Figure 4: The C-terminal domain of DNA polymerase β is targeted for proteasome-mediated degradation by ubiquitylation on K206/K244. | Nature Communications

Figure 4: The C-terminal domain of DNA polymerase β is targeted for proteasome-mediated degradation by ubiquitylation on K206/K244.

From: HSP90 regulates DNA repair via the interaction between XRCC1 and DNA polymerase β

Figure 4

(a) Immunoblot of WCLs from stable LN428 cell lines expressing Flag-Polβ(TM) after expression of HA-ubiquitin. Flag-Polβ(TM) was isolated by IP and probed for the HA-ubiquitin modification by immunoblot with the HA-Ab. The level of Flag-Polβ(TM) in the WCL and the input level of Flag-Polβ(TM) is detected using the M2 Flag-Ab. (b) The relative level of Flag-Polβ in WCLs from stable LN428 cell lines expressing Flag-Polβ(WT), Flag-Polβ(TM) and the indicated K206A, K244A or K206A/K244A mutants. The predicted ubiquitylation sites K206 and K244 reverse the instability of Flag-Polβ(TM), as determined by immunoblot analysis, as in Supplementary Fig. 4D. The result indicates mean±s.d. of two independent experiments. (c) Immunoblot of WCLs from stable LN428 cell lines expressing Flag-Polβ(TM) or Flag-Polβ(TM/K206A/K244A) after exposure to Cyclo for the times indicated. The immunoblot image depicts the levels of Flag-Polβ, XRCC1 and PCNA, before and after treatment with Cyclo, showing the rapid degradation of Flag-Polβ(TM) and the stability of Flag-Polβ(TM/K206A/K244A). A representative immunoblot image is shown. For quantified results, see Supplementary Fig. 4E. (d) Immunoblot of WCLs from stable LN428 cell lines expressing Flag-Polβ(TM) or Flag-Polβ(TM/K206A/K244A) after expression of HA-ubiquitin. Flag-Polβ(TM) or Flag-Polβ(TM/K206A/K244A) was isolated by IP and probed for the HA-ubiquitin modification by immunoblot with the HA-Ab, showing the ubiquitylation of Flag-Polβ(TM) but not Flag-Polβ(TM/K206A/K244A). The level of Flag-Polβ(TM) or Flag-Polβ(TM/K206A/K244A) in the WCLs and the input level of Flag-Polβ(TM) or Flag-Polβ(TM/K206A/K244A) are detected using the M2 Flag-Ab.

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