A RRM1 H2AX DAPI. RRM1 H2AX DAPI Merge. Cont. sirna RRM1

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1 A H2AX DAPI H2AX DAPI Merge Cont sirna Figure S1: Accumulation of RRM1 at DNA damage sites (A) HeLa cells were subjected to in situ detergent extraction without IR irradiation, and immunostained with the indicated antibodies. Scale bars, 5 μm. (B) HeLa cells were treated with control (Cont) or RRM1 (RRM1) sirnas and were exposed to IR at 1 Gy,. Cells were then subjected to in situ detergent extraction 5 min after IR irradiation and immunostained as in (A). Scale bars, 5 μm.

2 A H2AX C H2AX E H2AX Cont psiren RRM2 RRM1/RRM2 H2AX sirna: D Cont RRM2 H2AX psiren Tip60.1 psiren Tip60.2 sirna: Figure S2: Accumulation of RNR at DNA damage sites (A) GM02063 cells were treated with UVA microirradiation as in Fig. 1B and stained with the indicated antibodies without cell extraction by detergent. Arrows represent accumulated RRM1 along with microirradiation lines. (B) GM02063 cells were treated with UVA microirradiation in the absence of BrdU and stained with the indicated antibodies after cell extraction. (C) GM02063 cells were transfected with either control (Cont) or RRM1 (RRM1) sirnas, then treated with microirradiation and stained as in (B). (D) GM02063 cells were transfected with either control (Cont) or RRM1 (RRM1) sirnas, then treated with microirradiation and stained as in (B). (E) GM02063 cells were transfected with either control vector (psiren) or those expressing specific shrnas for Tip60 (psiren-tip60.1 and psiren-tip60.2), then treated with microirradiation and stained as in (B). Scale bars, 10 μm.

3 1/10 Input 1/10 Input A RFL 201-C 601-C 741-C 761-C 781-C N-60 N-20 RC1( ) RM1( ) TFL TN1 TN2 TM1 TM2 TC2 TC1 ATP-cone CHROMO Tip6 Binding CXXC Tip6 RRM Binding + + D C IP: -myc NLS-RC1-HA NLS-GFP-HA Tip6 WT 761-C 781-C S C S C S C GST-Tip6 Figure S3: Tip60 binds to the C-terminus region of To identify the minimal binding regions of RRM1 and Tip60, two-hybrid interaction assays were performed. The constructs used are schematically represented. (A) RRM1; RFL(1-792), 201-C, 601-C, 741-C, 761-C, 781-C, N-200, N-600, RC1( ), and RM1( ). (B) Tip60beta; TFL(1-494), TN1(1-150), TN2(1-249), TM1( ), TM2( ), TC1( ), and TC2( ). (C) Two-hybrid interaction assays between RRM2, RRM1, and full-length or TC1 mutant Tip60. (D) Interaction between HA-tagged RC1 with nuclear localization signal (NLS-RC1-HA) and Tip60-myc-His. HeLa cells were transfected with vectors expressing NLS-RC1-HA or NLS-GFP-HA as a control, and Tip60-myc-His and the lysates were immunoprecipitated with the indicated antibodies. The resulting precipitates were immunoblotted using the indicated antibodies. (E) In vitro binding of RC1 with Tip60. Purified MBP-RC1 produced in E.coli was incubated with insect cell lysates expressing GST-Tip60. The resultant complexes were precipitated using GST-beads (GST) or control-beads (Cont), and analyzed by immunoblotting using the indicated antibodies.* represents non-specific bands. (F) HeLa cells were transfected with expression vectors for wild-type (WT), 761-C, and 781-C RRM1-HA and Tip60-myc-His. The lysates were subjected to biochemical fractionation as in Fig. S2A and immunoblotted using the indicated antibodies. (S: soluble fraction, C: chromatin fraction) -HA E IgG -myc GST pull-down -Leu -Trp Input IgG -myc MBP-RC1 GST-Tip6 MBP-RC1 -Leu -Trp -His -Ade Cont GST F R1 Wt R2 Wt X X Tip60 Wt Tip60 Wt R1 Wt X R2 Wt * R1 Wt X Tip60 TC1 R2 Wt X Tip60 TC1

4 A Mnase: + S1+S2 P2 S1 S2 S3 P2 S3 P2 sirna: R1 C R1 C Tip6 IKK Chk1 Orc2 C Tip6 Control S1+S2 P2 S1+S2 P2 RRM1-HA Tip60-Myc/His D IR: Tip6 Orc2 S1 S2 P Figure S4: A part of RRM1 localized at chromatin fraction. (A) Chromatin binding of endogenous RRM1 and Tip60. HeLa cells were subjected to biochemical fractionation. Aliquots of P2 fractions were incubated with or without micrococcal nuclease (MNase). The resulting extracts and pellets were subjected to immunoblotting using anti-rrm1, anti-tip60, and anti-chk1 (as a control). S1: cytosolic fraction, S2: nuclear soluble fraction, P2: chromatin-enriched fraction, S3: solubilized chromatin fraction. (B) Knockdown of RRM1 protein in the soluble and chromatin-bound fractions by the specific sirna. HeLa cells were transfected with either control (C) or RRM1 (R1) sirna and fractionated as in (A). The extracts were subjected to immunoblotting using anti-rrm1, anti-ikk (control for the cytosolic fraction), and anti-orc2 (control for the chromatin fraction). (C) HeLa cells were transfected with either control (Control) or Tip60-Myc-His (Tip60), and RRM1-HA vectors. The lysates were subjected to biochemical fractionation as in (A) and immunoblotted using the indicated antibodies. (D) Amounts of RRM1 on chromatin were not affected by DNA damage. HeLa cells were exposed to IR (5 Gy) and harvested at 0.5 hours after exposure. The lysates were subjected to biochemical fractionation as in (A) and immunoblotting using the indicated antibodies.

