PIE1 ARP6 SWC6 KU70 ARP6 PIE1. HSA SNF2_N HELICc SANT. pie1-3 A1,A2 K1,K2 K1,K3 K3,LB2 A3, A4 A3,LB1 A1,A2 K1,K2 K1,K3. swc6-1 A3,A4.

Transcription

1 A B N-terminal SWC2 H2A.Z SWC6 ARP6 PIE1 HSA SNF2_N HELICc SANT C pie1-3 D PIE1 ARP6 5 Kb A1 200 bp A3 A2 LB1 arp6-3 A4 E A1,A2 A3, A4 A3,LB1 K1,K2 K1,K3 K3,LB2 SWC6 swc6-1 A1,A2 A3,A4 K1,K2 K1,K3 100 bp KU70 LB2 ku70-2 K1 K2 K3 UBC28 -RT 600 bp Supplemental Figure 1. Molecular and morphological characterization of novel T-DNA insertion mutants. (A) Components of the Arabidopsis thaliana SWR1 complex (adapted from March-Diaz et al. 2009). (B) Early flowering phenotype of 5-week-old plants of AtSWR1 mutants pie1-3, arp6-3 and swc6-1 compared to. (C) Scheme of PIE1, ARP6, SWC6 and KU70 genes, including introns (lines) and exons (boxes). T-DNA insertions are depicted as inverted triangles, and the arrows indicate the position and direction of primers used for genotyping and qrt-pcr of previously uncharacterized alleles. (D) Genotyping of T-DNA insertion mutants by PCR with primers upstream of the T-DNA insertion (as an amplification control), flanking the insertion sites, or with a primer combination of one flanking primer and one within the T-DNA border (see Supplemental table 1). (E) RT-PCR with primers located upstream of (A1/A2; K1/K2) or flanking (A3/A4; K1/K3) the T-DNA insertion. UBC28 was used as an amplification control and reference gene. In all mutant alleles, 5 transcripts were present but no amplification was obtained with primers flanking the T-DNA, indicating the absence of full-length transcripts.

2 arp6-3 C 10 d D 10 d HU 10 d E swc6-1 pie1-3 arp6-3 MMC 10 d 12 d swc6-1 pie1-3 swc6-1 arp6-3 pie1-3 ku70-2 swc6-1 arp6-3 pie1-3 ku70-2 swc6-1 arp6-3 pie1-3 A 10 d γ-ir 10d γ-ir 21d B 10 d BLEO 10 d BLEO 21 d UV-C 12 d UV-C 21 d Supplemental Figure 2. Examples of plant phenotypes after DNA damaging treatments. (A) γ-irradiation (γ-ir), (B) bleocin (BLEO), (C) mitomycin C (MMC), (D) hydroxyurea (HU), (E) ultraviolet light (UV-C).

3 A PIE1 ARP6 SWC6 HTA8 2 HTA9 2 HTA Time after BLEO treatment 2 B Gene Supplemental figure 3. Analysis of transcriptional response to bleocin treatment. (A) Transcript levels of genes encoding AtSWR1 subunits (PIE1, ARP6 and SWC6) and H2A.Zcoding genes (HTA8, HTA9 and HTA11) in, at different time points after bleocin treatment, relative to untreated set as 1. (B) Transcript levels of DNA damage-induced genes in, arp6-3 and swc6-1, 2 hours after exposure to 50 μg/ml bleocin for 1 hour, relative to levels in mock-treated controls. Transcript levels were measured by quantitative RT-PCR and normalized to UBC28. The values displayed represent the average of two or more biological replicates. Error bars correspond to the standard deviation of the mean (SD).

4 swc6 arp6 x brca1 MOCK x ku70 x brca1 BLEO 10d x ku70 Supplemental Figure 4. Examples of plant phenotypes of homozygous single (arp6-3, swc6-1, brca1-1, ku70-1) and double mutants (arp6 brca1, swc6 brca1, arp6 ku70, swc6 ku70), mock and bleocin-treated ( μg/ml) 10-day-old seedlings. = selfed plants, single mutants

5 Supplemental Figure 5. ASY1 localization and RAD51 foci in wild type, swc6 and arp6 mutants. Chromosome spreads of Arabidopsis wild type, swc6-1 and arp6-3 meiocytes stained with α-asy1 (green) and α-rad51 antibodies (red). Size bar: 10 µm.

