Browsing by Subject "DNA repair"
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Item DNA-Protein Cross-links: Formation in Cells and Tissues, Repair, and Inhibition of DNA Transcription(2019-04) Park, DaeyoonDNA is constantly damaged by exogenous and endogenous agents, generating a range of nucleobase lesions. It is important to understand the biological consequences and repair mechanisms of DNA adducts. Cellular proteins can become covalently trapped on DNA to generate DNA-protein crosslinks (DPCs). Because of their unusually bulky nature, DPCs are anticipated to block many cellular processes including replication, transcription, and repair. However, cellular effects of DPCs have not been fully elucidated. Chapter 1 of this thesis provides background information on the formation, biological consequences, and repair pathways of DPCs studied in previous studies. In Chapter 2, we employed a quantitative nanoLC-ESI+-MS/MS assay to investigate the formation of free radical-induced DPCs between thymidine in DNA and tyrosine sidechains of proteins. This methodology was used to examine the role of SPRTN protease and immunoproteasome in DPC repair in human cells and mouse models. In Chapter 3, a mass spectrometry based CTAB assay was used to study the effects of DNA-peptide crosslinks on transcription in human cells. We constructed plasmid molecules containing DPCs between C5 of dC and lysine sidechains of polypeptides in order to mimic conjugates that form endogenously at DNA epigenetic marks (5-formylc-dC). Lesion bearing and control plasmids were transfected into human cells, and the amounts of RNA transcripts were determined using a mass spectrometry based approach. Moreover, DNA lesion bearing plasmid models were used to determine the importance of NER pathway in DPC repair. In Chapter 4, we investigated in vivo formation of DPCs in cells exposed to monofunctional alkylating agent, methyl methanesulfonate (MMS). A mass spectrometry-based TMT proteomics approach was used to characterize MMS-induced DNA-protein cross-linking in Chinese hamster lung fibroblasts (V79). utilizing Our results revealed that DPCs can be produced via nucleophilic attack of proteins at the C8 position of N7-methylguanine (MdG). Our results revealed novel DPC formation mechanisms and the toxicities of monofunctional agent induced DPCs. In summary, mass spectrometry-based quantification was used to the amounts of free radical induced DPCs in cells, providing evidence for the role of DPC proteolysis in repair, while CTAB assay demonstrated the effect of endogenously formed DPCs on transcription. Moreover, a mass spectrometry-based methodology was applied to examine a novel DPC formation mechanism following treatment with monofunctional alkylating agents.Item Repair of DNA-protein crosslinks in mammalian cells(2018-07) Chesner, LisaThe work below describes a new assay called strand-specific primer extension-quantitative polymerase chain reaction (SSPE-qPCR) used to study the repair of DNA-protein crosslinks in mammalian cells. DNA-protein crosslinks (DPCs) are bulky lesions which disrupt important cell processes such as transcription and replication. They are formed by endogenous molecules such as formaldehyde and exogenous damaging agents such as ionizing radiation. However, the repair mechanisms associated with their repair are still unclear. Chapter 1 of this document provides background information on the formation, biological consequences, current models, and methods used to study DPC repair. Chapter 2 describes the SSPE-qPCR assay and its uses/limitations for studying the repair of plasmids containing DPCs or other polymerase-blocking adducts transfected into mammalian cells. Chapter 3 describes results generated using this assay to assess the role of nucleotide excision repair in DPC repair and highlights the versatility of the SSPE-qPCR assay. Chapter 4 extends observations made in Chapter 3 by using SSPE-qPCR to examine repair of DPC-containing plasmids in the presence of a homologous donor. It also provides evidence for homologous recombinational repair of DPCs in mammalian mitochondria. Overall, this work provides additional insight into the mechanisms of DPC repair in the nucleus and mitochondria using a quantitative, flexible assay that has not been available previously.Item The role of DNA repair & regulatory proteins in the maintenance of human telomeres and their control of cellular immortalization(2017-04) Harvey, AdamTelomeres are the nucleoprotein structures that protect the ends of linear chromosomes from recognition as a double-stranded DNA break (DSB). In the absence of proper telomere function, the ends of a chromosome fuse together, creating di-centromeric chromosomes, which can no longer properly segregate at mitosis. Thus, proper