we established that the protection of 3' overhangs is achieved through the transient formation of RNA-DNA hybrids. The DNA strand in the hybrids is the 3' ssDNA overhang, while the RNA strand is newly synthesized. RNA polymerase III (RNAPIII) is responsible for synthesizing the RNA strand. Furthermore, RNAPIII is actively recruited to DSBs by the MRN complex. CtIP and MRN nuclease activity is required for initiating the RNAPIII-mediated RNA synthesis at DSBs. A reduced level of RNAPIII suppressed HR, and genetic loss > 30bp increased at DSBs. Thus, RNAPIII is an essential HR factor, and the RNA-DNA hybrid is an essential repair intermediate for protecting the 3' overhangs in DSB repair.

Single Molecule Experiments Define the Role of Nbs1 and Xrs2 in DNA Tethering by MRN and MRX in DSB Repair

To ensure genome stability and hinder introduction of disease-causing changes, DNA double-strand breaks (DSB) need to be repaired. Homologous recombination (HR) is a key DSB repair pathway. At the start of HR, the MRE11-RAD50-NBS1 (MRN) complex finds the broken ends and upon stimulation of its nuclease activity by CtIP performs the DSB processing for repair. The repair process involves DNA tethering, which is poorly understood. in this study, we use single-molecule analysis to investigate how MRN interacts with long DNA. The method is based on confining long DNA molecules in nanochannels. in contrast to tethering one or both ends to a substrate, as in most common single DNA molecule methods, the DNA is completely free in the nano channels, meaning that reaction on the DNA ends can be studied. Single molecule experiments with MRN and DNA in nanochannels show direct evidence of a strong DNA interaction and tethering of distant DNA fragments by MRN. When comparing MRN and the yeast homologue Mre11-Rad50-Xrs2 (MRX), we observed key similarities. in both systems, the absence of NBS1/Xrs2, lowers the concatemer and circle formation dramatically. Data from single molecule experiments and EMSA show that NBS1 and Xrs2 alone have a strong DNA interaction. These findings strengthen the theory of Nbs1/Xrs2 playing a role in the localization of the MRN/X complex to DNA ends and mediate DNA annealing. Finally, the addition of the MRN/X co-factor CtIP and Sae2, respectively, shows little to no effect on the bridging by MRN/X, although both CtIP and Sae2 exhibit strong DNA bridging on their own. Thus the interaction between MRN/X and the co-factor that is crucial for the nuclease activity is potentially not important for DNA bridging.

Structure of the ATP-Free Mre11-Rad50 DNA Damage Repair Complex Bound to DNA Substrates

DNA double strand breaks, where both strands of the double-stranded DNA are broken, are among the most harmful forms of DNA damage because an intact template is not present for repair. The Mre11-Rad50–Nbs1 (Xrs2 in yeast) complex plays a central and critical role in the detection and repair of DSBs and is conserved in all kingdoms of life, as Mre11- Rad50 (MR) in prokaryotes and as MRN/X in eukaryotes. Although there are some structural models available for MR complex bound to DNA substrates, there are limitations in these structures. Therefore, our goal is to examine the MR complex bound to different DNA substrates using an integrated structural biology approach. Using uniformly deuterated, 13CH3-methyl labeled samples, we measured distance restraints for the ∼120 kDa P. furiosus MR complex bound to spin-labeled double-stranded or single-stranded DNA substrates via paramagnetic relaxation enhancements (PREs). Additional distance and global restraints were measured from Luminescence Resonance Energy Transfer (LRET) and Small Angle X-ray Scatter (SAXS) experiments. These data then served as restraints for calculating structural models using multi-body docking implemented in HADDOCK. Our models revealed novel modes of DNA binding for the ATP-free MR complex and provide the first depiction of a single-stranded DNA-bound complex. We tested our models through biochemical activity assays to determine the effect of disruption of key interactions, via mutation, in our models.

SPRTN protease-cleaved MRE11 decreases DNA repair and radiosensitises cancer cells

The human MRE11/RAD50/NBS1 (MRN) complex plays a crucial role in sensing and repairing DNA DSB. MRE11 possesses dual 3′−5′ exonuclease and endonuclease activity and forms the core of the multifunctional MRN complex. We previously identified a C-terminally truncated form of MRE11 (TR-MRE11) associated with post-translational MRE11 degradation. Here we identified SPRTN as the essential protease for the formation of TR-MRE11 and characterised the role of this MRE11 form in its DNA damage response (DDR). Using tandem mass spectrometry and site-directed mutagenesis, the SPRTN-dependent cleavage site for MRE11 was identified between 559 and 580 amino acids. Despite the intact interaction of TR-MRE11 with its constitutive core complex proteins RAD50 and NBS1, both nuclease activities of truncated MRE11 were dramatically reduced due to its deficient binding to DNA. Furthermore, lack of the MRE11 C-terminal decreased HR repair efficiency, very likely due to abolished recruitment of TR-MRE11 to the sites of DNA damage, which consequently led to increased cellular radiosensitivity. The presence of this DNA repair-defective TR-MRE11 could explain our previous finding that the high MRE11 protein expression by immunohistochemistry correlates with improved survival following radical radiotherapy in bladder cancer patients.