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A germline polymorphism of thymine DNA glycosylase induces genomic instability and cellular transformation.

Sjolund A, Nemec AA, Paquet N, Prakash A, Sung P, Doublié S, Sweasy JB - PLoS Genet. (2014)

Bottom Line: 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.The persistent accumulation of abasic sites in cells expressing G199S leads to the induction of double-strand breaks (DSBs).

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Yale University School of Medicine, New Haven, Connecticut, United States of America.

ABSTRACT
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.

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Related in: MedlinePlus

G199S has a slightly higher affinity for the 5-FU:A lesion and binds significantly more tightly to its abasic product.Increasing concentrations (0–5400 nM) of WT TDG (filled diamonds) or G199S (open circles) protein were incubated for 30 mins at room temperature with 32P-labeled oligonucleotides carrying a tetrahydrofuran moiety (THF) or 5-FU opposite template A (Table 1). Data were plotted as fraction of DNA bound versus TDG protein concentration to obtain the KD. A and B. The representative binding curve of WT TDG and G199S on 5-FU:A and THF:A, respectively. C. The dissociation constants (KD) for WT TDG and G199S on 5-FU:A and THF:A substrates.
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pgen-1004753-g003: G199S has a slightly higher affinity for the 5-FU:A lesion and binds significantly more tightly to its abasic product.Increasing concentrations (0–5400 nM) of WT TDG (filled diamonds) or G199S (open circles) protein were incubated for 30 mins at room temperature with 32P-labeled oligonucleotides carrying a tetrahydrofuran moiety (THF) or 5-FU opposite template A (Table 1). Data were plotted as fraction of DNA bound versus TDG protein concentration to obtain the KD. A and B. The representative binding curve of WT TDG and G199S on 5-FU:A and THF:A, respectively. C. The dissociation constants (KD) for WT TDG and G199S on 5-FU:A and THF:A substrates.

Mentions: The crystal structures of human TDG bound to DNA suggest (Figure 2) that Gly199 is an important residue for stabilizing the everted abasic site into the glycosylase active site pocket during lesion processing [9], [16]. Gly199 is located in a loop that approaches the DNA from the major groove. The glycine residue is within van der Waals distance of the abasic site product [9]. We hypothesized that alteration of amino acid residue 199 from a small, nonpolar glycine to a nucleophilic serine could affect product release by TDG since Gly199 is in close vicinity to the abasic site product formed after excision of the base. To test this hypothesis, we used purified protein in gel electromobility shift assays to characterize the ability of WT and G199S TDG to bind DNA containing either A:5-FU or tetrahydrofuran opposite template A (Table 1). G199S binds to its substrate slightly more tightly than WT (KD = 77±13 nM and 41±5 nM, WT and G199S, respectively) (Figure 3A and C). Most importantly, G199S binds over 4-fold more tightly to DNA containing tetrahydrofuran (KD = 153±33 nM and 35±7 nM, WT and G199S, respectively) (Figure 3B and C). These data show that G199S remains more tightly bound to its abasic product after base removal, which could prevent proper processing of the repair intermediate leading to DNA breaks and genomic instability.


A germline polymorphism of thymine DNA glycosylase induces genomic instability and cellular transformation.

Sjolund A, Nemec AA, Paquet N, Prakash A, Sung P, Doublié S, Sweasy JB - PLoS Genet. (2014)

G199S has a slightly higher affinity for the 5-FU:A lesion and binds significantly more tightly to its abasic product.Increasing concentrations (0–5400 nM) of WT TDG (filled diamonds) or G199S (open circles) protein were incubated for 30 mins at room temperature with 32P-labeled oligonucleotides carrying a tetrahydrofuran moiety (THF) or 5-FU opposite template A (Table 1). Data were plotted as fraction of DNA bound versus TDG protein concentration to obtain the KD. A and B. The representative binding curve of WT TDG and G199S on 5-FU:A and THF:A, respectively. C. The dissociation constants (KD) for WT TDG and G199S on 5-FU:A and THF:A substrates.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4222680&req=5

pgen-1004753-g003: G199S has a slightly higher affinity for the 5-FU:A lesion and binds significantly more tightly to its abasic product.Increasing concentrations (0–5400 nM) of WT TDG (filled diamonds) or G199S (open circles) protein were incubated for 30 mins at room temperature with 32P-labeled oligonucleotides carrying a tetrahydrofuran moiety (THF) or 5-FU opposite template A (Table 1). Data were plotted as fraction of DNA bound versus TDG protein concentration to obtain the KD. A and B. The representative binding curve of WT TDG and G199S on 5-FU:A and THF:A, respectively. C. The dissociation constants (KD) for WT TDG and G199S on 5-FU:A and THF:A substrates.
Mentions: The crystal structures of human TDG bound to DNA suggest (Figure 2) that Gly199 is an important residue for stabilizing the everted abasic site into the glycosylase active site pocket during lesion processing [9], [16]. Gly199 is located in a loop that approaches the DNA from the major groove. The glycine residue is within van der Waals distance of the abasic site product [9]. We hypothesized that alteration of amino acid residue 199 from a small, nonpolar glycine to a nucleophilic serine could affect product release by TDG since Gly199 is in close vicinity to the abasic site product formed after excision of the base. To test this hypothesis, we used purified protein in gel electromobility shift assays to characterize the ability of WT and G199S TDG to bind DNA containing either A:5-FU or tetrahydrofuran opposite template A (Table 1). G199S binds to its substrate slightly more tightly than WT (KD = 77±13 nM and 41±5 nM, WT and G199S, respectively) (Figure 3A and C). Most importantly, G199S binds over 4-fold more tightly to DNA containing tetrahydrofuran (KD = 153±33 nM and 35±7 nM, WT and G199S, respectively) (Figure 3B and C). These data show that G199S remains more tightly bound to its abasic product after base removal, which could prevent proper processing of the repair intermediate leading to DNA breaks and genomic instability.

Bottom Line: 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.The persistent accumulation of abasic sites in cells expressing G199S leads to the induction of double-strand breaks (DSBs).

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Yale University School of Medicine, New Haven, Connecticut, United States of America.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus