Schematic representation of the endogenous PALB2 locus and gene-disruption constructs

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1 Supplementary Figures Supplementary Figure 1. Generation of PALB2 -/- and BRCA2 -/- /PALB2 -/- DT40 cells. (A) Schematic representation of the endogenous PALB2 locus and gene-disruption constructs carrying the puro or bsr selection-marker gene. The solid boxes represent exons. Relevant HindIII sites are indicated. (B) Southern-blot analysis of genomic DNA digested by HindIII was performed using the probe DNA shown in (A). Positions of hybridizing fragments of wild-type (WT) and targeted loci are indicated. (C) RT-PCR analysis of PALB2 +/- and PALB2 -/- cells. Supplementary Figure 2. Generation of mutant BRCA2 allelic genes. Resulting cells carry the conditionally-inactivated null gene in one allele while the other allele carries either a deletion at the N-terminal segment (BRCA2 N ), both a deletion at the N-terminal segment and a BRC3 truncation (BRCA2 N+BRC3tr ) or a deletion in the DNA-binding domain (BRCA2 DBD ). (A) Schematic representation of the endogenous BRCA2 locus and mutation-insertion constructs carrying the Histidinol (His) or Blasticidin (Bsr) selection-marker genes. The solid boxes represent exons, and numbers inside the boxes represent the number of exons. Relevant BstXI and SacII sites are indicated. Note that the other allele is conditionally inactivated as previously described (1). The indicated SpeI was used to linearize the BRC3 truncation construct of BRCA2 gene prior to transfection into DT40 cells. The red line represents the stop codon and the transcription termination 1

2 sequence. (B) Southern-blotting of BRCA2 N, BRCA2 DBD, BRCA2 N+ DBD and BRCA2 N+BRC3tr cells. Genomic DNA digested by both BamHI and EcoRI was hybridized with the DNA probe shown in (A). The designation con(+) indicates the conditional allele that expresses the intact BRCA2, while con(-) indicates a conditionally inactivated allele. Positions of hybridizing fragments of con(+), N and N+BRC3tr are indicated. (C) RT-PCR analysis of BRCA2 N, BRCA2 DBD, BRCA2 N+ DBD, BRCA2 N+BRC3tr and BRCA2 -/- clones to verify the expected mutations. BRCA2 -/- represents null-mutant cells. BRCA2 -/ N, BRCA2 DBD, BRCA2 N+ DBD and BRCA2 -/ N+BRC3tr clones, all carrying the conditionally inactivated null gene in an allele, are described as BRCA2 N, BRCA2 DBD, BRCA2 N+ DBD and BRCA2 N+BRC3tr clones, respectively. (D) Western-blotting of the green fluorescence protein (GFP)-tagged BRCA2 BRC3tr and GFP-tagged BRCA2 N+BRC3tr expressed in wild-type cells. Wild-type cells were used as a control. (E) Western-blotting of proteins expressed from the GFP-tagged endogenous wild-type BRCA2, BRCA2 N, BRCA2 DBD and BRCA2 N+ DBD genes. BRCA2 -/- cells were used as a control. Supplementary Figure 3. Relative proliferation kinetics for cells carrying the indicated genotypes. The histogram is calculated based on cellular proliferation over an eight-hour period during which the number of wild-type cells doubles. Error bars show the standard error for at least three independent experiments. P values were calculated by Student s t-test. 2

3 Supplementary Figure 4. Cellular sensitivity of the indicated BRCA2 mutant cells to the DNA-damaging agents methyl methane sulfonate (MMS), an alkylating agent, and ultraviolet light (UV). The histograms show LC 30 values (the concentration of DNA-damaging agents that reduces cellular survival to 30% relative to cellular survival without exposure to DNA-damaging agents). Values shown are mean ± SD. Supplementary Figure 5. Quantification of RAD51 foci in individual cells of the indicated genotype. Data shown are the means of three experiments. Error bars indicate standard deviation. Statistical analysis was performed using the Student s t test. P <0.01 compared to wild-type. Supplementary Figure 6. BRCA2 mutant proteins stability and distribution. (A) Western-blotting of proteins expressed from the GFP-tagged endogenous wild-type BRCA2, BRCA2 N, BRCA2 DBD and BRCA2 N+ DBD genes. (B) Quantification of the amount of BRCA2 protein in (A). Data shown are the means of three experiments. Error bars indicate standard deviation. (C) Visualization of the distribution of BRCA2 in wild-type and mutant DT40 clones using immuno-staining with anti-gfp antibody (red) and DAPI (blue). Supplementary Figure 6. A diagram summarizes all the mutation examined. 3

4 Supplementary Material and Methods Generation of PALB2 -/- and BRCA2 -/- /PALB2 -/- cells The generation of PALB2 -/- and BRCA2 -/- /PALB2 -/- cells is shown in Supplementary Figure 1A. The PALB2 targeting construct was amplified from the genomic DNA of DT40 cells using primers 5 -agcattgctgaggagggagtacagcgagac-3 and 5 - gctgctgatcagcttcccatctcttagccc-3 and LA Taq polymerase (Takara Bio, Shiga, Japan). Puromycin- and Blasticidin-resistant genes were inserted into the BamHI site in exon 2 of the PALB2 targeting construct, resulting in 4.6 kb upstream and 2.6 kb downstream arms. A PALB2 probe was generated with primers 5 -gagccatgcaagagcagcaagtggaaatag-3 and 5 -cgtacagagtcagaatatgctcgatggcag-3 and targeted to exons 8 and 9 of the PALB2 gene. To generate PALB2 -/- and BRCA2 -/- /PALB2 -/- cells, we sequentially targeted PALB2-puro R and PALB2-Blasticidin R vectors into wild-type and BRCA2 -/cond DT40 cells. Deletion of N-terminal sequences in the BRCA2 gene The deletion of the N-terminal sequences in the BRCA2 gene is shown in Supplementary Figure 2A. The BRCA2 N-terminal deletion construct was generated by amplifying genomic DNA. The upstream arm was amplified using primers 5 - aggttgcccaaggaggctgtggat -3 and 5 - ggatggatgtgaggtctggtactgg-3 and the downstream arm using primers 5 - ttgagctctctctgggctcaccgt -3 and 5 - cttgagagaggtggctttatgcatagagc-3. PCR yielded a 4.6 kb fragment, which was digested with HindIII, resulting in a 2.6 kb 4

5 upstream arm and a 4.5 kb downstream arm fragment. A Histidinol drug-resistance cassette flanked with LoxP signals was inserted between the upstream and downstream arms. The probe (1.8 kb) was generated using primer pair 5 -ttgagaaatgcacaggagctctcaagtgcc-3 and 5 -agctgctgtacctcggagccctaaa-3. The probe was targeted to exon10 of the BRCA2 gene. To generate BRCA2 cond/ N cells, we sequentially targeted the vector into BRCA2 cond/wt DT40 cells. Genomic DNA was cut with BstXI. Generation of an allelic gene carrying BRC3 truncation We used the same cells described in (2). The cells had a BRC3 truncated allele and a conditionally deleted allele. Generation of an allelic gene carrying both the N-terminal deletion ( N) and BRC3 truncation (BRC3tr) The generation of an allelic gene carrying both the N-terminal deletion and BRC3 truncation is shown in Supplementary Figure 2A. Genomic DNA was amplified using primers 5 -gaatgcggccgcgggtagccagtttgaattcacac-3 and 5 -aatgcggccgcactattctccttccacactgacac-3 with Phusion polymerase (New England Biolabs). The PCR yielded a 1.1 kb fragment, which was inserted into the NotI site in puc119 (3) vector containing the Blasticidin-resistance gene. The resulting construct was linearized with SpeI. The probe was generated using primers 5 - ttgagaaatgcacaggagctctcaagtgcc -3 and 5 -agctgctgtacctcggagccctaaa-3 and targeted to 5

