Journal: Nucleic Acids Research

Article Title: Stability and sub-cellular localization of DNA polymerase β is regulated by interactions with NQO1 and XRCC1 in response to oxidative stress

doi: 10.1093/nar/gkz293

Figure Lengend Snippet: The T304I cancer mutation of Polβ disrupts the Polβ/XRCC1 interaction and induces the ubiquitylation and degradation of Polβ. ( A ) Polβ(T304I) location and the structure of Polβ interacting with XRCC1 shown in the reduced (left) and oxidized (right) form. Left panel: Cyan: 2FMS; Gray: XRCC1 in 3K75 (Polβ bound to reduced XRCC1); Wheat: Polβ in 3K75 (Polβ bound to reduced XRCC1). Right panel: Cyan: 2FMS; Gray: XRCC1 in 3lqc(Polβ: oxidized XRCC1); Slate: Polβ in 3K75(Polβ: oxidized XRCC1); Orange spheres: Water molecules. ( B ) Polβ(T304I) mutation disrupts the Polβ/XRCC1 interaction in HCT116 cells. Top panel: Flag (M2) immunoprecipitation (IP) followed by an XRCC1 immunoblot (IB) shows that Polβ(T304I) immunoprecipitates less XRCC1 than does Polβ(WT). Bottom panel: XRCC1-IP followed by a Flag (M2) IB shows that XRCC1 immunoprecipitates less Polβ(T304I) than Polβ(WT). The level of PARP1 binding to XRCC1/Polβ complex was also examined. The blots shown are an analysis of lysates from HCT116 cells (10 μl of IP eluates was loaded per lane). ( C ) Analysis of the interaction between Polβ and XRCC1 by OpenSPR shows that the Polβ(T304I) mutation (right) decreases the binding affinity (2.38–12.48 fold; mean = 6.18) to XRCC1 as compared to Polβ(WT) (left). The complete set of raw data (sensograms) and the repeat experimental datasets are shown in . ( D ) Polβ(T304I) mutation induces the degradation of Polβ. Top panel: a representative immunoblot image of cycloheximide (Cyc) treatment resulting in the enhanced degradation of Polβ(T304I) in HCT116 cells (left panel). Treatment with the proteasome inhibitor MG132 stabilizes the level of the Polβ(T304I) protein (right panel). The immunoblot from two independent experiments are shown in . Bottom panels: The relative level of Polβ and of PCNA was determined by densitometry and was quantified using Image Lab (Bio-Rad) and the ratio of band densitometry of Polβ/PCNA is shown. The ratios for each cell line at time 0 were normalized to 1 (25 μg of WCL was loaded per lane). Plots show the mean ± SD of three independent experiments. The relative level of Polβ(WT) was compared to Polβ(T304I) in HCT116 cells treated with Cyc ( P < 0.001) or Cyc+MG132; ( P > 0.05), as determined by regular two-way ANOVA. ( E ) Polβ(T304I) mutation promotes enhanced ubiquitylation of Polβ in HCT116 cells. HCT116 cells expressing EGFP, Flag-Polβ(WT) or Flag-Polβ(T304I) were transiently transfected with pcDNA-HA-ubiquitin and the Flag-tagged proteins were immunoprecipitates with Flag-M2 agarose. The ubiquitylated form of Polβ was examined by immunoblot using an HA antibody, as shown. The relative level of ubiquitylated Polβ (WT or T304I) was determined by densitometry and was quantified using ImageJ and the relative level of ubiquitylated Polβ was calculated by determining the ratio of ubiquitylated Polβ/loaded Polβ, as shown in the plot to the right (10 μl of IP eluates was loaded into each lane). The immunoblot from three additional independent experiments are shown in ; P < 0.05, unpaired t -test was used for the statistical analysis.

Article Snippet: Binding experiments were carried out in an OpenSPR localized surface plasmon resonance (LSPR) biosensor (Nicoya Lifesciences).

Techniques: Mutagenesis, Immunoprecipitation, Western Blot, Binding Assay, Expressing, Transfection, Ubiquitin Proteomics