Figure 1.
Chromosomal aberrations in P242R-expressing cells.
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.
Figure 2.
Accumulation of BER intermediates in MCF10A cells expressing P242R Pol β.
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 p<0.01 and 0.001, respectively. ∧ denotes p<0.05 comparing 0 vs 2 h recovery within each cell line. ∧∧∧ denotes p<0.001 comparing 30 or 60 min recovery to 0 recovery. D. *** denotes p<0.001 comparing WT+MMS to P242R+MMS in each phase of the cell cycle. ∧ and ∧∧∧ denote p<0.05 and 0.001, respectively, comparing WT+MMS+recovery to P242R+MMS+recovery in each phase of the cell cycle.
Figure 3.
Expression of P242R induces cellular transformation.
A. Representative image of crystal violet stained untransformed cells. Note the clear monolayer of cell growth. B. Representative image of a focus. Note the accumulation of cells as marked by dark staining. C–D. Focus formation assay with two clonal cell lines either inducing (NT, solid line) or not inducing (T, dashed line) exogenous (C.) WT or (D.) P242R Pol β. Mean foci per 105 cells plated (± standard deviation of plating replicates) plotted against passage number. E. Anchorage independent growth assay with P242R clonal cell lines from the focus formation assay. Cells per field (± standard error) are plotted on the Y-axis. F. Anchorage independent growth assay with MCF10A pools expressing WT or P242R. The number of colonies per field are plotted on the Y-axis. G. WT or P242R MCF10A pools were plated in 5 dishes at a density of 25,000 or 50,000 cells per dish. Cells were trypsinized and counted each day. Data are plotted as the mean ± SEM of the change in cell number (n = 3).
Figure 4.
P242R Pol β confers slight sensitivity to MMS compared to WT.
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).
Figure 5.
The P242R germline variant of Pol β is slow and binds DNA tightly.
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. Results for the different proteins are shown as in A.