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The helicase domain of Polθ counteracts RPA to promote alt-NHEJ

Nature Structural & Molecular Biology volume 24, pages 1116–1123 (2017)Cite this article

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Abstract

Mammalian polymerase theta (Polθ) is a multifunctional enzyme that promotes error-prone DNA repair by alternative nonhomologous end joining (alt-NHEJ). Here we present structure–function analyses that reveal that, in addition to the polymerase domain, Polθ-helicase activity plays a central role during double-strand break (DSB) repair. Our results show that the helicase domain promotes chromosomal translocations by alt-NHEJ in mouse embryonic stem cells and also suppresses CRISPR–Cas9- mediated gene targeting by homologous recombination (HR). In vitro assays demonstrate that Polθ-helicase activity facilitates the removal of RPA from resected DSBs to allow their annealing and subsequent joining by alt-NHEJ. Consistent with an antagonistic role for RPA during alt-NHEJ, inhibition of RPA1 enhances end joining and suppresses recombination. Taken together, our results reveal that the balance between HR and alt-NHEJ is controlled by opposing activities of Polθ and RPA, providing further insight into the regulation of repair-pathway choice in mammalian cells.

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Figure 1: Structure–function analysis reveals the role of Polθ-helicase in alt-NHEJ.
Figure 2: Polθ inhibition increases the efficiency of HR-mediated CRISPR–Cas9 gene targeting.
Figure 3: Polθ-helicase antagonizes RPA to promote DNA annealing and alt-NHEJ in vitro.
Figure 4: Mammalian RPA inhibits alt-NHEJ.
Figure 5: The interplay between Polθ and RPA determines the outcome of DSB repair.

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Acknowledgements

We thank R. Greenberg (University of Pennsylvania) and T. de Lange (The Rockefeller University) for providing key reagents. We are grateful to A. Pinzaru and R. Barry for providing comments on the manuscript. This work was supported by grants from Pershing Square Sohn cancer research alliance (A.S.), the V-foundation (A.S.), Pew-Stewart scholars award (A.S.), and the National Institutes of Health award 1R01GM115472-01 (R.T.P.). P.A.M.-G. is supported by a fellowship from the Department of Defense (BC134020). S.K.D. is supported by an award from The Leukemia & Lymphoma Society.

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Authors and Affiliations

  1. Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, New York, USA

    Pedro A Mateos-Gomez, Sarah K Deng & Agnel Sfeir

  2. Department of Cell Biology, New York University School of Medicine, New York, New York, USA

    Pedro A Mateos-Gomez, Sarah K Deng & Agnel Sfeir

  3. Temple University, Lewis Katz School of Medicine, Philadelphia, Pennsylvania, USA

    Tatiana Kent, Shane McDevitt, Ekaterina Kashkina, Trung M Hoang & Richard T Pomerantz

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  1. Pedro A Mateos-Gomez
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Contributions

Experiments were designed by A.S., R.T.P., and P.A.M.-G. P.A.M.-G. and S.K.D. performed in vivo experiments. T.K., S.M., E.K., and T.M.H. performed in vitro experiments. A.S. wrote the manuscript. All authors discussed the results and commented on the manuscript.

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Correspondence to Agnel Sfeir.

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Agnel Sfeir is a cofounder and shareholder in Repare Therapeutics.

Integrated supplementary information

Supplementary Figure 1 Polθ structure-function analysis

(a) Inactivating polymerase and helicase mutations do not alter the stability of Polθ. 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+/+, PolqΔPol and PolqΔHel. 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.

Supplementary Figure 2 The function of Polθ-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 γ-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 Polθ 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; not-significant (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 PolqΔPol and PolqΔHel mutants. *P < 0.05 and **P < 0.01. Two-tailed Student’s t-test.

Supplementary Figure 3 In vitro analysis of Polθ-helicase function

(a) Coomassie-stained SDS-page gel of purified Polθ–helicase (right) and the trimeric RPA complex (left) (b) Phosphorimager scan of thin layer chromatography plate containing results of Polθ ATPase assay in the presence of ssDNA. (c-d) Polθ–hel promotes DNA annealing independent of ATP in the absence of RPA. (c) Schematic of assay. (d) Non-denaturing gel showing Polθ-hel annealing of complementary ssDNA substrates in the presence and absence of ATP. (e-f) In the absence of RPA, Polθ-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 Polθ–hel concentration. (g-h) Polθ–hel performs limited annealing in the presence of E. coli SSB. (g) Schematic of assay. (h) Non-denaturing gel showing Polθ–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.

Supplementary Figure 4 In vitro analysis of Polθ end-joining activity

(a) Top: Schematic of assay. Polq-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 Polθ 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 Polθ 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 from1. Non-denaturing gel showing that Polθ end-joining products are susceptible to EcoRI digestion, which confirms end-joining mechanism. (d) Polθ–helicase promotes alt-NHEJ in the presence of RPA. Left: Schematic of Polθ–pol mediated alt-NHEJ assay in the presence of RPA and Polθ–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, 230–237, doi:10.1038/nsmb.2961 (2015).

Supplementary Figure 5 Polθ–pol template dependent activity is resistant to RPA binding of ssDNA.

(a) Top: Schematic of the assay. Bottom: Denaturing gel showing Polθ–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 Polθ–pol. (b) Top: Schematic of the assay. Bottom: Denaturing gel showing Polθ–pol primer extension on a gapped DNA template in the presence of the indicated amounts of RPA.

Supplementary Figure 6 Investigating the interplay between Polθ-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 TRF1F/FTRF2F/FLig4−/−Cre-ERT 2 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 TRF1F/FTRF2F/FLig4−/−Cre-ERT2 MEFs treated with shControl and shRPA1. (d) Western blot analysis for RPA1 in TRF1F/FTRF2F/FLig4−/−Cre-ERT2 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 TRF1F/FTRF2F/FLig4−/−Cre-ERT2 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-ERT2 (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 co-expressing 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.

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Mateos-Gomez, P., Kent, T., Deng, S. et al. The helicase domain of Polθ counteracts RPA to promote alt-NHEJ. Nat Struct Mol Biol 24, 1116–1123 (2017). https://doi.org/10.1038/nsmb.3494

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