SUPPLEMENTARY INFORMATION

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Beatrix Morgan
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NCS (ng/ml) Time (min) kd 500 500 0 30 0 30 IP: IP: 112 105 75 IB: ps407 IB: Mdm2 NCS + + IB: IB: tuulin IP input sup NCS (ng/ml) 50 100 500 Time (min) 0 15 30 60 120 15 30 60 120 15 30 60 120 IB:

tuulin Reconstituted AT cells 35 30 25 ATM ps407 NonReconstituted AT cells tuulin Figure S1 Chrcteriztion of the DNA dmge response detected y the αmdm2/ps407 ntiody.

Lympholstoid cells were treted with 500 ng/ml of the rdiomimetic drug NCS for 30 min nd the nlysis ws crried out using totl cellulr extrcts.

Wild type nd AT lympholstoid cells were incuted with NCS t the indicted doses nd cellulr extrcts were prepred t the indicted time points. c. Reconstitution of AT cells with ectopiclly expressed ATM reconstitutes the DSBinduced signl detected y the αmdm2/ps407 ntiody.

1 NCS (ng/ml) Time (min) kd IP: IP: IB: ps407 IB: Mdm2 NCS + + IB: IB: tuulin IP input sup NCS (ng/ml) Time (min) IB: ps kd Pounceu S stining Wild type Ectopic HA IB: ps kd NCS (200ng/ml) Pounceu S stining AT c NCS (ng/ml) Time (min) IB: ATM ps407 tuulin Reconstituted AT cells ATM ps407 NonReconstituted AT cells tuulin Figure S1 Chrcteriztion of the DNA dmge response detected y the αmdm2/ps407 ntiody.. Western lotting nlysis showing tht the gel migrtion pttern of the protein recognized y the αmdm2/ps407 ntiody following DSB induction is different from tht of Mdm2. Lympholstoid cells were treted with 500 ng/ml of the rdiomimetic drug NCS for 30 min nd the nlysis ws crried out using totl cellulr extrcts. The sme lot ws proed successively with the αmdm2/ps407 nd n αmdm2 ntiodies.. Time course nd ATM dependence of the rection of the αmdm2/ps407 ntiody with the elusive protein. Wild type nd AT lympholstoid cells were incuted with NCS t the indicted doses nd cellulr extrcts were prepred t the indicted time points. c. Reconstitution of AT cells with ectopiclly expressed ATM reconstitutes the DSBinduced signl detected y the αmdm2/ps407 ntiody. Immortlized AT firolsts (AT22IJE T) devoid of endogenous ATM were stly reconstituted with ectopic ATM expressed from n episoml vector 36. The cells were treted with the indicted NCS doses nd processed s descried ove. Figure S2 The protein whose phosphoryltion is detected y the αmdm2/ ps407 ntiody is.. Reciprocl immunoprecipittions of the protein were crried out using n ntiody ginst nd the αmdm2/ ps407 ntiody. Cellulr extrcts of HEK293 cells treted with 200ng/ ml of NCS for 30 minutes were used s source of proteins. The immune complexes were lotted consecutively with the αmdm2/ps407 nd αkap 1 ntiodies. Right pnel: Quntittive immunoprecipittion of y the αps824 ntiody using the sme experimentl conditions. Input: 10% of totl cell extrct; sup: the superntnt left fter immunoprecipittion. Note the depletion of y the αps824 ntiody.. Western lotting nlysis of totl cell extrcts of HEK293 cells in which endogenous ws replced y ectopic wild type or mutnt protein. Note the specificity of the α nd αps824 ntiodies. 1

5 1 2 4 6 8 UT 24 HSC70 HSC70 AT c HU (1mM) UT 0.

phosphoryltion in response to vrious types of

The experiment ws crried out s descried in Fig. 1c.

