Regulated Proteolysis of DNA Polymerase η during the DNA-Damage Response in C. elegans  Seung-Hwan Kim, W. Matthew Michael  Molecular Cell  Volume 32, Issue 6, Pages 757-766 (December 2008) DOI: 10.1016/j.molcel.2008.11.016 Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 1 GEI-17 Controls POLH-1 Protein Levels during the DNA-Damage Response (A) Early embryos were obtained by bleaching gravid N2 adults, and extracts were prepared as described (Kim et al., 2007). The samples were fractionated on SDS-PAGE and then probed, by immunoblotting, with antibodies against POLH-1. The lysates were also probed with antibodies against PCN-1, to ensure equal loading of the samples. Prior to extract preparation the animals were either treated, or not, with polh-1 RNAi by feeding, as indicated. (B) Early embryo extracts were prepared and then probed, by immunoblotting, with anti-POLH-1 antibodies, after SDS-PAGE. The lysates were also probed with anti-PCN-1 antibodies, to ensure equal loading of the samples, and with anti-GEI-17 antibodies, to monitor the effectiveness of the gei-17 RNAi. Prior to extract preparation, the animals were either treated, or not, with gei-17 RNAi and MMS, as indicated. MMS treatment was performed by inclusion of 0.005% MMS in the plate media and incubation of animals on these plates for 20 hr. We note that POLH-1 runs as doublet on this blot; this was not reproducibly observed, and we suspect that the lower band in the doublet represents a proteolytic fragment of POLH-1 that is occasionally produced during preparation of the extracts. (C) Early embryo extracts were prepared after optional gei-17 RNAi, and then probed, by immunoblotting, with antibodies against POLH-1, GEI-17, or PCN-1, after SDS-PAGE. Prior to extract preparation the animals were either exposed, or not, to 100 J/m2 of UV light, as indicated. The samples were harvested 5 hr after UV irradiation. (D) A POLH-1-GFP fusion protein was expressed in animals via stable transformation with a construct driving expression of POLH-1-GFP with the pie-1 promoter (pie-1::polh-1-GFP; see Experimental Procedures). These animals were treated with the indicated RNAi, and early embryos were examined for GFP signals by fluorescence microscopy. The samples were also stained with Hoechst 33258, to visualize the DNA. “GFP” indicates GFP-based fluorescence, “DNA” indicates Hoechst 33258-based fluorescence, and “merge” shows both signals together in the same image. The exposure times during image capture was identical for all of the samples shown. Where indicated, animals were treated with MMS, as in (B). Molecular Cell 2008 32, 757-766DOI: (10.1016/j.molcel.2008.11.016) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 2 POLH-1 Is Destroyed in a PIP Box- and CRL4-Cdt2-Dependent Manner in MMS-Exposed, GEI-17-Deficient Embryos (A) A PIP box deletion mutant (ΔPIP) was introduced into the pie-1::polh-1-GFP construct, and stable transformants were obtained via particle bombardment (see Experimental Procedures). Early embryo extracts were prepared, and the WT POLH-1-GFP or ΔPIP POLH-1-GFP proteins were probed by immunoblotting, after SDS-PAGE, with anti-GFP antibodies. The lysates were also probed with antibodies against PCN-1, to ensure equal loading of the samples, and with anti-GEI-17 antibodies, to monitor the effectiveness of the gei-17 RNAi. The animals were exposed, or not, to MMS, as in Figure 1B. (B) WT POLH-1-GFP worms were treated with gei-17/pcn-1 RNAi by soaking, as indicated, and then optionally exposed to MMS (as in Figure 1B). Early embryo extracts were prepared and the POLH-1-GFP protein was probed by immunoblotting, after SDS-PAGE, with anti-GFP antibodies. The lysates were also probed with antibodies against PCN-1 or GEI-17, to monitor the effectiveness of the gei-17/pcn-1 RNAi, as indicated. The intensity of the signals in the POLH-1-GFP blot were quantified using the histogram tool of Photoshop, and the values obtained were 52, 51, 49.5, and 51, respectively, for the samples present in lanes 1–4 of the blot. (C) Feeding RNAi, in various configurations, was performed on the WT POLH-1-GFP worm as indicated. The animals were optionally exposed to MMS (as in Figure 1B) and then embryo extracts were prepared. Extracts were fractionated on SDS-PAGE gels and then probed by immunoblotting for POLH-1-GFP (using anti-GFP antibodies) and GEI-17 and PCN-1, as indicated. (D) Early embryo extracts were prepared and POLH-1, GEI-17, or PCN-1 proteins were then detected by immunoblotting, after SDS-PAGE. Prior to extract preparation, the animals were either treated, or not, with gei-17 RNAi by feeding and MMS (as in Figure 1B), as indicated. Molecular Cell 2008 32, 757-766DOI: (10.1016/j.molcel.2008.11.016) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 3 GEI-17 Directly SUMOylates POLH-1 to Protect It from CRL4-Cdt2 (A) In vitro SUMOylation assays were performed as described previously (Holway et al., 2005; see also