DAPI H2AX RRM1 DAPI Cont siRNA RRM1 - Genes &...

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B A RRM1 H2AX DAPI RRM1 H2AX DAPI Merge Cont RRM1 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.

Transcript of DAPI H2AX RRM1 DAPI Cont siRNA RRM1 - Genes &...

Page 1: DAPI H2AX RRM1 DAPI Cont siRNA RRM1 - Genes & Developmentgenesdev.cshlp.org/content/suppl/2010/01/25/24.4.333.DC1/... · 2010. 1. 25. · B C TFL TN1 TN2 TC2 TC1 CHROMO Binding RRM1

B

ARRM1 H2AX DAPI

RRM1 H2AX DAPI Merge

Cont

RRM1

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.

Page 2: DAPI H2AX RRM1 DAPI Cont siRNA RRM1 - Genes & Developmentgenesdev.cshlp.org/content/suppl/2010/01/25/24.4.333.DC1/... · 2010. 1. 25. · B C TFL TN1 TN2 TC2 TC1 CHROMO Binding RRM1

A RRM1 H2AX C RRM1 H2AX

Cont

RRM1

B

RRM1 H2AX

pSIREN

pSIREN-Tip60.1

pSIREN-Tip60.2

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.

D

RRM1/RRM2 H2AX

RRM1

RRM2 Cont

RRM1

RRM2 H2AX

siRNA:

siRNA:

E

Page 3: DAPI H2AX RRM1 DAPI Cont siRNA RRM1 - Genes & Developmentgenesdev.cshlp.org/content/suppl/2010/01/25/24.4.333.DC1/... · 2010. 1. 25. · B C TFL TN1 TN2 TC2 TC1 CHROMO Binding RRM1

B

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TC2TC1

CHROMOBinding

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WT 761-C 781-C

S C S C S C

RRM1

Tip60

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A Binding

RFL201-C601-C

RC1(701-792)

RM1(601-700)

RRM1

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-

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-

+

RRM2

+

+

-

--

-

--

CXXC

741-C761-C781-C

ATP-cone

--

++

-

-

Figure S3: Tip60 binds to the C-terminus region of RRM1To 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(701-792), and RM1(601-700). (B) Tip60beta; TFL(1-494), TN1(1-150), TN2(1-249), TM1(151-249), TM2(151-363), TC1(250-494), and TC2(151-494). (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)

NLS-RC1-HA

IgG -m

yc

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ut

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-myc

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-HA

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Tip60 Wt

R1 WtX

R2 WtR2 Wt

XTip60 TC1

R1 WtX

Tip60 TC1

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Tip60 Wt

-Leu -Trp-Leu -Trp-His -Ade

F

GST-Tip60

Input

GST pull-down

MBP-RC1

MBP-RC1

GST-Tip60

*

Co

nt

GS

T

Page 4: DAPI H2AX RRM1 DAPI Cont siRNA RRM1 - Genes & Developmentgenesdev.cshlp.org/content/suppl/2010/01/25/24.4.333.DC1/... · 2010. 1. 25. · B C TFL TN1 TN2 TC2 TC1 CHROMO Binding RRM1

RRM1

siRNA: R1 C R1 C

S1+S2 P2

IKK

Orc2

RRM1

S1 S2 P2

Mnase: - +

Tip60

Chk1

S3 P2S3

S1+S2 S1+S2 P2P2

Tip60 Control

A B

C

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.

Tip60

RRM1

S1 S2 P2

IR:

Orc2

- + - + - +

D

Tip60-Myc/His

RRM1-HA

Page 5: DAPI H2AX RRM1 DAPI Cont siRNA RRM1 - Genes & Developmentgenesdev.cshlp.org/content/suppl/2010/01/25/24.4.333.DC1/... · 2010. 1. 25. · B C TFL TN1 TN2 TC2 TC1 CHROMO Binding RRM1

Figure S5: in vivo generation of a DSB by the expression of I-SceISTEFKu70-/-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).

(kb)0 13 25 36 48 0

EcoRIEcoRI+I-SceI

(h)

3

M

Page 6: DAPI H2AX RRM1 DAPI Cont siRNA RRM1 - Genes & Developmentgenesdev.cshlp.org/content/suppl/2010/01/25/24.4.333.DC1/... · 2010. 1. 25. · B C TFL TN1 TN2 TC2 TC1 CHROMO Binding RRM1

0

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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.

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old

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Wt Co

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-myc

Mut

LZ I-SceI

-Orc2

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A

Page 7: DAPI H2AX RRM1 DAPI Cont siRNA RRM1 - Genes & Developmentgenesdev.cshlp.org/content/suppl/2010/01/25/24.4.333.DC1/... · 2010. 1. 25. · B C TFL TN1 TN2 TC2 TC1 CHROMO Binding RRM1

Figure S7: Tip60 depletion or its overexpression did not affect expression of RRM1 and RRM2 proteinsHeLa 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.

RRM1

RRM2

Tip60

siRNA

Co

ntr

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Tip

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yc

Co

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-actin

Orc2

Page 8: DAPI H2AX RRM1 DAPI Cont siRNA RRM1 - Genes & Developmentgenesdev.cshlp.org/content/suppl/2010/01/25/24.4.333.DC1/... · 2010. 1. 25. · B C TFL TN1 TN2 TC2 TC1 CHROMO Binding RRM1

DAPIRRM1 Fragment Merge

NLS-RC1-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.

BA

NLS-RC1-HA

NLS-GFP-HA

RRM1 H2AX

Page 9: DAPI H2AX RRM1 DAPI Cont siRNA RRM1 - Genes & Developmentgenesdev.cshlp.org/content/suppl/2010/01/25/24.4.333.DC1/... · 2010. 1. 25. · B C TFL TN1 TN2 TC2 TC1 CHROMO Binding RRM1

Tet: +

Rel

ativ

e R

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act

ivit

y (%

)

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RRM1

RRM2

NLS-RC1-HAIg

G

Tet: - +- + +-Input

-RR

M1

IgG -R

RM

1

IP:

BA

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.

Page 10: DAPI H2AX RRM1 DAPI Cont siRNA RRM1 - Genes & Developmentgenesdev.cshlp.org/content/suppl/2010/01/25/24.4.333.DC1/... · 2010. 1. 25. · B C TFL TN1 TN2 TC2 TC1 CHROMO Binding RRM1

0

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S G1

LacZ

I-SeI

Ch

IP (

-fo

ld)

Figure S10: Cell cycle-independent of RRM1 recruitment at a DSB siteSTEFKu70-/-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).

Page 11: DAPI H2AX RRM1 DAPI Cont siRNA RRM1 - Genes & Developmentgenesdev.cshlp.org/content/suppl/2010/01/25/24.4.333.DC1/... · 2010. 1. 25. · B C TFL TN1 TN2 TC2 TC1 CHROMO Binding RRM1

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Figure S11: Expression of a mutant RRM1 lacking Tip60 binding sensitizes cells to Zeocin, but not to MMCKnockout-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).

A B

Su

rviv

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%)

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Page 12: DAPI H2AX RRM1 DAPI Cont siRNA RRM1 - Genes & Developmentgenesdev.cshlp.org/content/suppl/2010/01/25/24.4.333.DC1/... · 2010. 1. 25. · B C TFL TN1 TN2 TC2 TC1 CHROMO Binding RRM1

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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-CKockout-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).