6 A B C Supplemental Figure 6. Analysis of DNA damage sensitivity upon reduced H2A.Z expression. (A) RNAi construct targeting all three H2A.Z genes HTA8, HTA9, HTA11 (from Choi et al. 2007). (B) Relative transcript levels of all three H2A.Z genes in wild type and plants expressing the RNAi construct. Transcript levels were measured by quantitative RT-PCR, and values are presented relative to HTA8 in wild type. The asterisk indicates a significant difference according to a p value from unpaired t-tests between 1 and 5. (C) True leaf assay with seedlings of wild type and plants expressing the RNAi construct treated with bleocin (1 μg/ml).

7 Supplemental Table 1. Sequences of primers used to genotype T-DNA insertion lines. Primer names according to Supplemental Figure 1C. Primer name Alternative name Sequence (5 to 3 ) A1 ARP6-2R TCATCGTCATCAATGTCAAACA A2 ARP6-2F CTTTCTCTCCCCAACCTTATCC A3 ARP6-R GGATAAGGTTGGGGAGAGAAAG A4 ARP6-F TGTTTTGGTTAGCCTCAATGAA B1 BRCA1-F TTCTGCAGTTTCACTTTCGTGT B2 BRCA1-R AACGGAAGCATCTCTTACTTGC K1 KU70-F CCGGACGGAGTAAAGCATAA K2 KU70-2F TCTTCAGTTGGTCCTTCATGTCCTC K3 KU70q-F GGCAGTCAAACTGGTATTGTCTCAGATTC LB1 LB2-SAIL GCTTCCTATTATATCTTCCCAAATTACCAATACA LB2 LB WISC AATAGCCTTTACTTGAGTTGGCGTAAAAG LB3 LBb1 GCGTGGACCGCTTGCTGCAACT P1 PIE1-F CATAATGTCCTCCGTCCTTTCC P2 PIE1-R TCCTTCAAGTTCACACGCTGTT S1 SEF-R CTAAACATTTTCCGGGATGGAG S2 SEF-F AAGCTTTCGCTTGTCGAGTTTT

8 Supplemental Table 2. Sequences of primers used in qpcr analysis of transcript levels. Gene AGI Forward and Reverse primer sequence (5 to 3 ) ARP6 AT3G33520 TTAGGGATGAATCAGGCGGGACTTGC TGGGACAAGTGGTCGAAGCTCTCCTT BRCA1 HTA8 HTA9 HTA11 PARP1 PARP2 RAD17 RAD51 SWR1/ PIE1 SWC6/ SEF UBC28 At4g21070 At2g38810 AT1G52740 AT3G54560 At5g41140 At4g02390 At1g66130 At5g20850 At1g64230 At5g37055 At1g64230 GTTACGTGTGCAAAACTCATACCAGAATG GATACTTGTTTAGGCTGAGAGTGCAGTGG GCAGTGGCACCAACTCTTCCATGTG TCTAGCTGCGAAGACGACGGCAGCA AACCCAGCGGTAGCGACAAGGATAA TCGGCGAGATACGTTTCACCTTCAA CCAGTTGGACGAATTCACAGGCAAC GTGTGTCCAGCTCCTCATCTCCTCT GTGTAGCGAAAAGATCTTGAAAGGAGAGG CCATCCAGACAAACTTTCCAGTTCAGTAG ACATGGTTTACACCAGATGGGGAAGAG GGACTTGGGATGTGGGATAAACTCCTT CCTCGGTTGCTGTTCGTGGTGTGT ATCTGCCACTCTTGACCCCTCATAG CTCCGAGGAAGGATCTCTTGCAG GCTCGCACTAGTGAACCCCAG AGG TGCAACTGAGGCATACAGGCAGCAA TCGGCAGCCAGTGATCCTTTCTTCA CCAACGTTCGCCGGCAATCTAAAC GCTTCTAAAGCTTTCGCTTGTCGAG TCCAGAAGGATCCTCCAACTTCCTGCAGT ATGGTTACGAGAAAGACACCGCCTGAATA