Purification of an E3 ubiquitin ligase for Pol β and identification as Mule. In vitro ubiquitylation of Pol β (5 pmol) by (A) CHIP (15 pmol) or (B) active fraction purified from HeLa whole cell extracts in the presence of E1 (0.7 pmol), ubiquitin (0.6 nmol) and various E2 enzymes (9.5 pmol) analysed by 10% SDS–PAGE and immunoblotting using Pol β antibodies. (C, E) In vitro ubiquitylation of Pol β (5 pmol) by active fractions purified from HeLa whole cell extracts in the presence of E1 (0.7 pmol), H7 (9.5 pmol) and either ubiquitin (lanes 1–4, 0.6 nmol) or mutant ubiquitin (lane 5, 0.6 nmol) unable to form polyubiquitin chains. Samples were analysed by 10% SDS–PAGE and immunoblotting using Pol β antibodies. (D) Peptide sequences detected by nanoLC-MS/MS from the final chromatography fractions (C2 and C3) that correspond to the 482 kDa Mule protein (SwissProt Nr. Q7Z6Z7, Mascot Score: 192). (F) Analysis of final chromatography fractions purified from HeLa whole cell extracts by 10% SDS–PAGE and immunoblotting using Mule antibodies showing correlation with in vitro ubiquitylation activity. In vitro ubiquitylation of Pol β (5 pmol) by truncated Mule (3.5 pmol) in the presence of E1 (0.7 pmol), ubiquitin (0.6 nmol) and (G) H7 (9.5 pmol) or (H) various E2 enzymes (9.5 pmol) analysed by 10% SDS–PAGE and immunoblotting using Pol β antibodies. Molecular weight markers are indicated on the side of appropriate figures and the positions of ubiquitylated Pol β (Pol β_ub) are shown.

Identification of Mule ubiquitylation sites within Pol β. (A) Schematic diagram of the protein structure of Pol β showing the major sites (K41/K61/K81) of ubiquitylation by Mule that are present within the 8 kDa domain. In vitro ubiquitylation of (B) 8 kDa and (C) 31 kDa Pol β domains by active fraction purified from HeLa whole cell extracts in the presence of E1 (0.7 pmol), H7 (9.5 pmol) and ubiquitin (0.6 nmol) analysed by 10% SDS–PAGE and immunoblotting using Pol β antibodies. (D) In vitro ubiquitylation of 8 kDa Pol β domain by active fraction purified from HeLa whole cell extracts in the presence of E1 (0.7 pmol), H7 (9.5 pmol) and ubiquitin (0.6 nmol) analysed by Coomassie staining. The monoubiquitylated 8 kDa Pol β band was identified (8 kDa_ub; see arrow), excised and analysed by nanoLC-MS/MS to identify the sites of ubiquitylation (Supplementary Figure S2A–C). In vitro ubiquitylation of wild-type Pol β (WT) and various Pol β mutants using (E) active fraction purified from HeLa whole cell extracts or (F) purified truncated Mule (3.5 pmol) in the presence of E1 (0.7 pmol), H7 (9.5 pmol) and ubiquitin (0.6 nmol) analysed by 10% SDS–PAGE and immunoblotting using Pol β antibodies. Molecular weight markers are indicated on the left hand side of appropriate figures and the positions of ubiquitylated Pol β (Pol β_ub) and ubiquitylated 8 kDa domain Pol β (8 kDa_ub) are shown. (G) HeLa cells were transfected with mammalian vectors expressing wild type or K41/K61/K81 mutant FLAG-tagged-Pol β for 24 h, whole cell extracts were prepared and analysed by 10% SDS–PAGE and immunoblotting with FLAG or PCNA antibodies. The relative Pol β value is normalized to the amount of PCNA (average of three experiments).

Identification of monoubiquitylated Pol β in human cell extracts and dependence on Mule and ARF. (A) Whole cell extracts were prepared from HeLa cells in the presence and absence of 1 mM NEM and analysed by 10% SDS–PAGE and immunoblotting with Pol β and PCNA antibodies. (B, E) HeLa cells were grown in 6 cm² dishes for 24 h to 30–50% confluency and then treated with Lipofectamine transfection reagent (10 μl) in the absence and presence of (B) Pol β or (E) Mule and ARF siRNA (200 pmol) for a further 72 h. Cells were pelleted by centrifugation, cytoplasmic (C) and nuclear (N) fractions were prepared and 40 μg protein in the C fraction and an equal volume of the N fraction were analysed by 10 % SDS–PAGE and immunoblotting with the antibodies indicated. (C) The ∼47 kDa protein suspected to be ubiquitylated Pol β was partially purified from calf thymus by phosphocellulose and Mono Q chromatography and fractions crossreacting with Pol β antibodies were also probed with ubiquitin antibodies. (D) HeLa cells were transfected with a mammalian vector (1 μg) expressing FLAG-tagged-Pol β in the presence or absence of a mammalian vector expressing His-tagged-ubiquitin (1 μg) for 24 h, whole cell extracts were prepared, ubiquitylated proteins were precipitated with Ni-agarose beads and analysed by western blotting using FLAG antibodies. Molecular weight markers are indicated on the side of appropriate figures and the positions of ubiquitylated Pol β (Pol β_ub) are shown. NS corresponds to a non-specific protein recognized by the Pol β antibodies.

