A Germline Polymorphism of Thymine DNA Glycosylase Induces Genomic Instability and Cellular Transformation

et al. (2014) A Germline Polymorphism of Thymine DNA Glycosylase Induces Genomic Instability and Cellular Transformation. PLoS Genet 10(11): e1004753. doi:10.1371/journal.pgen.1004753 A Germline Polymorphism of Thymine DNA Glycosylase Induces Genomic Instability and Cellular Transformation Ashley Sjolund 0 Antonia A. Nemec 0 Nicolas Paquet 0 Aishwarya Prakash 0 Patrick Sung 0 Sylvie Doublie 0 Joann B. Sweasy 0 Nancy Maizels, University of Washington School of Medicine, United States of America 0 1 Department of Genetics, Yale University School of Medicine, New Haven, Connecticut, United States of America, 2 Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, Connecticut, United States of America, 3 Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut, United States of America, 4 Department of Microbiology and Molecular Genetics, University of Vermont , Burlington, Vermont , United States of America Thymine DNA glycosylase (TDG) functions in base excision repair, a DNA repair pathway that acts in a lesion-specific manner to correct individual damaged or altered bases. TDG preferentially catalyzes the removal of thymine and uracil paired with guanine, and is also active on 5-fluorouracil (5-FU) paired with adenine or guanine. The rs4135113 single nucleotide polymorphism (SNP) of TDG is found in 10% of the global population. This coding SNP results in the alteration of Gly199 to Ser. Gly199 is part of a loop responsible for stabilizing the flipped abasic nucleotide in the active site pocket. Biochemical analyses indicate that G199S exhibits tighter binding to both its substrate and abasic product. The persistent accumulation of abasic sites in cells expressing G199S leads to the induction of double-strand breaks (DSBs). Cells expressing the G199S variant also activate a DNA damage response. When expressed in cells, G199S induces genomic instability and cellular transformation. Together, these results suggest that individuals harboring the G199S variant may have increased risk for developing cancer. - Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by grant number PO1 CA098993 from the National Institutes of Health (http://nih.gov/). JBS received this funding. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Thymine DNA glycosylase (TDG) is a monofunctional DNA glycosylase that functions in base excision repair (BER), the pathway responsible for repairing up to 20,000 endogenous lesions/cell/day [1]. This glycosylase is well known for its ability to remove T from G:T mispairs and can also excise a variety of other bases, some of which include U opposite A and 5-fluorouracil (5FU) paired with A or G [25]. More recent work has implicated TDG in an active demethylation pathway with the ten-eleven translocation (TET) protein family [6]. It has been shown both biochemically and biologically that TDG can remove TETgenerated 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC) in a process that requires BER to regenerate unmodified C [69]. Because both DNA repair and DNA methylation dynamics are vital processes to the maintenance of genomic stability, aberrant activity of either of these processes could contribute to cancer development. The rs4135113 single nucleotide polymorphism (SNP) of TDG, where G is mutated to A at position 818, has a minor allele frequency of approximately 10%, is most commonly found in African and East Asian populations and is usually heterozygous (www.ncbi.nlm.nih.gov/SNP/). This mutation leads to the substitution of glycine to serine at amino acid residue 199. G199 is located in a loop that appears to stabilize the flipped out abasic site within the active site pocket of TDG and serves to prevent flipping of the residue back into the helix [10]. We hypothesize that mutating this residue could affect the stability of the everted abasic site in the active site pocket. There is limited available evidence on TDG polymorphisms in relation to cancer or biomarkers of cancer. One study found G199S to be associated with increased likelihood of micronuclei in Chinese workers who had been exposed to vinyl chloride, suggesting individuals carrying G199S are more susceptible to chromosomal damage [11]. Other studies have found that G199S is not associated with esophageal squamous cell carcinoma or gastric adenocarcinoma in a Chinese population [12], and is not associated with increased risk of non-melanoma skin cancer, lung and rectal cancer [13,14]. More recent work found that TDG expression levels are upregulated in colorectal carcinoma (CRC) and that TDG serves to regulate Wnt signaling, a key driver for CRC [15]. Interestingly, depletion of TDG significantly inhibited cancer cell proliferation and tumor formation in this study, suggesting TDG is required for CRC growth and may serve as a biomarker. In this study, we tested the hypothesis that expression of G199S has as functional phenotype that could induce cellular transformation. We report herein that while the purified G199S protein has a glycosylase activity similar to WT, it binds significantly more DNA repair is vital to the survival and propagation of cells. It helps protect DNA from becoming permanently damaged and prevents cells from becoming cancerous. The base excision repair (BER) pathway is responsible for the removal of up to 20,000 lesions/cell/day. Thymine DNA glycosylase (TDG) is one of the DNA glycosylases that initiates BER. There is a germline variant of TDG that is found in 10% of the global population, where amino acid residue glycine 199 is mutated to serine. Here, we provide evidence that TDG variant G199S binds significantly more tightly to its abasic product and leads to increased DNA strand breaks in cells. We go on to show that G199S induces genomic instability, in the form of chromosomal aberrations, and leads to cellular transformation, both hallmarks of tumorigenesis. Collectively, our work suggests that a germline variant of TDG can drive carcinogenesis. tightly to its abasic product. Expression of G199S in human breast epithelial cells results in the accumulation of DNA double-strand breaks and activates a DNA damage response in cells progressing through the S- and G2/M-phases of the cell cycle. Expression of G199S also leads to the induction of chromosomal aberrations and cellular transformation. Our results suggest that carriers of the rs4135113 SNP may have an increased cancer predisposition. Germline variant G199S has similar catalytic activity to the WT protein The rs4135113 SNP encodes the G199S variant TDG protein. To determine if al (...truncated)