6 exon 10 of the BRCA2 gene. To generate BRCA2 con/ N+BRC3tr cells, we sequentially targeted the vector into BRCA2 cond/ N DT40 cells. Genomic DNA from the Blasticidin-resistance clones was digested with BstXI and SacII restriction enzymes. Generation of allelic genes carrying N-terminal deletion ( N), DNA-binding domain deletion ( DBD) and deletion of both ( N+DBD) The generation of an allelic gene carrying either DBD or both the N and DBD mutants is shown in Supplementary Figure 2A. Genomic DNA was amplified using primers 5 -tttcagtggatggaaaagacatcagctggc-3 and 5 -ttgaaatatacagtactgtacatgtgca-3. The PCR yielded a 6.7 kb fragment which was sequenced to confirm the identity of the genomic DNA sequence and then inserted into the site in topozero blunt vector. The DBD mutation was created by site-directed mutagenesis based on PCR product cloned in topozero blunt vector, described above. One pair of primers, 5 -gtacagttatcagcaacaaag-3 and 5 -ctgcgatgttgacagctggcc-3, was used to generate the first deletion (3009AA-3017AA) and another pair, 5 -tatttatctgatgaaagctataatctt-3 and 5 - ggatattacaactccaactacat-3, was used for the second deletion (3074AA-3083AA). The Blasticidin-resistance gene was inserted in the PacI site inside the mutated genomic DNA. The probe was generated using primers 5 - tgtacgtacgccactggaagccccagattccc-3 and 5 - accgtgcgcatcgcgtgtttgctgc-3. To generate BRCA2 DBD and BRCA2 N+ DBD mutants, we targeted vector linearized by SacI into 6

7 wild-type and BRCA2 N DT40 cells. The genomic DNA was digested with BamHI restriction enzyme and analyzed by Southern-blotting. Insertion of the GFP tag gene into the C-terminal end of the endogeneous BRCA2 To insert the GFP tag gene into the C-terminal end of the endogeneous BRCA2 wild-type or mutant ( N, DBD or N+ DBD) allelic genes, we generated a GFP knock-in construct as follows. Genomic DNA was amplified using two sets of primers, 5 - cggcggccgcctaggagctcttggttttatactgatatgacttatggtgagga-3, 5 -gggcggccgccggcgtagcatttgcgggatttgcgcct-3, and 5 - gggctactaacttatctggtgatacaagag-3, 5 -gtacttctctgcaatgtgtgactgttgctc-3 with Phusion polymerase. The PCR yielded a 1.7 kb fragment upstream arm, which was inserted into the SacI and SmaI sites of pefgp-n2 vector, and a 9.2 kb fragment downstream arm. The upstream arm, tagged with the GFP gene, was excised off the pefgp-n2 vector with SacI and AflIII digestion and transferred to pbluescript SK+ vector by inserting it into the SacI and BstXI sites. In the final step of generating the GFP knock-in construct, the downstream arm, with a Puromycin drug-resistance cassette flanked with LoxP signals, was inserted into the KpnI site of the resulting plasmid. To generate BRCA2 wild-type-gfp, BRCA2 N-GFP, BRCA2 DBD-GFP and BRCA2 N+ DBD-GFP mutant cells, we linearized the GFP knock-in construct with SacII and then transfected the DNA into wild-type, BRCA2 N, BRCA2 DBD and BRCA2 N+ DBD DT40 cells. The probe was generated using primers 5 -gcagatctccggcattttgctatcgaa-3 and 7

8 5 -ctcttaggtaaaatgggtgtaagtgagcaatttc-3. PCR amplification yielded a 1 kb fragment. The genomic DNA was digested with XbaI restriction enzyme for Southern-blot analysis. Southern blotting showed a 10 kb band for the wild-type allele and a 6 kb band for the knock-in allele. Conditional generation of a null mutation in the endogenous BRCA2 allele (the cond(+) allele) To generate BRCA2 -/- /PALB2 -/-, BRCA2 -/ N, BRCA2 -/BRC3tr, BRCA2 -/ N+BRC3tr, BRCA2 -/ DBD and BRCA2 -/ N+ DBD mutant cells, we inserted mutations into an intact BRCA2 allele in cells (called BRCA2 +/con(+) cells) carrying the conditional BRCA2 mutation in the other allele (the cond(+) allele). To generate BRCA2 -/- /PALB2 -/- cells, we disrupted the PALB2 gene in the BRCA2 +/con(+) cells. The cond(+) allele of the resulting BRCA2 -/cond(+) /PALB2 -/-, BRCA2 cond(+)/ N, BRCA2 cond(+)/brc3tr, BRCA2 cond(+)/ N+BRC3tr, BRCA2 cond(+)/ DBD and BRCA2 cond(+)/ N+ DBD clones can be excised by the Cre recombinase upon exposure of the cells to Tamoxifen (1). To confirm the excision of the cond(+) allele, we performed Southern blotting of genomic DNA from subclones derived from TAM-treated cells (Supplementary Figure 2A). Generation of BRC3-truncated BRCA2 transgenes having W31C and W31R mutations 8

