Fig 2: Human Rad17 R53–Q63 and R37–S47 residues encode the destruction box.A, amino acid sequences in Rad17 R53–Q63 and R37–S47 showed similarity to the canonical destruction box. The putative destruction boxes from Rad17 proteins were aligned with the destruction boxes known in mammalian proteins. White letters on black background indicate the consensus residues, which are R55/L58 and K39/P42 in human Rad17 (isoform 1). Letters in frames also indicate the conserved residues. B, Rad17 R55A/L58A and K39A/P42A mutants showed increases in protein amount. The same experiments as in Figure 3. COS-1 cells were transfected with flag-Rad17 R55A/L58A, K39A/P42A, and K39A/P42A/R55A/L58A (KPRL/4A) mutants, and SDS-lysate was prepared. The signal ratio of anti-FLAG and NPT2 blots is shown in a graph. The graph represents the result from three independent experiments. The p-values were calculated using Welch’s t test. C–E, Rad17 has two putative destruction boxes on the N-terminus. Recombinant proteins of GST-Cdh1 and flag-Rad17 M1–D82 peptides were separately expressed in bacteria. The lysates were mixed and incubated. The flag-Rad17 M1–D82 peptide was precipitated, and coprecipitation of GST-Cdh1 was examined with anti-GST blot. Mutation in K39A/P42A/R55A/L58A (C), but not R55A/L58A (D) or K39A/P42A (E), abolished the interaction. The representative results from more than two independent experiments are shown. F, the Rad17 S35–G66 peptide was fused with flag tag, EGFP, and the E295–D380 peptide and was expressed in COS-1 cells (flag-S35–G66-EGFP-E295–D380). Forty-eight hours after transfection, the SDS lysate was prepared and probed with the indicated antibodies. The graph shows the signal ratios of anti-FLAG and NPT2 blots in three independent experiments. The p-values were calculated using the paired t test. The S35–G66 wild-type was used as 100% standard. E295–D380 only, flag-EGFP-E295–D380. S35–G66 wild-type, flag-S35–G66-EGFP-E295–D380 with the wild-type destruction boxes. S35–G66 KPRL/4A, flag-S35–G66-EGFP-E295–D380 with the mutated destruction boxes. n indicates the number of samples. **p < 0.01. ***p < 0.001. At, Arabidopsis thaliana; Dr, Danio rerio; Gg, Gallus gallus; Hs, Homo sapiens; Mm, Mus musculus; Xl, Xenopus laevis.

Fig 3: A proposed model. A schematic model of nuclear translocation and proteasomal degradation of Rad17. In the cytoplasm, Rad17 changes its conformation to associate with the 9-1-1 complex upon ATP binding and translocates into the nucleus. In the nucleus, Rad17 is degraded through interaction between Cdh1 and the N-terminal destruction boxes. Alternatively, Rad17 releases ATP/ADP and is exported from the nucleus.

Fig 4: Rad17?230–270and R240A/L243A mutants have a structural abnormality in the ATPase domain.A and B, Rad17 ?230–270 and R240A/L243A mutants were deficient, and K/R359–363A and 3SD mutants were proficient in the interaction with the 9-1-1 complex. flag-Rad17 was expressed in COS-1 cells, and a low-salt extract was prepared. flag-Rad17 was precipitated, and the coprecipitation of endogenous Rad1 was examined. The signal intensity of endogenous Rad1 was normalized to that of flag-Rad17 and is shown in graphs. The graphs represent results from two independent experiments. n indicates the number of samples. Rad17 ?230–270 and R240A/L243A mutants did not coprecipitate endogenous Rad1. Rad17 K132E was used as negative control. C, structural modeling of ATPase domain of human Rad17. Structure of the ATPase domain of Rad17 was modeled based on the homology with RFC1. The drawing represents a snapshot structure of the R240 and L243 residues at the last of the 100 ns MD simulation. The numbering of a-helices and ß-strands was assigned according to reference (25).

Fig 5: ATR-dependent RAD17 phosphorylation at S635 enhances SMG7 interaction. (a) The FH-SMG7-H1299 cells were treated for 4 h with 10 µM ATM (KU-55933) or ATR (VE-822) inhibitor. The total cell lysates and a-Flag immunoprecipitates were analyzed by western blot using a-SMG7, a-RAD17 and a-RAD17-pS635 antibodies. Relative levels of immunoprecipitated RAD17 are quantitated in Fig. S3a. (b) Cells treated with or without ATR inhibitor (VE-822, 10 µM for 4 h) were exposed to 10 Gy IR and harvested 1 h later. The total cell lysates and a-Flag immunoprecipitates were analyzed by western blot using a-SMG7, a-RAD17 and a-RAD17-pS635 antibodies as in (a). Relative levels of immunoprecipitated RAD17 and RAD17-pS635 are quantitated in Fig. S3b. (c) GST and GST-SMG7 fragment fusion proteins were incubated with lysates from 293FT cells transiently expressing HA-RAD17, and RAD17 pulled down by the GST proteins was analyzed by western blot with a-RAD17 antibody. (d) Alignment of the amino acid sequences adjacent to the serine residues within the SQ motif of RAD17, UPF1 and p53 (top). The FH-RAD17 and FH-RAD17-S635A proteins purified from transfected 293FT cells were analyzed by western blot using a-RAD17-pS635 and a-RAD17 antibodies (bottom). (e) GST or GST-SMG7 fragments were incubated with purified FH-RAD17 or FH-RAD17-S635A protein from (d) in GST pull-down assays. RAD17 and RAD17-pS635 were detected by western blotting with the antibodies indicated. Relative levels of RAD17 binding and RAD17-pS635 binding are quantitated in Fig. S3f. (f) H1299 cells were co-transfected with plasmids expressing FH-SMG7 and HA-RAD17 WT, S635A, S645A or S635/645A, and the total cell extracts and a-Flag immunoprecipitated materials were examined by western blot analysis using a-SMG7, a-RAD17 and a-RAD17-pS635 antibodies. Relative levels of RAD17 and RAD17-pS635 binding to FH-SMG7 are quantitated in Fig. S3g.