Experimental Procedures). POLH-1 was produced by transcription/translation in vitro using rabbit reticulocyte lysate and was radiolabeled by virtue of inclusion of [35S]methionine in the translation reaction. The reactions were then supplemented with recombinant forms of UBC-9- and SUMO-activating enzyme (abbreviated as “E1,” this is a heterodimer of the SAE1 and SAE2 proteins), SUMO (SUMO 1 and 3), or GEI-17, as indicated. Reactions were then fractionated on SDS-PAGE and visualized via phosphorimaging. (B) Early embryo extracts were immunoprecipitated with anti-POLH-1 antibodies. These immune complexes were fractionated on SDS-PAGE and then probed, by immunoblotting, with either anti-POLH-1 or anti-SUMO antibodies, as indicated. Prior to embryo extract preparation, the animals were either treated, or not, with MMS, as in Figure 1B. (C) Chromatin fractions from total embryo extracts were prepared as described (Kim et al., 2007), and blots of these fractions were probed by immunoblotting with anti-GEI-17 antibodies. The starting extract used to prepare the chromatin fractions (“total embryo extract”) was also examined. Prior to chromatin isolation, the animals were either treated, or not, with MMS, as in Figure 1B. The PCN-1 signal confirms that equivalent amounts of the chromatin fractions were loaded on the gel, and the antitubulin signal confirms that the chromatin fractions were not contaminated with cytoplasmic proteins. (D) In vitro SUMOylation assays were performed as in (A). For these experiments, all assays contained the full complement of components (i.e., recombinant E1, UBC-9, SUMO 1/3, and GEI-17). Two different substrates, wild-type POLH-1 and POLH-1 K85R/K260R double mutant, were included in the assays. (E) The K85R/K260R double mutant was introduced into the pie-1::polh-1-GFP construct, and transformants were obtained via particle bombardment (see Experimental Procedures). These animals, and pie-1::polh-1-GFP animals, were then treated, or not, with MMS as in Figure 1B, and early embryo extracts were prepared. The POLH-1-GFP derivatives were then detected by immunoblotting, after SDS-PAGE, with anti-GFP antibodies. The lysates were also probed with antibodies against PCN-1, to ensure equal loading of the samples. Molecular Cell 2008 32, 757-766DOI: (10.1016/j.molcel.2008.11.016) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 4 The Sole Purpose of GEI-17-Mediated SUMOylation of POLH-1 Is to Protect It from CRL4-Cdt2 Animals were treated with the indicated RNAi and were optionally exposed to MMS, as in Figure 1B. Adults were then bleached and the embryos were collected. One thousand embryos for each sample were then cultured in media containing [3H]thymidine. Thirty minutes later samples were collected, lysates were prepared, and tritium incorporation was measured as described (Hartman et al., 1991). Samples were taken again at 60, 120, and 240 min, and tritium incorporation was again assessed. The amount of tritium incorporated by the wild-type, undamaged sample at the 240 min time point was set to 100, and all other values were adjusted accordingly. Molecular Cell 2008 32, 757-766DOI: (10.1016/j.molcel.2008.11.016) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 5 DNA Damage Triggers CRL4-Cdt2-Mediated POLH-1 Destruction in Wild-Type Embryos (A) Early embryo extracts were prepared and the POLH-1 protein was detected by immunoblotting, after SDS-PAGE, with anti-POLH-1 antibodies. The lysates were also probed with antibodies against PCN-1, to ensure equal loading of the samples. Prior to extract preparation, the animals were optionally depleted of cdt-2 by RNAi, and either exposed, or not, to 0.005%, 0.02%, and 0.025% MMS. Embryonic lethality was also determined for the different concentrations of MMS, and is displayed under the blot. (B) Animals that were pretreated with the indicated RNAi were exposed to 750 J/m2 of UV light. At the indicated time early embryos were collected, extracts were prepared, and POLH-1 and PCN-1 proteins were detected by western blotting. Embryonic lethality (emb) under these conditions was also determined, and is displayed to the left of the blots. Molecular Cell 2008 32, 757-766DOI: (10.1016/j.molcel.2008.11.016) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 6 A Model for GEI-17/CRL4-Cdt2-Mediated Regulation of POLH-1 during the DNA-Damage Response (A) When GEI-17 is inactivated (GEI-17−), then POLH-1 is destroyed by CRL4-Cdt2 (CDT-2 in the figure) before it can perform its function in TLS. The model proposes that a single lesion (red square) can catalyze multiple rounds of POLH-1 destruction, as designated by the blue arrow. (B) The model proposes that, in the wild-type condition, GEI-17 SUMOylates POLH-1, and this prevents CRL4-Cdt2-mediated destruction until after POLH-1 has performed its role in TLS. Molecular Cell 2008 32, 757-766DOI: (10.1016/j.molcel.2008.11.016) Copyright © 2008 Elsevier Inc. Terms and Conditions