Browsing by Subject "DNA Repair"
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Item Discovering GCNA: A Novel Regulator of Germline Genomic Stability(2018-10-15) Bhargava, Varsha; Mendell, Joshua T.; Buszczak, Michael; Olson, Eric N.; Tu, BenjaminGerm cells transfer genetic information across generations. Any change in germ line DNA is inherited by succeeding generations. Therefore, germ cell DNA must be protected from both internal and external assault. An advantage of sexual reproduction stems from the ability to generate variation by exchange of chromosomal segments during meiosis. During meiosis, hundreds of double-stranded DNA breaks are initiated at once, which if generated in most other cell types would introduce chromosomal aberrations. Germ cells, however, execute the formation of these breaks while preventing their deleterious effects from becoming pervasive throughout the genome. The mechanisms underlying the robustness of germ cells in the face of DNA damage, however, are poorly understood. We initiated an in vivo CRISPR-Cas9 knockout screen for genes highly enriched in the Drosophila female germ line. From this screen, we identified Germ Cell Nuclear Acidic Peptidase (GCNA) as a conserved regulator of genome stability across multiple species. Loss of GCNA results in replication stress, chromosomal instability, and an accumulation of DNA-protein crosslinks (DPCs). Disruption of GCNA leads to an accumulation of nuclear Top2 and Top2 DPCs. This work shows GCNA protects germ cells from damage and provides novel insights into the conserved networks that promote genome integrity across generations.Item Downregulation of the Cytosolic Iron-Sulfur Assembly Pathway in Cancer by an E3 Ubiquitin Ligase(2017-06-19) Weon, Jenny Linda; Tu, Benjamin; Potts, Patrick Ryan; Minna, John D.; Liu, YiIron-sulfur (Fe-S) clusters are considered to be one of the oldest cofactors utilized by proteins and are essential for life from bacteria to mammals. Multiple processes in the cell require Fe-S cofactors, such as electron transfer in mitochondrial respiration, enzymatic reactions, and as structural components in DNA repair enzymes. We describe here the first post-translational mechanism to regulate Fe-S assembly and delivery through the ubiquitination and degradation of a key cytosolic iron-sulfur cluster assembly (CIA) pathway component by a MAGE-RING ligase (MRL). The MAGE protein family consists of ~40 members in humans that function in complex with E3 ubiquitin ligases to enhance ubiquitination activity, alter E3 subcellular localization, and/or specify E3 targets. Using biochemical and cellular approaches we have discovered that the MAGE-F1-NSE1 ligase disrupts Fe-S cluster delivery through ubiquitination and degradation of the CIA pathway protein MMS19. MMS19 is a substrate specifying, late-acting component of the CIA pathway that facilitates Fe-S transfer from the multi-component cascade of assembly proteins to specific recipient apoproteins. Notably, many MMS19 targets are enzymes involved in DNA repair. We found that MAGE-F1 directs the E3 ligase NSE1 to target MMS19 for ubiquitination and degradation. Knockdown of MAGE-F1 stabilized MMS19 and overexpression of MAGE-F1 decreased MMS19 levels without affecting MMS19 mRNA levels. We further confirmed MAGE-F1 inhibits Fe-S incorporation into known MMS19-dependent Fe-S proteins, such as FANCJ, POLD1, RTEL1, XPD, and DPYD, but not MMS19-independent Fe-S proteins, such as PPAT. Loss of Fe-S incorporation leads to decreased DNA repair capacity of cells, exemplified by decreased homologous recombination rates and altered sensitivity to DNA damaging agents. Importantly, numerous cancer types harbor copy-number amplification of MAGE-F1, including lung squamous carcinoma and head and neck squamous carcinoma. Consistent with MAGE-F1 inhibitory activity on Fe-S incorporation into key DNA repair enzymes, MAGE-F1-amplified tumors bear a significantly greater mutational burden than non-MAGE-F1-amplified cancers and the expression of MAGE-F1-NSE1 correlates with poor patient prognosis. In summary, we provide the first evidence for post-translational regulatory control of Fe-S cluster assembly and a novel mechanism by which a broad spectrum of DNA repair enzymes can be regulated and lead to genomic instability in cancer.Item Functional Analysis of the Human SMC5/6 Complex in Homologous Recombination and Telomere Maintenance(2008-05-13) Potts, Patrick Ryan; Yu, HongtaoDNA repair is required for the genomic stability and well-being of an organism. The structural maintenance of chromosomes (SMC) family of proteins has been implicated in the repair of DNA