Supplementary Figure 1. Nature Structural & Molecular Biology: doi: /nsmb.3494
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1 Supplementary Figure 1
2 Pol structure-function analysis (a) Inactivating polymerase and helicase mutations do not alter the stability of Pol. Flag epitopes were introduced using CRISPR/Cas9 gene targeting at the C-terminal of Polq in mouse ES cells (CCE) with the following genotype: Polq +/+, Polq Pol and Polq Hel. Three independent clones were isolated for each genotype. ve lysates were obtained from non-targeted Polq +/+ cells. (b) Sequence analysis of the targeted CCE-mES cells with the indicated genotypes. (c) Relative quantification of BRCA1 mrna in CCE-mES cells treated with shcontrol and shbrca1. (d) Western-blot analysis for Flag-Cas9 in cells with the indicated genotype. (e) Examples of junction sequence for Der (6) retrieved from several translocation events and classified according to the presence of insertions, deletions and micro-homology. Junctions that fall in the category of alt-nhej signature are indicated with a check.
3 Supplementary Figure 2
4 The function of Pol -helicase and polymerase during homologous recombination (a) Accumulation of Rad51 in response to 1 Gy ionizing radiation of mes (CCE) cells with the indicated genotypes. Four hours post-irradiation, cells were fixed and co-stained with anti-rad51 and -H2AX. Graph represents quantification of cells with > 5 RAD51 foci. (Mean ± SD, n=4 two independent experiments, each carried out with two independent clonal cell lines, *p<0.05, **p<0.01). Two-tailed Student s t-test. n.s; not-significant (b) Scheme depicting the method used to investigate the impact of Pol on the efficiency of gene targeting by HR. (c) Sequence analysis of the CCEs following CRISPR/Cas9 targeting to generate Polq knockout allele. (d) Schematic of the strategy employed to target the Sox2 locus. Primers used for genotyping are indicated. (e) FACS analysis to assess the frequency of CCE cells expressing Zsgreen from the Sox2 locus following CRISPR/Cas9 targeting. Three independent population of cells were identified. (f) Genotyping PCR on sorted cells from (e) with the indicated genotypes. (g) Quantification of CRISPR/Cas9- mediated targeting of Sox2 using Cas9 nuclease in Polq +/+ and Polq -/- mes cells (CCE). As a control, DNA- Pki increases the efficiency of targeting. Bars represent the average of six independent experiments for (-) DNA-PKi and four experiments in the case of (+) DNA-PKi ± S.D. Two-tailed Student s t-test. n.s; notsignificant (h) Quantification of CRISPR targeting with Cas9 nickase of Sox2 in CCE cells with the indicated genotypes. Bars represent mean ± S.D. from six independent experiments in the case of Polq -/- and Polq +/+ and four with Polq Pol and Polq Hel mutants. *P < 0.05 and **P < Two-tailed Student s t-test.
5
6 Supplementary Figure 3 In vitro analysis of Pol -helicase function (a) Coomassie-stained SDS-page gel of purified Pol helicase (right) and the trimeric RPA complex (left) (b) Phosphorimager scan of thin layer chromatography plate containing results of Pol ATPase assay in the presence of ssdna. (c-d) Pol hel promotes DNA annealing independent of ATP in the absence of RPA. (c) Schematic of assay. (d) Non-denaturing gel showing Pol -hel annealing of complementary ssdna substrates in the presence and absence of ATP. (e-f) In the absence of RPA, Pol -helicase stimulates DNA synapsis independently of ATP hydrolysis. (e) Schematic of assay. Increasing amounts of Polq-helicase are mixed with the indicated cy3 and cy5 end-joining model substrates containing 3 overhangs with 4 nt microhomology in the presence of ATP or AMPPNP. (f) Plot showing relative cy5 fluorescence intensity in the presence of indicated nucleotide and Pol hel concentration. (g-h) Pol hel performs limited annealing in the presence of E. coli SSB. (g) Schematic of assay. (h) Non-denaturing gel showing Pol hel annealing of ssdna substrates pre-bound by the indicated amounts of RPA and E. coli SSB. Percent annealing indicated. Percent annealing calculated by dividing the intensity of the upper band by the sum of the intensities of the upper and lower bands. (i) Comparison of RPA and E. coli SSB affinity for ssdna (26 nt). Plots showing fluorescence anisotropy following incubation of the indicated amounts of RPA and E. coli SSB with fluorescein-labeled ssdna. Since the affinity for ssdna is similar for RPA and SSB, the helicase is likely to displace RPA due to a specific mechanism rather than lower binding affinity.
