SUPPLEMENTARY INFORMATION

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1 Supplementary Table 1 Components of the RAD6 damage tolerance pathway in yeast and humans S. cerevisiae Homo sapiens Functions Rad6 HHR6A and HHR6B Ubiquitin conjugating enzyme (E2) Rad18 RAD18 Ubiquitin ligase (E3) Ubc13 UBC13 Ubiquitin conjugating enzyme (E2) Mms2 MMS2/UEV2 E2 variant Rad5 HLTF and SHPRH E3 ligase and SWI/SNF2 type helicase Rev1, Polz, Polh Y-family DNA polymerases TLS polymerases Pol30 PCNA DNA clamp. In both yeast and humans, same lysine residue (K164) is modified with ubiquitin. 1

2 Supplementary Table 2 DNA damage checkpoint and homologous recombination proteins in yeast and humans S. cerevisiae Homo sapiens Functions Mec1 ATR PI3-like kinase Ddc2/Lcd1/Pie1 ATRIP ATR-binding partner Tel1 ATM PI3-like kinase Rad24 Rad17 RFC-like protein Ddc1 Rad9 PCNA-like protein Rad17 Rad1 PCNA-like protein Mec3 Hus1 PCNA-like protein Mrc1 Claspin Mediator Rad9 53BP1 Mediator Chk1 Chk1 Effector kinase Rad53 Chk2 Effector kinase Spo11 Spo11 Meiotic DSB formation Mre11-Rad50-Xrs2 (MRX complex) Mre11-Rad50-Nbs1 (MRN complex) DSB end processing and damage checkpoint Sae2/Com1 CtIP DSB end processing Rad51 Rad51 Recombinase Rad52 Rad52 Recombinase mediator Rad54 Rad54 Swi2/Snf2 family of ATPdependent DNA translocase Tid1/Rdh54 Rad55-Rad57 complex Rad54B Rad51 paralogues Rad54-like protein Recombinase mediator Mei5-Sae3 complex Sfr1-Swi5 complex Recombinase mediator Dmc1 Dmc1 Meiosis-specific recombinase No homologue Brca2 Recombinase mediator Mus81-Mms4 Mus81-Ems1 Structure-specific endnuclease 2

3 Supplementary Table 3 Saccharomyces cerevisiae strains used in this study strain Genotype Source BY4741 MATa leu2 0 met15 0 ura3 0 his3 1 ATCC TH551 BY4741 rev1 :: KanMX rad30 ::HIS3 rev3 :: KanMX this study TH560 a BY4741 mec1 :: KanMX sml1 ::LEU2 this study TH561 a BY4741 rad53 ::URA3 sml1 ::LEU2 this study TH411 BY4741 TUB1-GFP::HIS3 1 TH412 BY4741 :: KanMX TUB1-GFP::HIS3 this study TH413 BY4741 :: KanMX TUB1-GFP::HIS3 this study TH562 a BY4741 ::KanMX mec1 ::KanMX sml1 ::LEU2 this study TH563 a BY4741 ::KanMX rad53 ::URA3 sml1 ::LEU2 this study TH564 BY4741 ::KanMX rad9 ::KanMX this study TH565 BY4741 ::KanMX tel1 :: KanMX this study TH566 BY4741 ::KanMX mrc1 ::KanMX this study TH567 BY4741 :: KanMX chk1 ::KanMX this study TH571 BY4741 ::KanMX mec1 ::KanMX sml1 ::LEU2 this study TH572 BY4741 ::KanMX rad53 ::URA3 sml1 ::LEU2 this study TH573 BY4741 ::KanMX rad9 ::KanMX this study TH574 BY4741 ::KanMX tel1 ::KanMX this study TH575 BY4741 ::KanMX mrc1 ::KanMX this study TH576 BY4741 ::KanMX chk1 ::KanMX this study TH450 BY4741 RAD53-9myc::KanMX this study TH451 BY4741 ::KanMX RAD53-9myc::KanMX this study TH452 a TH451, mec1 ::KanMX sml1 ::LEU2 this study TH453 TH451, rad9 ::KanMX this study TH552 BY4741 ::KanMX rad52 ::KanMX this study 3

4 TH590 BY4741 rad5k538a this study TH595 BY4741 pol30k164r this study YYK20 b MATa bar1 ::hisg can1-100 CDC45-3HA::TRP1 his3-11, 15 leu2-3, 112 SLD3-9myc::LEU2 trp1-1 ura3-1 2 TH900 b YYK20, ::KanMX this study TH901 b MATa can1-100 MCM7-3HA::TRP1 his3-11, 15 leu2-3, 112 trp1-1 ura3-1 this study TH905 b TH901, ::KanMX this study W C b W C b W C b MATa RAD52-YFP MATa RFA-YFP MATa DDC2-YFP, RAD52-CFP TH1190 b W C, ::KanMX this study TH1191 b W C, ::KanMX this study TH1192 b W C, ::KanMX this study TH1193 b W C, ::KanMX this study TH1194 b W C, ::KanMX this study TH1195 b W C, ::KanMX this study a A deletion of SML1 suppresses the lethality without suppressing their checkpoint defect. b These strains are derivatives of W303. Supplementary References Huh, W. K. et al. Global analysis of protein localization in budding yeast. Nature 425, (2003). Kamimura, Y., Tak, Y. S., Sugino, A. & Araki, H. Sld3, which interacts with Cdc45 (Sld4), functions for chromosomal DNA replication in Saccharomyces cerevisiae. Embo J. 20, (2001). Lisby, M., Barlow, J. H., Burgess, R. C. & Rothstein, R. Choreography of the DNA damage response: spatiotemporal relationships among checkpoint and repair proteins. Cell 118, (2004). 4

