"Chemical tools for the detection and regulation of DNA damage and repair"
The DNA in our cells is constantly under threat from both endogenous and exogeneous factors. To protect the integrity of our genome, nature has evolved a collection of DNA repair enzymes that can recognize and reverse harmful DNA lesions. DNA damage and (misregulated) DNA repair have been implicated in many diseases. Accordingly, targeting DNA repair pathways has been proposed as a promising strategy for cancer prevention and therapy. However, many unanswered questions remain regarding how DNA repair enzymes can be regulated and how DNA damage occurs. This presentation will cover two related stories on efforts toward developing chemical and analytical tools aimed to address some of these questions.
First, I will describe the discovery and optimization of small molecule activators of the DNA repair enzyme SMUG1. SMUG1 is a DNA glycosylase responsible for excising deaminated cytosine from genomic DNA, which accounts for one of the most frequent mutations in many cancers. Upregulating SMUG1 activity thus can potentially lower mutation rates and prevent cancer development. We report the first discovery of a potent SMUG1 activator, identified through screening of a kinase inhibitor library and optimized via iterative structure-activity relationship studies. Our data suggest that the lead compound accelerates SMUG1 turnover, and experiments in model cell lines show that it significantly reduces the cytotoxicity of the SMUG1-specific lesion 5-hmdU. This study demonstrates the feasibility of chemically upregulating a DNA glycosylase and provides insight on possible implications of accelerating base excision repair in human cells.
The second topic focuses on the metabolic incorporation of damaged nucleosides into genomic DNA. While much of the existing research has focused on DNA damage that occurs within a cell, relatively little attention has been dedicated to extracellular sources of lesions. We aim to determine whether externally supplied oxidized pyrimidine nucleosides can be metabolically incorporated into genomic DNA. I will first discuss the synthesis of four isotopically labeled nucleosides and our efforts on improving the existing synthetic methodology. Next, after feeding these compounds to mice and human cell lines, we use LC-MS/MS to quantify the uptake of these lesions into the genome. Through these isotope tracing experiments, we provide the first direct evidence that extracellular damaged nucleosides can be incorporated into genomic DNA, highlighting a previously overlooked source of DNA damage.
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