double-strand breaks (DSBs) by homologous recombination (HR). The SMC1/3 cohesin complex promotes HR by localizing to DSBs where it holds sister chromatids in close proximity to allow HR-induced strand invasion and exchange. The SMC5/6 complex is also required for DNA repair, but the mechanism by which it accomplishes this has been unclear. We have characterized the role of the human SMC5/6 complex in HRmediated DNA damage repair. The yeast SMC5/6 complex has been shown to be composed of the SMC5-SMC6 heterodimer and six non-SMC element (NSE) proteins. We show that the human homolog of one of these NSE proteins, MMS21/NSE2, is a ligase for small ubiquitin-like modifier (SUMO). Depletion of MMS21 by RNA interference (RNAi) sensitizes cells toward DNA damageinduced apoptosis. This hypersensitization of MMS21-RNAi cells is not due to a defect in DNA damage-induced cell cycle checkpoint, but rather in the kinetics of DNA damage repair. Since the yeast SMC5/6 complex has been implicated in HR-mediated DSB repair, we investigated the role of the human SMC5/6 complex in HR-mediated DSB repair. RNAi-mediated knockdown of the SMC5/6 complex components specifically decreases sister chromatid HR, but not non-homologous end-joining (NHEJ) or intra-chromatid, homologue, or extrachromosomal HR. We show that one potential mechanism by which the SMC5/6 complex specifically promotes sister chromatid HR is by facilitating the recruitment of the SMC1/3 cohesin complex to DSBs. We next examined whether the SMC5/6 complex is also required for sister chromatid HR of telomeres. Specific types of cancer cells, known as alternative lengthening of telomeres (ALT) cells, rely on telomere recombination for telomere lengthening and unlimited replicative potential. We show that the SMC5/6 complex promotes telomere recombination and lengthening in ALT cells by MMS21-dependent sumoylation of telomere-binding proteins. Sumoylation of these telomere-binding proteins relocalizes telomeres to nuclear PML bodies where HR proteins facilitate telomere recombination. These studies identify the human SMC5/6 complex and SUMO modification as critical mediators of sister chromatid HR.Item GCNA: Guardian of the Genome(2020-05-01T05:00:00.000Z) Goldstein, Courtney DaVee; Abrams, John M.; Buszczak, Michael; Brekken, Rolf A.; Olson, Eric N.The propagation of species depends on the ability of germ cells to protect their genome in the face of numerous exogenous and endogenous threats. While germ cells employ a number of know repair pathways, specialized mechanisms that ensure high-fidelity replication, chromosome segregation, and repair of germ cell genomes remain incompletely understood. Here, we identify Germ cell nuclear acidic peptidase (GCNA) as a conserved regulator of genome stability in flies, worms, zebrafish and human germ cell tumors. GCNA contains an acidic intrinsically disordered region (IDR) and a protease-like SprT domain. In addition to chromosomal instability and replication stress, Gcna mutants accumulate DNA-protein crosslinks (DPCs). GCNA acts in parallel with a second SprT domain protein Spartan. Structural analysis reveals that while the SprT domain is needed to limit meiotic and replicative damage, much of GCNA's function maps to its IDR. This work shows GCNA protects germ cells from various sources of damage, providing novel insights into conserved mechanisms that promote genome integrity across generations.Item Mechanistic Link Between DNA Damage Response (DDR) Signaling & Immune Activation(2018-11-26) Bhattacharya, Souparno; Story, Michael; Shay, Jerry W.; Sadek, Hesham A.; Aroumougame, AsaithambyProper maintenance of an intact genome is crucial for cellular homeostasis. To combat threats posed by DNA damage, cells have evolved sophisticated mechanisms - collectively termed as the DNA-damage response (DDR) signaling -, which detect DNA lesions, signal their presence, and promote their repair. Contribution of proper DDR signaling in not just confined to prevention of genomic instability and carcinogenesis, as emerging evidence indicates crosstalk exists at different levels between DDR signaling machinery and our immune system. In my dissertation work, using innovative models and techniques, I deciphered how RAD51, a protein normally associated with repair and replication of DNA, regulates innate immune response. Besides detection and repair of damaged DNA, proper DDR signaling also enables checkpoint activation, which prevents cell cycle progression with unrepaired DNA lesions. In my thesis work, I have proved how failure to arrest cells in the G2-M boundary after genotoxic stress, leads to generation of micronuclei, present