2 UV (25J/m²) UT UT 24 HSC70 AT HSC70 MMS (200µM) UT UT 24 HSC70 HSC70 AT c HU (1mM) UT UT 24 HSC70 HSC70 AT Figure S3 phosphoryltion in response to vrious types of genotoxic stress. The experiment ws crried out s descried in Fig. 1c. The cells were treted with UVirrdition t dose of 25 J/M 2 (), or with 200 µm of MMS, () or with 1 mm of hydroxyure (c). 2

3 Time (hr) HA UT c Time (hr) HA UT (12 hr) UT 48 hr Irrelevnt Irrelevnt ATM ATM d G1/G2 rtio Time (hr) Irrelevnt ATM G1/G2 rtio Time (hr) Irrelevnt ATM Figure S4 phosphoryltion is not involved in DNA dmgeinduced ctivtion of the cell cycle checkpoints.. Flow cytometry profiles of cell cycle distriution in U2OS cells with different constitutions during the first few hours fter NCS tretment (100 ng/ml). DNA content (PI) is indicted t the x xis, wheres the cell count is mesured t the y xis.. Expression of the dt shown in () s G1/G2 rtios. c nd d. Similr nlysis t extended time periods fter tretment. Note the mrked difference etween the cell cycle distriution ptterns in ATMdeficient cells nd ll other genotypes. 3

4 GFP NCS (Time) GFP H2AX GFP NCS (Time) GFP 53BP1 4

5 HA irrelevnt S824D IB: NCS ps1981/atm ps957/smc1 / Ns1 ps15/p53 tuulin Figure S5 phosphoryltion is not required for the erly steps in the DNA dmge response or the ctivtion of ATM nd downstrem pthwys. Experiments were crried out in U2OS cells in which GFPtgged, wild type or mutnt () replced the endogenous protein.. Immunofluorescence nlysis of H2AX phosphoryltion (upper pnel) nd formtion of nucler foci y the 53BP1 protein (lower pnel) fter tretment with 100 ng/ml NCS for the indicted time points. Scle rs: 5µm.. Western lotting nlysis of cellulr extrcts crried out fter tretment with 200ng/ml NCS for 30 min. Phosphoryltion of Ser957 of the Smc1 protein nd Ser15 of the p53 protein were monitored y specific ntiphospho ntiodies. Ns1 phosphoryltion ws monitored y following the typicl ndshift of the phosphorylted protein (see ref. 2 nd references therein). 5

GFP H2AX 2 min 8 min 12 min Figure S6 Temporry stlling of GFPtgged t DSB sites.

In the first few minutes fter dmge induction smll frction of ccumultes t the dmge sites ut this

Presumly molecules tht re lredy phosphorylted nd trverse the dmge sites do not stll for

6 GFP H2AX 2 min 8 min 12 min Figure S6 Temporry stlling of GFPtgged t DSB sites. Sptilly loclized DSBs were induced s previously descried in U2OS cells expressing GFPtgged. In the first few minutes fter dmge induction smll frction of ccumultes t the dmge sites ut this phenomenon is not further oserved s higher frction of ecomes phosphorylted. Presumly molecules tht re lredy phosphorylted nd trverse the dmge sites do not stll for phosphoryltion hence the trnsient ccumultion of t these sites is detectle only t erly time points fter dmge induction. Scle rs: 10µm. 6

Bleched region R1 R2 Prelech 0 5 20 60 120 Postlech (s) HlfFRAP on Kp1GFP Pre NFU 1.2 1 0.8 0.6 0.4 0.

4 % ± 3.1 Unound 5.3 % ± 4.2 5.5 % ± 1.6 Fst 26.8 % ± 0.9 2.1 s. ± 0.1 28.0 % ± 0.6 2.2 s. ± 0.1 Slow 64.1 % ± 0.8 27.1 s. ± 0.9 63.2 % ± 0.5 29.3 s. ± 0.3 Figure S7 Intrnucler moility nd chromtin inding properties of KAP 1.

A region (R1) spnning hlf of the nucleus ws exposed to single lech pulse (see Supplementry Methods).

The GFP signl uniformly recovers cross the leched comprtment (R1), implying tht the nucleus resemles wellmixed comprtment for.

Normlized fluorescence intensities for the leched nd unleched res from 10 cells were clculted nd plotted long the timescle of the experiments (180 s).