9 The generation of BRC3-truncated BRCA2 transgenes is illustrated in Supplementary Figure 2A. The truncated BRCA2 transgene was amplified using primers 5 -tactcgagatggcttacaaatctgggaaaagacctactttc-3 and 5 -agtcgactctccttccacactgacactatttgtcttttg-3, using a chicken cdna library as a template and Phusion polymerase. The amplified DNA was 4382 bp in length. It was base sequenced and inserted into the XhoI and SalI restriction sites of pegfp-n1 expression vector. The W13R and W13C mutations were created by site-directed mutagenesis (4) based on the pegfp-n1 BRC3-truncated BRCA2 vector, which was constructed as explained above. For the site-directed mutagenesis, primers 5 -ctgtaagtcttgaccggtttgaagagctca-3 and 5 -tgagctcttcaaaccggtcaagacttacag-3 were designed to generate the W31C mutation and primers 5 -ctgtaagtcttgactgctttgaagagctca-3 and 5 -tgagctcttcaaagctgtcaagacttacag-3 were designed to generate the W31R mutation. We determined the nucleotide sequences of the resulting DNA products to confirm the creation of the desired mutations. RT-PCR Total RNA was extracted with Sepasol (Nacalai Tesque, Kyoto, Japan) and then converted to cdnas with SuperscriptIII using oligo(dt) primer (Invitrogen, CA). RT-PCR of chicken beta-actin and PALB2 genes was amplified using primer pairs 5 -aggtatcctgaccctgaagtacc-3, 5 -catggctggggtgttgaaggtctc-3 and 5 -agcaaacatgagggatccag-3, 5 -gggagggaggagaacatcac-3, respectively. To confirm the 9

10 ( N) mutation in the DT40 cells, RT-PCR of the BRCA2 -/ N, BRCA2 -/ N+BRC3tr and BRCA2 -/ N+ DBD genes was carried out using primers 5 - atggcttacaaatctgggaaaagacctact -3 and 5 - cggacttgcacacacaggttttac -3. To confirm the (BRC3tr) mutation, RT-PCR of BRCA2 BRC3tr and BRCA2 N+BRC3tr genes was carried out using primers 5 -atggcttacaaatctgggaaaagacctact -3 and 5 - atctctttccccacattgtacgttgagatc-3. To confirm the ( DBD) mutation, RT-PCR of BRCA2 DBD and BRCA2 N+ DBD genes was carried out using primers 5 -tacagaatctgccagctgtcaaca-3 and 5 - tgccacaagattatagctttcatcag-3. Reference 1. Qing Y, Yamazoe M, Hirota K, et al. The epistatic relationship between BRCA2 and the other RAD51 mediators in homologous recombination. PLoS Genet. United States. p. e Hatanaka A, Yamazoe M, Sale JE, et al. Similar effects of Brca2 truncation and Rad51 paralog deficiency on immunoglobulin V gene diversification in DT40 cells support an early role for Rad51 paralogs in homologous recombination. Mol Cell Biol. United States; p Hirota K, Tanaka K, Watanabe Y, Yamamoto M. Functional analysis of the C-terminal cytoplasmic region of the M-factor receptor in fission yeast. Genes Cells 2001;6(3): Zhang Y, Riesterer C, Ayrall AM, Sablitzky F, Littlewood TD, Reth M. Inducible site-directed recombination in mouse embryonic stem cells. Nucleic Acids Res 1996;24(4):