5 no UV CLUV apn1 apn2 ntg1 ntg2 Supplementary Figure 1. Sensitivity to chronic UV irradiation of the BER mutants. Ten-fold serial dilutions of stationary-phase cells were spotted onto plates. DNA damage was induced by CLUV (0.18 J/m 2 /min) exposure for 2 days. 5

6 16 Chronic 250 Acute Relative intensity Relative intensity Time (h) UV dose (J/m 2 ) Supplementary Figure 2. Thymine dimer accumulation was measured as relative intensities of dot spots. Asynchronous cultures of and rad18! cells were exposed either to CLUV for the indicated times or to acute UV. DNA was extracted and CPD (anti-tdm2) and total DNA was quantified as described in Methods. CPD a mounts were measured as relative intensity to the initial amount of damage. Initial CPD amounts were t he same value in both chronic and acute UV experiments. Open and closed circles represent wild type and the rad18! strains, respectively. 6

7 rad6 pol30k164r tls no UV UV (20 J/m 2 ) UV (40 J/m 2 ) CLUV (<0.1 J/m 2 /min) Supplementary Figure 3. Sensitivity to acute and chronic UV irradiation of the RAD6 pathway mutants. Ten-fold serial dilutions of stationary-phase cells were spotted onto plates. DNA damage was induced by acute UV irradiation (20 or 40 J/m 2 ) or CLUV (< 0.1 J/m 2 /min) exposure for 2 days. In case of CLUV (< 0.1 J/ m 2 /min), the exact UV irradiance was n ot determined because it was outside of range of the UV meter (UVX-25, Ultraviolet Products). 7

8 Cdc45 W S Ch W S Ch nouv MCM7 Tubulin Cdc45 CLUV MCM7 Tubulin Supplementary Figure 4. Chromatin binding of Cdc45 and Mcm7. Asynchronous cultures of and rad18! cells were treated with CLUV for 3 h and used for chromatin-binding experiments. Whole cell extract (W), supernatant (S) and chromatin-enriched fractions (Ch) were separated by 8% SDS-PAGE. Western blots were probed with monoclonal 12CA5 antibody to detect the HA-tagged Cdc45 and Mcm7, and monoclonal B antibody to detect tubulin. 8

9 a no UV CLUV mrc1 tel1 chk1 rad53 rad9 mec Viable cells Time (h) b mec1 mec1 mec1 no UV CLUV Supplementary Figure 5. Effect of DNA checkpoint mutations on the CLUV sensitivities of rad5 mutants. a, Asynchronous log-phase cells were grown in rich media under CLUV irradiation and samples were taken every 3 h to de termine plating efficiency. Viable cells are represent as relative colony forming units (Time 0=1), which was obtained from at least three independent experiments. The error bars indicate the standard deviations. b, Cells were exposed to CLUV for 6 h, harvested and stained with DAPI. 9

10 no UV CLUV (3 h) DAPI Ddc2-YFP DAPI Ddc2-YFP Cells with Foci (%) no UV CLUV (3 h) Supplementary Figure 6. CLUV induces Ddc2-YFP foci in RAD6 error-free PRR deficient cells. Asynchronous cultures of, rad18! and rad5! cells were exposed to CLUV for 3 h and examined by fluorescence microscopy. 10

11 no UV CLUV (3 h) DAPI RPA-YFP DAPI RPA-YFP 100 Cells with Foci (%) no UV CLUV 3h Supplementary Figure 7. CLUV induces RPA-YFP foci in RAD6 error-free PRR deficient cells. Asynchronous cultures of, rad18! and rad5! cells were treated with CLUV for 3 h and examined by fluorescence microscopy. 11

12 no UV CLUV (3 h) DAPI Rad52-YFP DAPI Rad52-YFP Cells with Foci (%) no UV CLUV (3 h) Supplementary Figure 8. CLUV induces Rad52 foci in RAD6 error-free PRR deficient cells. Asynchronous cultures of, rad18! and rad5! cells were exposed to CLUV for 3 h and examined by fluorescence microscopy. 12

13 no UV CLUV (6 h) rad52 rad52 mec1 Supplementary Figure 9. rad18! rad52! double mutant cells are checkpoint proficient. Cells were incubated to early log phase and then exposed to CLUV for 3 h. DNA content of asynchronous cultures of the indicated strains was measured by FACS. 13

14 rad % of cells large bud Hours after release from CLUV exposure no or small bud multiple or protruded bud Supplementary Figure 10. rad18! rad52! double mutant cells are unable to recover from CLUV-induced G 2 arrest. Cells were synchronized at G 1 with -factor and released into S phase under CLUV conditions. Samples were taken at the indicated times after terminating CLUV and processed for morphological analysis. 14