Notly, no significnt differences were oserved etween the nucler moility of GFP in irrdited nd mocktreted ce

7 Bleched region R1 R2 Prelech Postlech (s) HlfFRAP on Kp1GFP Pre NFU FLIP mock FRAP mock FLIP 10 Gy FRAP 10 Gy Popultions Mock Gmm Reltive Aundnce Assocition T men Reltive Aundnce Assocition T men Immoile 3.5 % ± % ± 3.1 Unound 5.3 % ± % ± 1.6 Fst 26.8 % ± s. ± % ± s. ± 0.1 Slow 64.1 % ± s. ± % ± s. ± 0.3 Figure S7 Intrnucler moility nd chromtin inding properties of KAP 1.. A typicl exmple of FRAP/FLIP ssy performed in U2OS cells in which endogenous ws replced y GFPtgged. A region (R1) spnning hlf of the nucleus ws exposed to single lech pulse (see Supplementry Methods). Susequently, the fluorescence intensities within the leched region (R1) nd in the control nonleched region (R2) were monitored for the indicted time. The GFP signl uniformly recovers cross the leched comprtment (R1), implying tht the nucleus resemles wellmixed comprtment for.. The sme cells s in () were exposed to 10 Gy of IR or left untreted, nd sujected to the hlf FRAP ssy s in (). Normlized fluorescence intensities for the leched nd unleched res from 10 cells were clculted nd plotted long the timescle of the experiments (180 s). The convergence of the fluorescence curves representing the leched nd control regions indictes tht the frction of immoile is very low (etween 36%; see elow). Notly, no significnt differences were oserved etween the nucler moility of GFP in irrdited nd mocktreted cells. c. Chromtinssocition times of distinct GFP popultions were clculted ssuming mthemticl model tht ccounts for two trnsiently intercting pools of the protein (see Supplementry Methods). A slowmoving frction with men ssocition time of pproximtely 27 seconds ws the most undnt one, nd smller nd fster popultion ws oserved with men ssocition time of pproximtely 2 seconds. These vlues nd their distriutions re rodly reminiscent of those of numer of chromtinssocited proteins (see ref. 2 in the Supplementry Methods). Consistent with the fluorescence redistriution curves shown in (), neither of these vlues ws significntly ltered fter IR, indicting tht s moility remins lrgely unltered fter DNA dmge. 7

8 NCS (200ng/ml) + 1U MNse (time min) Irrelevnt ATM NCS 200ng/ml M M c N6 N5 N4 N3 N2 N1 OD N7 UT 30 min 60 min ATKD OD N6 N5 N4 N3 N2 N1 8

d DNse I (Units/rx) NCS 200ng/ml 10 12 15 18 20 + + + + + + Figure S8 Chromtin relxtion in response to DSBs.

ATM ws knocked down using RNAi in neurolstom cells LAN5 17. Chromtin ccessiility to MNse ws mesured following tretment with 200 ng/ml of NCS.

9 d DNse I (Units/rx) NCS 200ng/ml Figure S8 Chromtin relxtion in response to DSBs.. Time course of prtil MNse digestion of chromtin otined from HEK293 cells, untreted (UT) or treted with 500 ng/ml of NCS for 60 min.. ATM dependence of DSBinduced chromtin relxtion. ATM ws knocked down using RNAi in neurolstom cells LAN5 17. Chromtin ccessiility to MNse ws mesured following tretment with 200 ng/ml of NCS. Note the extent nd rpid kinetics of chromtin relxtion in these cells compred to HEK293 cells nd the cler ATM dependence of this process. c. Scn profiles of the gel in Fig. S8, UT: untreted cells. N1N7 denotes the oligonucleosome sizes. d. Prtil digestion with DNseI of chromtin otined from HEK293 cells, which were untreted or treted with 200 ng/ ml NCS for 45 min. Equl loding of DNA ws monitored y short time gel electrophoresis (top pnel), nd the sme gel ws then run for dditionl 48 hr (lower pnel). Note incresed DNAseI ccessiility fter DNA dmge. References for Supplementry Informtion Phir, R. D, Gorski, S. A. & Misteli, T. Mesurement of dynmic protein inding to chromtin in vivo, using photoleching microscopy. Methods Enzymol. 375, (2004). Phir, R.D. et l. Glol nture of dynmic proteinchromtin interctions in vivo: threedimensionl genome scnning nd dynmic interction networks of chromtin proteins. Mol. Cell. Biol. 24, (2004). Essers, J. et l. Nucler dynmics of RAD52 group homologous recomintion proteins in response to DNA dmge. EMBO J. 21, (2002). 9

SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION 1 1 μm c d EGF + TPA + e f Intensity 1.8 1.6 1.4 1.2 1.8.6.4.2 2 4 8 2 4 8 (Hours) 2 4 6 8 1 Time (Hours) Reltive luciferse ctivity 4 3 2 1 + CAMEK1 FRE reporter Figure S1 inhiitor incresed protein expression