Browsing by Subject "Mutagenesis"
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Item Delineating the APOBEC3 enzymes responsible for the APOBEC mutation signature in cancer(2021-08) Jarvis, MatthewMutations 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).Item Structure-function relationship of plant sucrose transporters (SUTs)(2012-07) Sun, YeSucrose transporters (SUTs or SUCs) are membrane proteins that transport sucrose and H+ into the cytoplam at a ratio of 1:1. They are important for the long-distance transport of sucrose in plants. However, little is known about the structure-function relationship of SUTs. In this thesis, the transport activity and substrate specificity of rice SUTs were measured using [14C] sucrose yeast uptake and oocyte electrophysiology. More importantly, a 3D structural model of rice sucrose transporter OsSUT1 was built using known crystal structures of transporters from E.coli as templates. Based on the predicted model, six charged amino acids in transmembrane spans were selected for mutagenesis, five of which turned out to be essential for the SUT transport function. One mutant, R188K, caused a complete loss of sucrose transport activity, and showed a H+ leak that could be blocked by sucrose. Based on electrophysiology experiments results, a putative binding interaction between Arg188 of OsSUT1 and hydroxyl groups of sucrose was proposed. A role of Arg188 in the substrate transport process was also suggested. In addition, methods to identify amino acids important for SUT substrate specificity were explored.