Modulation of BER by Mule and ARF. (A) HeLa cells were grown in 6 cm² dishes for 24 h to 30–50% confluency and then treated with Lipofectamine transfection reagent (10 μl) in the absence and presence of Mule siRNA (200 pmol) for a further 72 h. Cells were pelleted by centrifugation, cytoplasmic (C) and nuclear (N) fractions were prepared and 40 μg protein in the C fraction and an equal volume of the N fraction were analysed by 10% SDS–PAGE and immunoblotting with the antibodies indicated. HeLa and WI-38 cells were grown in 6 cm² dishes for 24 h to 30–50% confluency and then treated with Lipofectamine (10 μl) in the absence and presence of Mule (B) or ARF (C) siRNA (200 pmol) for a further 72 h. Whole cell extracts were prepared and analysed by 10% SDS–PAGE and immunoblotting with the antibodies indicated. Alternatively, after 72 h with Lipofectamine or (D) Mule or (E) ARF siRNA the cells were treated with 20 μM hydrogen peroxide for 5 min, allowed to repair for 0–120 min and the levels of single strand breaks and alkali labile sites then analysed by the alkaline single cell gel electrophoresis (Comet) assay. Shown are the mean % tail DNA values with standard deviations from at least three independent experiments. Statistically significant results comparing Lipofectamine and siRNA-treated cells are represented by ^*P<0.02, ^**P<0.005 and ^***P<0.001, as analysed by Student's t-test.

Modulation of BER by Mule depends on Pol β. Isogenic Pol β-proficient (Pol β^+/+) and Pol β-deficient (Pol β^−/−) cells were grown in 6 cm² dishes for 24 h to 30–50% confluency and then treated with Lipofectamine transfection reagent (10 μl) in the absence and presence of Mule siRNA (200 pmol) for a further 48 h. (A) Whole cell extracts were prepared and analysed by 10% SDS–PAGE and immunoblotting with the antibodies indicated. (B) Alternatively, cells were treated with 30 μM hydrogen peroxide for 5 min, allowed to repair for 0–120 min and the levels of single strand breaks and alkali labile sites then analysed by the alkaline single cell gel electrophoresis (Comet) assay. Shown are the mean % tail DNA values with standard deviations from at least three independent experiments. Statistically significant results comparing Lipofectamine and siRNA-treated cells are represented by ^***P<0.001, as analysed by Student's t-test.

Ubiquitylation of Pol β by Mule stimulates CHIP-dependent ubiquitylation and degradation. (A) In vitro ubiquitylation of Pol β (5 pmol) by truncated Mule (3.5 pmol) and/or CHIP (15 pmol) in the presence of E1 (0.7 pmol), ubiquitin (0.6 nmol) and H5c (9.5 pmol) analysed by 10% SDS–PAGE and immunoblotting using Pol β antibodies. (B) HeLa cells were grown in 6 cm² dishes for 24 h to 30–50% confluency, treated with Lipofectamine transfection reagent (10 μl) in the absence and presence of CHIP siRNA (200 pmol) for a further 48 and 72 h and then whole cell extracts prepared and analysed by immunoblotting with Pol β antibodies to identify Pol β_ub. (C) HeLa cells were grown in 6 cm² dishes for 24 h to 30–50% confluency and then treated with Lipofectamine transfection reagent (10 μl) in the presence and absence of Mule siRNA (200 pmol), CHIP siRNA (200 pmol) or both for 72 h, whole cell extracts were prepared and analysed by western blotting using Pol β or tubulin antibodies. Relative Pol β values are normalized to the amount of tubulin. (D) HeLa cells were grown in 6 cm² dishes for 24 h to 90–95% confluence, treated with Lipofectamine transfection reagent (10 μl) in the absence and presence of CHIP expressing plasmid (1.2 pmol) for a further 24 h. Whole cell extracts were then prepared and analysed by immunoblotting with Pol β or FLAG antibodies. LC corresponds to a loading control.

Proposed model for the regulation of Pol β steady-state levels by Mule, ARF and CHIP. If not required for DNA repair, Pol β is ubiquitylated by Mule that is then a target for CHIP mediated polyubiquitylation and subsequent degradation by the proteasome (left side of scheme). However, after detection of DNA damage ARF is released from the nucleoli into the cytoplasm in which it inhibits the activity of Mule, thus reducing Pol β degradation and upregulating DNA repair (right side of scheme). The repair of DNA damage will result in a decreased release of ARF, with a concomitant increased activity of Mule that will downregulate Pol β levels. A new adjustment cycle will therefore begin on the detection of increased levels of DNA damage.