Browsing by Subject "DNA Damage"
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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 The Function and Mechanism of RNA Interference in Neurospora(2009-01-14) Lee, Heng-Chi; Liu, YiRNA interference (RNAi) is a conserved gene silencing mechanism important for various biological processes, including developmental timing, genome defense, and heterochromatin formation. RNAi is triggered by double stranded RNA (dsRNA), which is processed by Dicer to siRNA. siRNA is loaded onto RNA-induced silencing complex (RISC), in which an Argonaute family protein, guided by a siRNA, mediates the cleavage of homologous RNAs. In the filamentous fungus Neurospora, we show that dsRNA not only trigger RNAi, it also transcriptionally activates several key components of RNAi pathway, including qde-2 (an Argonaute) and dcl-2 (a Dicer). A genome wide identification of dsRNA activated genes suggests that RNAi is part of a broad ancient host-defense response against viral and transposon infections. Our research on qde-2 regulation also suggests a role of RNAi during DNA damage. We show that DNA damage induces qde-2 expression, and the purification of QDE-2 bound RNAs identifies a novel class of small RNAs named qiRNAs. qiRNAs are averaged 21 nt in length and are mostly derived from ribosomal DNA (rDNA) locus. Importantly, qiRNA biogenesis requires RNAi components and RNAi mutants exhibit increased sensitivity to DNA damage, suggesting a role for qiRNAs during DNA repair. Further analysis suggests that the qiRNA contributes to the DNA damage checkpoints by inhibiting protein translation after DNA damage. To trigger RNAi against transgenes, it has been proposed that transgene- specific aberrant RNA (aRNA) is made and converted into dsRNA by RNA dependent RNA polymerase (RdRP). How aRNA is produced and specifically recognized by RdRP is not known. We show that QDE-1, a RdRP is also the DNA-dependent RNA polymerase (DdRP) that produces aRNA from ssDNA. QDE-1 is recruited to ssDNA by Replication Protein A (RPA) and QDE-3 (an RecQ helicase), both of them are also essential for aRNA production. Moreover, QDE-1 can produce dsRNA from ssDNA, a process facilitated by RPA. Our results provide a molecular mechanism of aRNA production in RNAi pathway.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 Generation of a Novel D. melanogaster Platform to Elucidate Oncogenic Activity of Common Human p53 Missense Mutants(2014-02-04) Jakubowski, Brandon; D'Brot, Alejandro; Abrams, JohnThe tumor suppressor p53 prevents uncontrolled cell growth by three separate mechanisms: inducing apoptosis, initiating cell-cycle arrest, and activating DNA repair mechanisms in response to cell damage. Due to its central role in tumor eradication, it is unsurprising that p53 mutations are found in over half of human cancers. Unlike all other tumor suppressors however, 75% of these are missense mutations, with just six of them accounting for a third of all mutations found in the DNA binding domain of p53. Recent findings indicate that mutations in these "hotspot" locations may encode gain of function oncogenic activity to p53. Given their high prevalence, these mutations suggest a previously underappreciated selective advantage. We sought to decode this novel oncogenic activity of human p53 mutations by exploiting the Drosophila model system. This organism shares a similar p53 regulatory network with humans, as well as many of the same DNA repair and pro-apoptotic target genes. We recently showed that human p53, despite millions of years of evolutionary distance, complements loss of function mutations in the native fly p53 gene. We used six humanized p53 Drosophila strains previously generated in the lab; these contain a human p53 gene insertion, each with one of the six most commonly found missense mutations in patients. To study these mutations, we first profiled the expression patterns of wild type and mutant hp53 in the fly and their ability to rescue dp53 function. Expression levels of p53 were determined by immunofluorescence, while biological function was determined by the use of a GFP biosensor that specifically reports dp53 activity and acridine orange staining to identify dying cells in irradiated embryos. Expression studies demonstrate that the reporter is activated within stem cells in region 1 of the germarium, while its activation was absent in p53 null mutants. This phenotype was recovered with a dp53 insertion rescue. Additionally, two separately generated hp53+ strains show unusually elevated levels of expression compared to the wild type strains, whereas all mutant strains show diminished reporter activation in the region 1 stem cells. Functional studies in the embryo and the wing disc demonstrate that both wild type flies and the dp53 rescue promote cell death after irradiation, while the p53 null mutant does not. The two hp53+ strains rescued the wild type phenotype in the embryo; however, one of the hp53+ strains, named B2, was unable to induce cell death in the wing disc. The missense mutant strains do not exhibit IR-induced apoptosis in the embryo, but preliminary imaging shows they may be able to in the wing disc. We also discovered that, unlike the six hotspot mutants, wild type human p53 localizes to unidentified subnuclear compartments. Importantly, this may allow us to stratify and characterize p53 mutations according to functional differences.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 Regulation of the Insulin-like Growth Factor 1-Secretory Clusterin Expression Axis in Genomic Instability and Cell Stress(2009-09-04) Goetz, Eva Marie; Boothman, David A.Secretory clusterin (sCLU) is a pro-survival factor that is up-regulated in human tumors and after exposure to cell stress. Understanding the regulation of sCLU expression in cancer, and after exposure to therapeutic agents, could reveal new therapeutic targets for cancer treatment. A DNA damage induced signaling cascade leading from ATM to sCLU expression mediated by IGF-1/IGF-1R/MAPK activation was uncovered. IGF-1 ligand promoter activity, mRNA, and protein expression induced after exposure to ionizing radiation (IR), hydrogen peroxide, or topoisomerase I and II-alpha poisons matched sCLU expression. Elevated basal IGF-1-sCLU signaling was noted in genomically unstable cells, whether they were deficient in DNA repair factors or telomerase function. ATM function was necessary for induction of sCLU after IR, and for maintaining elevated expression of sCLU in genomically unstable cells. p53 suppressed IGF-1 promoter activity, leading to decreased mRNA and protein expression, and abrogated induction of IGF-1 and sCLU by IR. Loss of p53 by knockdown or knockout enhanced IGF-1 and sCLU induction. Mutations in the p53 DNA binding domain found in cancer did not repress IGF-1 and sCLU. An NF-Y binding site in the IGF-1 promoter was essential for p53 suppression, and both p53 and NF-YA bound to the IGF-1 promoter. Nutlin-3, an Mdm2-p53 inhibitor, stabilized p53 expression, leading to dramatically decreased sCLU expression. Nutlin-3 treatment sensitized wild-type p53 cells to IR exposure. Finally, exogenous IGF-1 exposure led to serine 1981 auto-phosphorylation of ATM, and enhanced DNA damage repair and abrogated cell death after IR exposure. These studies uncovered key molecules important for the regulation of IGF-1-sCLU expression axis after IR exposure, and supported the use of IGF-1 or sCLU expression inhibitors for cancer chemotherapy.