Although uracil (U) is a canonical nucleobase in RNA, it has mutagenic potential in DNA. U lesions are corrected by the DNA base excision repair (BER) pathway. BER is initiated by a glycosylase specific to the lesion that removes the nucleobase, such as a uracil DNA glycosylase. The resulting abasic site then is processed by downstream enzymes to restore the DNA. Human cells express three uracil glycosylases belonging to the uracil DNA glycosylase (UDG) superfamily: (1) uracil DNA glycosylase (UNG); (2) single-strand selective monofunctional uracil DNA glycosylase (SMUG1); and (3) thymine DNA glycosylase (TDG). All three enzymes are capable of excising U as well as the chemotherapeutic 5-fluorouracil (5FU). Hence all three superfamily members have been implicated in the response of cells to 5FU treatment. UNG, SMUG1, and TDG also have known or candidate sites of lysine acetylation. Preliminary studies have demonstrated that UDG superfamily glycosylases display drastically different excision of U from DNA when packaged into a nucleosome core particle (NCP), consisting of 145 base pairs wrapped around a core of eight histone proteins. These results have led to three fundamental questions: (1) does post-translational acetylation modulate the activity of UNG, SMUG1, and TDG? (2) If the glycosylase activities are drastically different for excision of U from NCP, does this difference in activity extend to other lesions, such as 5FU? And (3) does post-translational acetylation determine the cellular response to 5FU? This proposal seeks to test the hypothesis that modulation of UDG superfamily glycosylase activity by post-translational acetylation affects the cellular response to 5FU.
In Aim 1, the effect of lysine acetylation on UDG superfamily glycosylase activity will be determined. Using biochemical tools, quantitative measures of binding and catalysis on DNA substrates containing both U and 5FU will be obtained.
In Aim 2, the contribution of the UDG superfamily glycosylases to 5FU response will be examined. Using a series of cell lines, glycosylase expression will be knocked down and the effect of acetylation will be tested by regulating expression of the histone/protein deacetylase SIRT1. These cell lines will be treated with 5FU and cell viability will be measured. Furthermore, the DNA occupancy for each of these enzymes will be probed by sequencing following chromatin immunoprecipitation (ChIP-Seq). These experiments will elucidate how these glycosylases can be used in assessing the tumor sensitivity to a chemotherapeutic in addition to identifying novel mechanisms for enhanced tumor killing. The long-term goal for this award is to transition into a career as a physician-scientist, exploring the intersection of metabolic disease and mitochondrial DNA repair. A sponsor team has been assembled with expertise in enzymology, DNA repair, cellular and molecular biology, and metabolic disease. This team also will mentor in the transition to clinical work. Further training will be acquired from workshops, courses, and clinical Grand Rounds offered both at and outside of Brown University coupled with opportunities to attend and present at conferences. These tools are essential for technical training and career development.
The chemotherapeutic agent 5-fluorouracil (5FU) is used to treat a wide variety of cancers. This project seeks to identify the role of three DNA glycosylases?uracil DNA glycosylase (UNG), single strand-selective monofunctional uracil DNA glycosylase (SMUG1), and thymine DNA glycosylase (TDG)? and the effect of their post-translational acetylation in the cellular response to 5FU. Elucidating the roles of these enzymes and the effect of acetylation will identify potential biomarkers for successful treatment with 5FU and also novel pathways and strategies to enhance tumor killing.
