Overall structure of the complex of human TDG with DNA containing a G:5hmU mismatch (PDBID code 4FNC; [9]) and close-up of the resulting abasic site (AP) everted from the double helix.

<p>TDG (yellow) is shown as a cartoon representation and the DNA as a stick model. As shown in the magnified part of the active site, Gly199 is located in a loop that makes a close approach to the everted region of the DNA.</p

Expression of G199S delays S phase progression and activates a DNA damage response.

<p>A. MCF10A cells expressing WT or G199S were synchronized and treated with 10 µM 5-FU for 24 hrs and allowed to recover for 0 or 12 hrs. Cells were pulsed with BrdU before collection, fixed and stained with propidium iodide to determine the cell cycle phase and analyzed by flow cytometry. Data are graphed as mean ± SEM. B. MCF10A cells expressing WT or G199S were treated with or without 10 µM 5-FU for 24 hrs and allowed to recover for up to 24 hrs in drug-free media. Quantification of pChk1 induction is found below. Chk1 (middle panel) was used to normalize the amounts of protein in the extracts and β-actin (lowest panel) was used as a loading control. C. MCF10A cells expressing WT or G199S were treated with in the presence or absence of the ATR inhibitor VE-821 for 2 hrs prior to the addition of 10 µM 5-FU for 24 hrs and allowed to recover for up to 24 hrs. pChk1 status was analyzed by flow cytometry. Data are graphed as mean ± SEM.</p

G199S has similar catalytic activity to WT TDG.

<p>Equal concentrations of WT (<i>filled diamonds</i>) or G199S (<i>closed circles</i>) protein (75 nM) were incubated with 5 nM of ³²P-labeled oligonucleotides (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004753#pgen-1004753-t001" target="_blank">Table 1</a>) containing a G:T or A:5-FU mispair for up to 20 mins at 37°C. Reactions were terminated with NaOH, heated and mixed with formamide loading dye. A and B. Representative data with the G:T substrate. A. The first lane is a no protein control while the following lanes have protein with increasing incubation time up to 20 mins. S represents substrate; P represents product. B. Data were plotted as product formed <i>versus</i> time and fit to a single exponential equation. C. Catalytic rates (<i>k_st</i>) for WT and G199S on G:T and A:5-FU substrates.</p

G199S has a slightly higher affinity for the 5-FU:A lesion and binds significantly more tightly to its abasic product.

<p>Increasing concentrations (0–5400 nM) of WT TDG (<i>filled diamonds</i>) or G199S (<i>open circles</i>) protein were incubated for 30 mins at room temperature with ³²P-labeled oligonucleotides carrying a tetrahydrofuran moiety (THF) or 5-FU opposite template A (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004753#pgen-1004753-t001" target="_blank">Table 1</a>). Data were plotted as fraction of DNA bound <i>versus</i> TDG protein concentration to obtain the K_D. A and B. The representative binding curve of WT TDG and G199S on 5-FU:A and THF:A, respectively. C. The dissociation constants (K_D) for WT TDG and G199S on 5-FU:A and THF:A substrates.</p

DNA substrates.

<p>DNA substrates.</p

Expression of G199S leads to induction of chromosomal aberrations.

<p>Representative image of metaphase spreads of MCF10A pools expressing (A) WT or (B) G199S. Chromosomal breaks are noted with arrows and fusions are noted with *. C. Graph illustrating the average number of aberrations per metaphase. A total of at least 50 metaphases were scored for each cell line. Data are graphed as mean ± SD. ** and * denote p<0.01 and P<0.05, respectively.</p