in the cytoplasm and subsequent immune activation. Work emanating from my thesis projects will add to the growing body of literature showing how different DDR factors' roles in modulating immune signaling are most often a consequence of their inherent ability to sense, repair and signal in response to DNA damage. Finally, our improving understanding of DDR has already provided new avenues for disease management (e.g. Use of PARP inhibitors in treating BRCA mutant tumors). A more precise understanding of mechanisms by which DDR factors are involved in regulation of cellular immunity can also be exploited to redirect the immune system for both preventing and treating variety of human pathologies including cancer, autoimmune diseases and age related disorders.Item Novel Insights into DNA Double-Strand Break Repair and Its Cancer Implications(2016-07-27) Hardebeck, Molly Catherine; Shay, Jerry W.; Brekken, Rolf A.; Bachoo, Robert; Burma, SandeepDespite the aggressive treatment with DNA damage-inducing agents, glioblastomas (GBM) inevitably develop therapy resistance, leading to relapse and patient mortality. Cancer cells that survive therapy acquire additional damage-induced oncogenic changes that likely facilitate therapy resistance and tumor recurrence. To understand which damage-induced oncogenic alterations may promote tumor recurrence, we previously irradiated brains of mice harboring deletions of key tumor suppressors frequently lost in GBM. The most significant acquired alteration was amplification of the Met tyrosine kinase. We find that Met-expressing cells display cancer stem cell properties, augmented tumorigenesis, up-regulation of numerous DNA damage response (DDR) proteins, and an extended G2/M arrest. We hypothesize that Met expression drives therapy resistance and may be a potential target for radiosensitizing GBM. An alternative sensitization approach could involve direct inhibition of key DDR proteins, specifically in the homologous recombination (HR) double-strand break (DSB) repair pathway which is implicated in radioresistance of GBM stem cells. One indispensable step of HR is DNA-end resection, primarily executed by the exonuclease EXO1. We found that an EXO1 construct lacking the C-terminus and containing only the nuclease domain does not localize to DSBs, causing severe resection and repair defects. We hypothesized that the C-terminus of EXO1 serves as a platform for proteins to regulate EXO1's function. We found that the C-terminus interacts with BLM helicase, and it contains four Ser/Thr-Pro sites that are phosphorylated by CDKs1/2 to promote resection. We are currently examining whether CDK phosphorylation of EXO1 modulates the duration of the G2/M checkpoint since proper DNA repair requires a halt in the cell cycle. We are using CRISPR technology to generate EXO1 knock-out cells that will be complemented with WT or CDK-mutant EXO1 for checkpoint studies. We hypothesize that CDK phosphorylation of EXO1 serves to regulate resection and sustain the G2/M checkpoint. To further elucidate the role of EXO1 in maintaining genomic stability, we examined a cancer-associated SNP in EXO1 and found that it causes resection and DSB repair defects which may contribute to genomic instability and cancer progression. Overall, we provide novel insights into multiple aspects of DSB repair and identify potential targets for cancer therapy.Item NQO1-Bioactivatable Drugs at the Interface of Cancer Metabolism and the DNA Damage Response(2015-05-07) Chakrabarti, Gaurab; Burma, Sandeep; Brekken, Rolf A.; DeBerardinis, Ralph J.; Boothman, David A.Increased levels of reactive oxygen species (ROS) have been observed in multiple cancer types, where they are crucial for tumor biology. Concomitantly, tumor cells also have enhanced expression of antioxidant pathway proteins to detoxify excess ROS. Thus, a challenge for anti-cancer therapeutics is to fine-tune this delicate balance from ROS protection, to ROS production while sparing normal tissue from toxicity. The phase II detoxification enzyme, NAD(P)H:quinone oxidoreductase-1, NQO1, is dramatically overexpressed in many solid tumor types, including pancreatic ductal adenocarcinoma (PDA) and non-small cell lung cancer (NSCLC). The Boothman laboratory has demonstrated that NQO1 bioactivates a unique class of quinones, such as ß-lapachone (ß-lap) and deoxyniboqunine (DNQ), through a futile redox cycle to generate massive levels of superoxide radical to induce extensive DNA oxidative base damage, single strand breaks and poly(ADP-ribose) polymerase 1 (PARP1)-driven depletion of intracellular NAD+. However, tumor cell NADPH and glutathione (GSH) biogenesis can attenuate the efficacy of this class of drugs by blunting the ROS formation produced from the futile redox cycle. Therefore, it is increasingly important to identify and target tumor specific antioxidant defenses to sensitize cancer cells, but not normal tissue, to NQO1 bioactivatable drugs. The data presented in the first half of this dissertation demonstrate that targeting glutamine dependent transamination reactions depletes antioxidant defenses in PDA and sensitizes tumors, but not normal tissue, to ß-lap-induced programmed necrosis in vitro and in vivo. Downstream of ROS formation, another mechanism by which tumors can attenuate ß-lap efficacy is through the repair of DNA lesions, specifically through base excision repair (BER). The latter half of this thesis focuses on inhibiting BER in combination with ß-lap as a mechanism to drive PARP1 hyperactivation and synergistic killing of NQO1-expressing PDA, but not associated normal tissue.Item The Role of RUVBL1/RUVBL2 and Their Potential as Therapeutic Targets in Non-Small Cell Lung Cancer(2019-07-31) Yenerall, Paul Matthew, II; DeBerardinis, Ralph J.; Minna, John D.; Kittler, Ralf; Mangelsdorf, David J.; Kraus, W. LeeBehind heart disease, cancer is the leading cause of death today in Americans. Among cancers, lung cancer is the deadliest, killing as many individuals as next 3 most lethal cancer types combined. Approximately 80% of lung cancers are a type known as non-small cell lung cancer (NSCLC), and despite numerous advances in the treatment of NSCLC, only 18% of all NSCLC patients live 5 years after their initial diagnosis. To identify new therapeutic targets in NSCLC, we performed a viability-based RNA interference (RNAi) screen targeting nuclear receptors, their coregulators and chromatin remodelers. This screen identified RUVBL1 and RUVBL2 (collectively referred to as RUVBL1/2) as differentially required for the viability of NSCLC. We show that RUVBL1/2 require their ATPase activity to support NSCLC viability and have developed an orally bioavailable, potent and specific inhibitor of RUVBL1/2 ATPase activity, known as Compound B. Multiple unbiased analyses suggested that RUVBL1/2 may have roles in DNA replication in NSCLC, and inhibition or depletion of RUVBL1/2 in sensitive NSCLC lines delays S-phase progression and ultimately results in cancer cell death via replication catastrophe. While Compound B treatment in vivo produces modest anti-tumor activity, only a subset of NSCLC cell lines show a therapeutically meaningful response. To enhance the efficacy of Compound B, we searched for therapies that may synergize with Compound B. Various analyses indicated that RUVBL1/2 may have roles in the response to ionizing radiation (IR), and indeed, genetic depletion or pharmacological inhibition of RUVBL1/2 radiosensitized NSCLC cell lines and patient tumors both in vitro and in vivo. Interestingly, Compound B, did not radiosensitize models of non-transformed cells, potentially because key DNA damage proteins such as ATM and DNA-PKcs were more stable after Compound B treatment in normal cells than in tumor cells. The combined necessity of RUVBL1/2 for NSCLC viability and the recovery from radiation, specifically in tumor cells, make RUVBL1/2 an attractive target for future preclinical development as a radiosensitizer in NSCLC.Item The Sec6/8 (a.k.a. Exocyst) Complex Supports DNA Repair Fidelity(2014-04-14) Torres, Michael Jason 1982-; Brekken, Rolf A.; Cobb, Melanie H.; Burma, SandeepThe exocyst complex, first described in yeast, is a heterooctomeric complex that serves as a signaling platform to mediate cellular responses to diverse spatial and temporal cues. Evidence suggests that the exocyst might contribute to oncogenesis, potentially by disrupting spatial and temporal regulation of pathways critical to determining cell survival vs. apoptosis. Our work investigated how cancer cells subvert the exocyst to upregulate the AKT (v-akt murine thymoma viral oncogene) pro-survival pathway through the innate immune protein TBK1 (TANK-binding kinase 1). siRNA-mediated depletion of TBK1 in pancreatic and breast cancer cell lines results in apoptosis, which is mediated through the AKT pathway. Pharmacological inhibition of TBK1 recapitulates the apoptotic phenotype in mouse orthotopic models. Additionally, my work uncovered exocyst participation in the regulation of DNA repair. The isolation of multiple components of the DNA damage response (DDR) within the human exocyst protein-protein interaction network, together with the identification of Sec8 as a suppressor of the p53 response, prompted an investigation of functional interactions