telomere maintenance is absolutely essential for all eukaryotic life. Unfortunately, maintaining telomeres at a size that is protective is problematic. For example, as a consequence of “the end-replication problem,” telomeres shorten incrementally during every cell cycle. These short telomeres can, in turn, function to regulate the lifespan of any given cell. Perhaps not surprisingly, therefore, humans have evolved a vast array of genes to enable telomere stability, in order to counteract any premature ageing or cell death. In order to ensure that offspring may begin their life with a default telomere length that is sufficient for stability during the organism’s lifespan, stem cells must not be subjected to overall telomere shortening. Thus, all telomere shortening that a stem cell occurs during its eternal proliferation must be correspondingly compensated for by a lengthening event. This telomere elongation mechanism in essence confers cellular immortality. The most well-characterized of these cellular immortality pathways is controlled by the enzyme telomerase, which precisely elongates telomeres in a stochastic way to maintain a telomere length equilibrium. Unfortunately, this functional, essential pathway can also be conscripted to perform pathological reactions. In human cancer, all malignant growths must enable cellular immortalization to allow for their characteristic uncontrolled proliferation. In most cases this is achieved simply by the reactivation of telomerase. Interestingly, 5 to 15% of all human cancers are telomerase negative. These cancers can be described as ALT cancers, as the Alternative Lengthening of Telomeres pathway enables their immortality. ALT, which is specific to cancer, achieves telomere elongation by aberrant recombination between telomeres. My research has found that DNA repair proteins, such as PARP1, (poly ADP ribose polymerase 1) are critical for both the maintenance of the genome and specifically for proper telomere maintenance. Furthermore, my research has demonstrated that the mutation of a single gene, ATRX, (alpha thalassemia mental retardation on the X chromosome) is an active repressor of ALT immortalization. In summary, I have contributed to the understanding of human telomere length maintenance and these studies have implications for human aging and the genesis of cancer.Item The Role of Fanconi Anemia Proteins in DNA Repair, Replication Stress and Genome Stability(2017-10) Thompson, ElizabethFanconi anemia (FA) is a genetic chromosomal instability disorder characterized by progressive bone marrow failure and a strong predisposition to cancer. The FA proteins work together in a cellular pathway for the repair of DNA interstrand crosslinks (ICLs). Currently 22 different FA genes are implicated in this disease and contribute to the heterogeneity in symptoms and severity. Using gene targeting techniques, we successfully created a set of isogenic knockout cell lines to represents all 3 groups of proteins within the FA pathway to characterize protein function and identify differences that help explain FA disease heterogeneity. In Chapter 2 we investigate the FA group 2 proteins, FANCI and FANCD2 that that form a heterodimer called the ID complex. We characterized the FANCI, FANCD2 and FANCI/FANCD2 double knockout cell lines and identified non-overlapping functions in the replication stress response. In fact, we found that only FANCD2 is required for restart of stalled replication forks and FANCI may even inhibit restart when FANCD2 is absent. In addition, FANCD2 has a more vital role in homologous recombination, and FANCI promotes apoptosis in the absence of FANCD2 with replication stress. In Chapter 3 we investigate FANCN, an FA group 3 protein that is associated with more severe FA disease. We used FANCN conditional knockout cells to determine that FANCN is essential for viability and genome stability. In addition, we evaluated FANCN FA-associated mutations and breast cancer-associated variants of unknown significance (VUS) mutations. We confirmed that the BRCA1/2 binding domains of FANCN are not essential for viability and identified two VUS mutations as potentially pathogenic. In conclusion, we have demonstrated alternative functions of FA proteins in response to replication stress as a potential source of FA disease heterogeneity. In addition, we have demonstrated that FANCN is essential for viability whereas FANCI and FANCD2 are not, providing insight into both the frequency of occurrence and severity of FA disease associated with these different genes. Finally, we created isogenic cell lines that are a valuable asset for the FA and breast cancer fields for further investigations into protein function, characterization of patient mutations, and screening novel therapeutics.