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. 2014 Mar 25;289(20):13708–13716. doi: 10.1074/jbc.M114.550400

FIGURE 5.

FIGURE 5.

S229L has impaired BER activity and induces the accumulation of BER intermediates. A, the LPSD DNA substrate was treated with UDG and incubated with Pol β−/− extract containing purified WT or S229L protein. B, quantification of n+1 product is shown as a function of total DNA. Data are presented as mean ± S.E. (error bars; n = 3). C, clonogenic survival assays were conducted with Pol β−/− MEFs expressing either WT or S229L Pol β or empty vector. Data are plotted as mean ± S.E. (n = 3–4). D, Western blotting was performed of Pol β in cell lines. Representative Western blots show expression of S229L Pol β in Pol β+/+ MEFs and Pol β−/− MEFs. Endogenous Pol β was used as a loading control in Pol β+/+ MEFs. Tubulin was used as a loading control in the Pol β−/− MEFs. The ratios of exogenously expressed Pol β protein to loading control are shown below the images of the blots. E, Pol β+/+ MEFs were left untreated or treated with 2 mm MMS for 30 min. Following MMS exposure, cells were allowed to recover for 0 or 30 min. Single-strand breaks were quantified by the alkaline comet assay. The percentage of tail DNA is plotted on the y axis. F, Pol β+/+ MEFs were left untreated or treated with 2 mm MMS for 2 h. Following MMS exposure, cells were allowed to recover for 0 or 2 h, stained with γH2AX antibody, and analyzed by flow cytometry to assess the levels of double-strand breaks. *, **, and *** denote p < 0.05, 0.01, and 0.001, respectively, comparing S229L versus WT within the same treatment group. ∧∧ and ∧∧∧ denote p < 0.01 and 0.001, respectively, comparing the treatment group versus untreated cells.