A Germline Polymorphism of DNA Polymerase Beta Induces Genomic Instability and Cellular Transformation

<div><p>Several germline single nucleotide polymorphisms (SNPs) have been identified in the <em>POLB</em> gene, but little is known about their cellular and biochemical impact. DNA Polymerase β (Pol β), encoded by the <em>POLB</em> gene, is the main gap-filling polymerase involved in base excision repair (BER), a pathway that protects the genome from the consequences of oxidative DNA damage. In this study we tested the hypothesis that expression of the <em>POLB</em> germline coding SNP (rs3136797) in mammalian cells could induce a cancerous phenotype. Expression of this SNP in both human and mouse cells induced double-strand breaks, chromosomal aberrations, and cellular transformation. Following treatment with an alkylating agent, cells expressing this coding SNP accumulated BER intermediate substrates, including single-strand and double-strand breaks. The rs3136797 SNP encodes the P242R variant Pol β protein and biochemical analysis showed that P242R protein had a slower catalytic rate than WT, although P242R binds DNA similarly to WT. Our results suggest that people who carry the rs3136797 germline SNP may be at an increased risk for cancer susceptibility.</p> </div

Chromosomal aberrations in P242R-expressing cells.

<p>Representative metaphase spread of MCF10A expressing (A.) WT or (B.) P242R Pol β. Chromosomal fusions are shown with the gray arrow and fragments are shown with black arrows. C. Number of aberrations per metaphase. A total of at least 50 metaphases were scored for each cell line.</p

Accumulation of BER intermediates in MCF10A cells expressing P242R Pol β.

<p>A. MCF10A pools expressing WT or P242R Pol β were treated with 2 mM MMS for 30 minutes and allowed to recover for 0, 30, or 60 min and single-strand breaks (SSBs) were analyzed by comet assay. The percentage of tail DNA is plotted on the Y-axis. B. MCF10A pools expressing WT or P242R Pol β were treated with 2 mM MMS for 30 min and allowed to recover for 0, 30, or 60 min, stained with γH2AX antibody, and analyzed by flow cytometry. Cells were treated for 120 min as a positive control. C–D. MCF10A pools expressing WT or P242R Pol β were treated with 2 mM MMS for 2 h and allowed to recover for 0 or 2 hours. Cells were stained with γH2AX antibody and propidium iodide to assess the levels of double-strand breaks (DSBs) and the cell cycle phase, respectively, and analyzed by flow cytometry. Data are plotted as the mean ± SEM. Data are plotted as the mean ± SEM (n = 3). A and C. ** and *** denote <i>p</i><0.01 and 0.001, respectively. ∧ denotes <i>p</i><0.05 comparing 0 vs 2 h recovery within each cell line. ∧∧∧ denotes <i>p</i><0.001 comparing 30 or 60 min recovery to 0 recovery. D. *** denotes <i>p</i><0.001 comparing WT+MMS to P242R+MMS in each phase of the cell cycle. ∧ and ∧∧∧ denote <i>p</i><0.05 and 0.001, respectively, comparing WT+MMS+recovery to P242R+MMS+recovery in each phase of the cell cycle.</p

The P242R germline variant of Pol β is slow and binds DNA tightly.

<p>A. Representative results from a presteady-state burst assay. Results for the WT are shown as filled squares fit with a solid curve. Results for the P242R are shown as open triangles fit with a dashed curve. The assay was repeated four times for each protein. B. Representative results from a gel electrophoretic mobility shift assay. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003052#s2" target="_blank">Results</a> for the different proteins are shown as in A.</p

P242R Pol β confers slight sensitivity to MMS compared to WT.

<p>Clonogenic survival assays were conducted with (A.) Pol β^−/− MEFs, (B.) Pol β^+/+ MEFs, or (C.) MCF10A pools expressing WT or P242R Pol β. Filled circles represent results from pools expressing empty vector, filled squares represent pools expressing WT Pol β, and filled triangles represent pools expressing P242R Pol β. Data are plotted as the mean ± SEM (n = 3).</p

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

Expression of G199S induces cellular transformation.

<p>A. Representative image of anchorage-independent growth of MCF10A pools expressing WT TDG at passage 13 at 20× magnification. B. Representative image of anchorage-independent growth of MCF10A pools expressing G199S at passage 13 at 20× magnification. Note several large colonies have formed. C. The average number of colonies per field are plotted on the Y-axis. Cells were scored at passages 5, 9, 13 and 17 after 5 weeks of growth in soft agar. ** and *** denote p<0.01 and p<0.001, respectively.</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