between the exocyst and the DDR. We found that exocyst perturbation resulted in a radioresistance phenotype to ionizing radiation (IR) that was associated with accelerated resolution of DNA damage. This occurred at the expense of genomic integrity, as enhanced recombination frequencies correlated with the accumulation of aberrant chromatid exchanges. Exocyst-dependent modulation of the DDR is, at least in part, through restraint of the associated chromatin modifiers ATF2 and RNF20. Exocyst perturbation resulted in aberrant accumulation of ATF2 and RNF20; the promiscuous accumulation of DDR-associated chromatin marks; and IR-induced increased Rad51 repairosomes. Thus, the exocyst indirectly supports DNA repair fidelity by limiting formation of repair chromatin in the absence of a DNA damage signal. This newly revealed regulation of DNA repair by the exocyst may provide additional insight into the emerging observations of DNA damage protein involvement in pathways not canonically associated DNA repair, such as the host cytokinesis, host defense response, and maintenance of cilia. This work further substantiates the importance of the exocyst in normal cell biology and gives insight into how disruption of exocyst function can result in disease.Item [Southwestern News](1999-10-12) Steeves, Susan A.Item Targeting DNA Double-Strand Break Repair to Potentiate Radio- and Chemo Therapy of Glioblastoma(2015-08-03) Gil del Alcazar, Carlos Rodrigo; Bachoo, Robert; Burma, Sandeep; Boothman, David A.; White, Michael A.Surgical resection followed by radiation and adjuvant temozolomide (TMZ) is the standard of care for glioblastoma (GBM). Not all GBMs respond to therapy, and most of them quickly acquire resistance to TMZ and recur. In order to develop more effective and rational treatments for GBM, it is crucial to understand the mechanisms underlying radio- and chemo-resistance. We find that protracted TMZ treatment of mice bearing orthotopic tumors (generated by xenografting GBM9 neurospheres) leads to acquired-TMZ resistance resulting in tumor recurrence. In order to understand the basis for therapy-driven TMZ resistance, we generated and functionally characterized ex-vivo cultures from the primary and recurrent tumors. We found that cell lines derived from recurrent (TMZ-treated) tumors were more resistant to TMZ in vitro compared to cell lines derived from primary (untreated) tumors. We also found that the increased resistance to TMZ was due to the augmented repair of TMZ-induced DNA double strand breaks (DSBs). TMZ induces DNA replication-associated DSBs that are repaired primarily by the homologous recombination (HR) pathway. We found that cell lines from recurrent cultures exhibited faster resolution of Rad51 foci and higher levels of sister chromatid exchanges (SCEs), which indicated that a higher level of HR was contributing to TMZ resistance in these lines. We have recently shown that CDKs 1 and 2 promote HR in S and G2 phases of the cell cycle, in part, by phosphorylating the exonuclease EXO1. We hypothesized, therefore, that blocking CDKs 1 and 2 might be a viable strategy for re-sensitizing recurrent tumors to TMZ. Indeed, we found that CDK inhibitors, AZD5438 and Roscovitine, could attenuate HR in the recurrent TMZ-resistant cell lines resulting in significant chemo-sensitization to TMZ. While HR is primarily involved in the repair of TMZ-induced DSBs, ionizing radiation induced DSBs would be mainly repaired by the non-homologous end joining (NHEJ) pathway. We, therefore, developed another approach to sensitize these tumors to both radiation and TMZ by using a dual PI3K/mTOR inhibitor, NVP-BEZ235, to block both DNA-PKcs and ATM, key enzymes in the NHEJ and HR pathways, respectively. We found that NVP-BEZ235 inhibited both DNA-PKcs and ATM in tumors generated in mice from GBM9 neurospheres, thus blocking the repair of TMZ- and IR-induced DSBs, and resulting in significant chemo- and radio-sensitization. Next, we established that NVP-BEZ235 can cross the blood-brain barrier and recapitulated its radiosensitizing effects in an intracranial GBM model. We found that NVP-BEZ235, administered with IR, can attenuate DSB repair in these intracranial tumors, attenuate tumor growth, and extend survival of tumor-bearing mice. Importantly, attenuation of DSB repair was more pronounced in tumor cells compared to normal brain cells, thereby providing a larger therapeutic window. In sum, this study indicates that augmented DNA repair may underlie therapy resistance in GBM and provides support for the potential use of DNA repair inhibition as an effective strategy for improving the efficacy of GBM therapy.