7
8 Supplementary Figure 4 In vitro analysis of Pol end-joining activity (a) Top: Schematic of assay. Pol pol promotes DNA end-joining in vitro via alt-nhej. Following annealing of complementary DNA, the polymerase extends each 3 minimally paired overhang by using the opposing overhang as a template in trans, resulting in strand displacement synthesis followed by limited terminal transferase activity. Middle: Denaturing gel showing Pol end-joining product and 48 nt marker. Bottom: Non-denaturing gel showing Polq end-joining product and 55 bp dsdna marker. Lower molecular weight products are due to Pol terminal transferase activity on 3 overhangs. (b) Sequence of the DNA substrates using in end-joining assay. (c) Top: Schematic of control experiment used to validate the end-joining in vitro assay. Schematic adapted from 1. Non-denaturing gel showing that Pol end-joining products are susceptible to EcoRI digestion, which confirms end-joining mechanism. (d) Pol helicase promotes alt- NHEJ in the presence of RPA. Left: Schematic of Pol pol mediated alt-nhej assay in the presence of RPA and Pol hel. Non-denaturing gels from two independent experiments showing alt-nhej products in reactions with the indicated proteins and ATP (right). 1 Kent, T., Chandramouly, G., McDevitt, S. M., Ozdemir, A. Y. & Pomerantz, R. T. Mechanism of microhomology-mediated end-joining promoted by human DNA polymerase theta. Nature structural & molecular biology 22, , doi: /nsmb.2961 (2015).
9 Supplementary Figure 5 Pol pol template dependent activity is resistant to RPA binding of ssdna. (a) Top: Schematic of the assay. Bottom: Denaturing gel showing Pol pol primer-template extension in the presence of the indicated amounts of RPA which were pre-incubated with the primer-template. Black asterisk marks template-independent extension (terminal transferase activity) of duplexed DNA by Pol pol. (b) Top: Schematic of the assay. Bottom: Denaturing gel showing Pol pol primer extension on a gapped DNA template in the presence of the indicated amounts of RPA.
10 Supplementary Figure 6
11 Investigating the interplay between Pol -helicase and RPA during alt-nhej (a) Alignment of RPA1 protein sequence from human, mouse, S. cerevisiae and S. pombe. Highlighted is the conserved aspartic acid residue at positions 228, 258, 228, and 223, respectively. (b) Representative metaphase spreads from TRF1 F/F TRF2 F/F Lig4 -/- Cre-ER T2 MEFs, with the indicated shrna treatment, 96 h after Cre expression. CO-FISH assay was performed using a FITC-OO-(CCCTAA)3 PNA probe (green) and a Tamra-OO-(TTAGGG)3 PNA probe (red). DAPI in blue. Examples of alt-nhej mediated fusion are indicated by white arrows. Scale bars, 10 μm. (c) Western-blot analysis of Myc-RPA1, total RPA1 and tubulin in TRF1 F/F TRF2 F/F Lig4 -/- Cre-ER T2 MEFs treated with shcontrol and shrpa1. (d) Western blot analysis for RPA1 in TRF1 F/F TRF2 F/F Lig4 -/- Cre-ER T2 treated with different amounts of shrpa1. Knock-down efficiency is quantified with image lab (Biorad). (e) Quantification of telomere fusions in shelterin-free cells treated with shrpa1 as in (d). (f) Western-blot analysis for the depletion of Rad51 in TRF1 F/F TRF2 F/F Lig4 -/- Cre-ER T2 MEFs. (g) Quantification of telomere fusion (alt-nhej) and T-SCE (HR) in the indicated cells treated with shrnas against Rad51 or control shrna. Bars represent mean ± S.D. from three independent experiments. *P < 0.05 and **P < 0.01; two-tailed Student s t-test. (h) Representative images of U2OS cells expressing an inducible TRF1-FokI-ER T2 (mcherry) and co-transfected with Myc-RPA1-IRES-GFP and Flag- Polq-Helicase-IRES-TdTomato (wt or K121M). The percentage of cells expressing RPA1, and those coexpressing RPA1 and Polq-Helicase was determined prior to induction of TRF1-FokI. Mean ± S.D. from three independent experiments. Scale bars, 100 μm. (i) Western blot analysis of cells with the indicated treatment.
12 Supplementary Table 2A Translocation Junctions in Polq +/+ cells Der(6) Chr-11 Chr nt
13 Supplementary Table 2B Translocation Junctions Polq +/+ cells Der(6) Chr-11 Chr-6
14 Supplementary Table 2C Translocation Junctions in Polq +/+ cells Der(6) Chr-11 Chr nt 256 nt 130 nt
15 Supplementary Table 2D Translocation Junctions in Polq ΔPol cells Der(6) Chr-11 Chr-6
16 Supplementary Table 2E Translocation Junctions in Polq ΔPol cells Der(6) Chr-11 Chr-6
17 Supplementary Table 2F Translocation Junctions in Polq ΔHel cells Der(6) Chr-11 Chr-6
18 Supplementary Table 2G Translocation Junctions in Polq ΔHel cells Der(6) Chr-11 Chr nt 143 nt 143 nt
19 Supplementary Table 2H Translocation Junctions in Polq ΔRad51 cells Der(6) Chr-11 Chr nt 295 nt
20 Supplementary Table 2I Translocation Junctions in Polq ΔRad51 cells Der(6) Chr-11 Chr nt Supplementary Tables 2A-I: Sequences of Der (6) breakpoint junction from CCE mes cells with the indicated genotype. Reference sequence is highlighted on top. Lines represent individual translocations recovered by PCR and subject to Sanger sequencing. Nucleotide insertions are marked in red. Gaps in the sequence represent nucleotide deletions. Microhomology is denoted by blue boxes.
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