5 (kb) 3 M EcoRI EcoRI+ I-SceI (h) Figure S5: in vivo generation of a DSB by the expression of I-SceI STEFKu70 -/- phprt-dr-gfp cells were infected with adenoviruses expressing I-SceI. Cellular DNAs were prepared at the indicated times and subjected to Southern blotting using the specific primers shown in Fig. 2A (left panel). Band intensities of a 3.1 kb EcoRI fragment and a 2.6 kb EcoRI/I-SceI fragment were quantitated and the results are shown as percentages of the production of the EcoRI/I-SceI fragment (right panel).

6 A ChIP (-fold) 2 1 LZ I-SceI Vector: Wt Mut Adeno: -myc -Orc2 LZ I-SceI LZ I-SceI Control ChIP (-fold) Tip60: Wt Mut LZ I-SceI Tip6 Tip6 Caffeine None Figure S6: Recruitment of RRM1 to a DSB site is independent of the histone acetylase activity (HAT) of Tip60 and activation of checkpoint signalings (A) Cells containing an I-SceI cassette depleted endogenous Tip60 were transfected with vectors expressing either wild-type or its mutant Tip60-myc lacking HAT activity. Aliquots of cell lysates were subjected to immunoblotting using anti-myc and anti-orc2 antibodies (right panels). The resultant cells were infected with adenoviruses and ChIP analysis was performed anti-rrm1 antibodies as in Fig. 2B (left panel). (B) Cells containing an I-SceI cassette were treated with 10 mm caffeine for 24 hours. The resultant cells were infected with adenoviruses and ChIP analysis was performed anti-rrm1 and anti-tip60 antibodies as in Fig. 2B.

7 Tip6 Orc2 RRM2 -actin sirna Tip6 Control Control Tip60-Myc Figure S7: Tip60 depletion or its overexpression did not affect expression of RRM1 and RRM2 proteins HeLa cells were transfected with Tip60 sirna (Tip60-1), control sirna, or vectors expressing Tip60 or mock. Cells were then harvested 48 hours after transfection and the chromatin fractions (for Tip60 and Orc2) and the lysates (for RRM1, RRM2, and -actin) were subjected to immunoblotting using the indicated antibodies.

8 A Fragment Merge DAPI H2AX NLS RC1-HA NLS RC1-HA NLS GFP-HA NLS GFP-HA Figure S8: Expression of NLS-RC1-HA suppresses accumulation of endogenous RRM1 at DNA damage sites (A) Ectopic expression of NLS-RC1-HA suppresses formation of nuclear foci of endogenous RRM1 upon DNA damage. HeLa cells were transfected with vectors expressing either NLS-RC1-HA or NLS-GFP-HA as a control. Cells were exposed to IR and subjected to immunostaining with anti-rrm1 or anti-ha antibodies. Nuclear DNA was counterstained with DAPI. (B) GM02063 cells were transfected with either NLS-GFP-HA or NLS-RC1-HA, and then treated with microirradiation and stained as in Fig. 1B. Scale bars, 10 μm.

9 A IP: RRM2 IgG Tet: - IgG - Input NLS-RC1-HA Relative RNR activity (%) Tet: + Figure S9: Induction of NLS-RC1-HA does not affect complex formation and activity of endogenous RNR (A) Tet-on HeLa cells expressing NLS-RC1-HA were treated with (+) or without (-) doxycyclin (1 μg/ml) and harvested 24 hours after treatment. Cell lysates were subjected to immunoprecipitation using the indicated antibodies. The resultant precipitates and input lysates were then analyzed by immunoblotting using the indicated antibodies. (B) RRM1 immunoprecipitates from (A) were subjected to RNR assay as in Fig. 2E.

10 ChIP (-fold) LacZ I-SeI S G1 Figure S10: Cell cycle-independent of RRM1 recruitment at a DSB site STEFKu70 -/- phprt-dr-gfp cells infected with I-SceI or LacZ adenoviruses were synchronized as described in Materials and Methods. Cells were then subjected to ChIP analysis using the anti-rrm1 antibodies as in Fig. 2B. Data are shown as relative increases in PCR products from cells expressing I-SceI (I-SceI) relative to those from cells expressing Lac Z (LZ). Data are means ± standard deviation (n=3).

11 A Survival (%) WT AC Survival (%) WT AC Zeocin (mg/ml) 5 1 MMC (μg/ml) Figure S11: Expression of a mutant RRM1 lacking Tip60 binding sensitizes cells to Zeocin, but not to MMC Knockout-knockin HeLa cells expressing wild-type (WT: filled circles) or A776C 781-C (AC: open circles) were exposed to the indicated concentrations of Zeocin (A) or MMC (B). Viabilities of treated cells were examined by XTT assay 48 hours (Zeocin) and 24 hours (MMC) after treatment. Data are means ± standard deviation (n=3).

12 Tail moment G1 WT AC S WT AC Time (h) Time (h) Figure S12: Supply of excess amounts of dadp, dgdp, dcdp, and dump in the culture medium restores impaired DNA repair during G1 phase in cells expressing A776C 781-C Kockout-knockin HeLa cells expressing either wild-type (WT: filled bars) or A776C 781-C (AC: open bars) RRM1-HA were synchronized at G1 (G1) and S (S) phase as described in Materials and Methods. Excess amounts of dadp, dgdp, dcdp, and dump (250 μm of each) were supplied in the culture medium of synchronized cells. Synchronized cells were then released into G1 phase or S phase and exposed to IR (4 Gy) as described in Fig. 4E. DNA repair was evaluated as in Fig. 4E. Data are means ± standard deviation (n=3).