Browsing by Subject "DNA damage"

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    Delineating the APOBEC3 enzymes responsible for the APOBEC mutation signature in cancer
    (2021-08) Jarvis, Matthew
    Mutations drive the initiation and progression of cancer. The leading druggable source of mutation in cancer, cytosine deamination by a subset of the nine-membered APOBEC family of DNA deaminase enzymes, leaves a distinct mutation signature on the cancer genome. This signature is characterized as C-to-T and C-to-G mutations in a TCA/T trinucleotide context, and thus APOBEC-dependent mutations can be resolved computationally from other processes of mutation in clinical next-generation tumor sequencing datasets. While specific APOBEC3 (A3) enzymes have been implicated as the main progenitors of this mutation signature (namely, APOBEC3A, APOBEC3B, and APOBEC3H, abbreviated A3A, A3B, and A3H), the literature is full of conflicting data and it is not clear which of these enzymes contributes most prominently, and whether other A3 enzymes may also contribute to mutation in cancer. In this thesis, we aim to definitively characterize the A3 enzymes that can contribute to genomic mutation in a mammalian cell, and potentially be involved in cancer mutagenesis. To accomplish this, we utilized bioinformatic approaches to understand mutational profiles in >1000 cancer cell models, the capacity of individual A3s to generate a cellular damage response and genomic mutation in culture, and the carcinogenic action of APOBECs in multiple animal systems of cancer initiation and progression. Taken together, these analyses indicate that both A3A and A3B have the capacity to generate a mutation signature in mammalian cells, and that A3A has the ability to initiate tumor formation in vivo. These novel advancements in the APOBEC biology field could prove invaluable in the design and implementation of future therapies and diagnostics targeting the A3s in cancer. An understanding of enzyme-specific mutational capacity will improve the development of targeted therapies, which could span to small molecule inhibition of enzymatic activity, synthetic lethal strategies, or immunotherapy-based approaches to selectively kill A3-expressing tumor cells, with the ultimate goal of attenuating or exploiting this mutational process to improve poor clinical outcomes (including drug resistance and metastasis).
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    DNA Replication And Telomere Maintenance Require Pcna-K164 Ubiquitination
    (2020-09) Leung, Wendy
    Genome integrity relies on a robust DNA replication program to ensure faithful duplication of genetic material, free from sequence mutations, deletions or rearrangements. There is an estimated 10 quadrillion (1x1016) cell divisions that occur in the average lifetime of a human being (Weinberg 2014). Thus, cells rely on a global DNA damage response (DDR) network to sense and repair errors that occur during replication to prevent the perpetuation of mutations (Ciccia and Elledge 2010). Although the DDR is highly efficient, some errors may escape repair and interfere with the progression of replication forks. In this scenario, cells utilize DNA damage tolerance (DDT) pathways to bypass errors/lesions encountered during replication and promote replication fork restart (Friedberg 2005, Chang and Cimprich 2009, Ghosal and Chen 2013). A major regulator of DDT pathways is proliferating cell nuclear antigen (PCNA) (Hoege et al. 2002). Ubiquitin modification at the conserved lysine residue 164 (K164) is crucial to DDT pathway choice – mono-ubiquitination activates error-prone translesion synthesis (TLS), while poly-ubiquitination activates error-free template switching (TS) (Shcherbakova and Fijalkowska 2006, Lehmann et al. 2007, Branzei 2011, Sale et al. 2012). However, whether PCNA ubiquitination regulates other genome maintenance mechanisms is unclear. The ends of chromosomes, known as telomeres, are origin-poor and present multiple challenges for the replication machinery including the propensity to form guanine (G)-quadruplexes and RNA-DNA hybrids (Sfeir et al. 2009, Maestroni et al. 2017). Because telomeres are intrinsically “difficult to replicate”, these regions are particularly sensitive to replication stress (Özer and Hickson 2018). In addition to the canonical replication machinery, additional proteins are needed to properly replicate the telomeric duplex. One of these proteins, the TLS polymerase η, functions to alleviate telomeric replication stress (Pope-Varsalona et al. 2014, Garcia-Exposito et al. 2016). The recruitment of TLS polymerases, including Pol η, to DNA lesions occurs through the direct interaction with mono-ubiquitinated PCNA (Bienko et al. 2005). These observations suggest a direct role for PCNA ubiquitination in the replication of telomeres. However, several reports have suggested that TLS can operate in the absence of PCNA ubiquitination (Haracska et al. 2006, Acharya et al. 2007, Parker et al. 2007, Edmunds et al. 2008, Nikolaishvili-Feinberg et al. 2008, Hendel et al. 2011, Krijger et al. 2011), thus it is not clear whether this modification is involved in telomere maintenance. While the role of PCNA-K164 ubiquitination for normal DNA replication and DDT pathway activation has been extensively studied in model systems of yeast, chicken, and mouse, how this modification functions in maintaining human genome stability is still not understood. This thesis addresses several critical functions of K164 ubiquitination in human cells. Studies in PCNAK164R mutants reveal that PCNA ubiquitination is required for gap-filling on the lagging strand behind progressing replication forks (Thakar et al. 2020). Additionally, we provide evidence that K164 ubiquitination functions to resolve late replicating intermediates (LRIs) through mitotic DNA synthesis (MiDAS) and promote efficient origin licensing in the subsequent G1 phase. Finally, we find that post-translational modification of PCNA at K164 regulates telomere maintenance specifically in transformed cells. Together, these studies show that the functions of PCNA-K164 go well beyond progressive DNA synthesis and DDT activation and extend to MiDAS and telomere maintenance.
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    DNA-Protein Cross-links: Formation in Cells and Tissues, Repair, and Inhibition of DNA Transcription
    (2019-04) Park, Daeyoon
    DNA 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.