Abstract
Timely repair of chromosomal double-strand breaks is required for genome integrity and cellular viability. The polymerase theta-mediated end joining pathway has an important role in resolving these breaks and is essential in cancers defective in other DNA repair pathways, thus making it an emerging therapeutic target1. It requires annealing of 2–6 nucleotides of complementary sequence, microhomologies, that are adjacent to the broken ends, followed by initiation of end-bridging DNA synthesis by polymerase θ. However, the other pathway steps remain inadequately defined, and the enzymes required for them are unknown. Here we demonstrate requirements for exonucleolytic digestion of unpaired 3′ tails before polymerase θ can initiate synthesis, then a switch to a more accurate, processive and strand-displacing polymerase to complete repair. We show the replicative polymerase, polymerase δ, is required for both steps; its 3′ to 5′ exonuclease activity for flap trimming, then its polymerase activity for extension and completion of repair. The enzymatic steps that are essential and specific to this pathway are mediated by two separate, sequential engagements of the two polymerases. The requisite coupling of these steps together is likely to be facilitated by physical association of the two polymerases. This pairing of polymerase δ with a polymerase capable of end-bridging synthesis, polymerase θ, may help to explain why the normally high-fidelity polymerase δ participates in genome destabilizing processes such as mitotic DNA synthesis2 and microhomology-mediated break-induced replication3.
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Data availability
NGS data are available at Bioproject under assession PRJNA881334. Analysed STORM localization coordinates and relevant ROI are available on Mendeley Data (treated, https://data.mendeley.com/datasets/mg6n5r87cp/1; untreated, https://data.mendeley.com/datasets/ymrs93b9c9/1).
Code availability
MH finder code used to identify candidate TMEJ products is available on GitHub (https://github.com/aluthman/Ramsden-Lab/tree/main/Stroik_et_al). The relevant code for the NGS data analysis is available at https://github.com/aluthman/Ramsden-Lab/tree/main/Stroik_et_al. Code used to analyse STORM images is available at https://github.com/d-in-crtl/SMLM.
References
Ramsden, D. A., Carvajal-Garcia, J. & Gupta, G. P. Mechanism, cellular functions and cancer roles of polymerase-theta-mediated DNA end joining. Nat. Rev. Mol. Cell Biol. 23, 125–140 (2022).
Minocherhomji, S. et al. Replication stress activates DNA repair synthesis in mitosis. Nature 528, 286–290 (2015).
Costantino, L. et al. Break-induced replication repair of damaged forks induces genomic duplications in human cells. Science 343, 88–91 (2014).
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. Nat. Struct. Mol. Biol. 22, 230–237 (2015).
Wyatt, D. W. et al. Essential roles for polymerase theta-mediated end joining in the repair of chromosome breaks. Mol. Cell 63, 662–673 (2016).
Yousefzadeh, M. J. et al. Mechanism of suppression of chromosomal instability by DNA polymerase POLQ. PLoS Genet. 10, e1004654 (2014).
Carvajal-Garcia, J. et al. Mechanistic basis for microhomology identification and genome scarring by polymerase theta. Proc. Natl Acad. Sci. USA 117, 8476–8485 (2020).
Eckstein, F. Nucleoside phosphorothioates. Annu. Rev. Biochem. 54, 367–402 (1985).
Arana, M. E., Seki, M., Wood, R. D., Rogozin, I. B. & Kunkel, T. A. Low-fidelity DNA synthesis by human DNA polymerase theta. Nucleic Acids Res. 36, 3847–3856 (2008).
Schmitt, M. W. et al. Active site mutations in mammalian DNA polymerase delta alter accuracy and replication fork progression. J. Biol. Chem. 285, 32264–32272 (2010).
Weedon, M. N. et al. An in-frame deletion at the polymerase active site of POLD1 causes a multisystem disorder with lipodystrophy. Nat. Genet. 45, 947–950 (2013).
Hu, Z., Perumal, S. K., Yue, H. & Benkovic, S. J. The human lagging strand DNA polymerase delta holoenzyme is distributive. J. Biol. Chem. 287, 38442–38448 (2012).
Lancey, C. et al. Structure of the processive human Pol delta holoenzyme. Nat. Commun. 11, 1109 (2020).
Fleury, H. et al. The APE2 nuclease is essential for DNA double-strand break repair by microhomology-mediated end joining. Mol. Cell 83, 1429–1445.e8 (2023).
Bennardo, N., Cheng, A., Huang, N. & Stark, J. M. Alternative-NHEJ is a mechanistically distinct pathway of mammalian chromosome break repair. PLoS Genet. 4, e1000110 (2008).
Hedglin, M., Pandey, B. & Benkovic, S. J. Stability of the human polymerase delta holoenzyme and its implications in lagging strand DNA synthesis. Proc. Natl Acad. Sci. USA 113, E1777–E1786 (2016).
Hussmann, J. A. et al. Mapping the genetic landscape of DNA double-strand break repair. Cell 184, 5653–5669 e5625 (2021).
Schimmel, J., Kool, H., van Schendel, R. & Tijsterman, M. Mutational signatures of non-homologous and polymerase theta-mediated end-joining in embryonic stem cells. EMBO J. 36, 3634–3649 (2017).
Feng, W. et al. Marker-free quantification of repair pathway utilization at Cas9-induced double-strand breaks. Nucleic Acids Res. 49, 5095–5105 (2021).
Lee, K. & Lee, S. E. Saccharomyces cerevisiae Sae2- and Tel1-dependent single-strand DNA formation at DNA break promotes microhomology-mediated end joining. Genetics 176, 2003–2014 (2007).
Meyer, D., Fu, B. X. & Heyer, W. D. DNA polymerases delta and lambda cooperate in repairing double-strand breaks by microhomology-mediated end-joining in Saccharomyces cerevisiae. Proc. Natl Acad. Sci. USA 112, E6907–E6916 (2015).
Villarreal, D. D. et al. Microhomology directs diverse DNA break repair pathways and chromosomal translocations. PLoS Genet. 8, e1003026 (2012).
Takata, K. I. et al. Analysis of DNA polymerase nu function in meiotic recombination, immunoglobulin class-switching, and DNA damage tolerance. PLoS Genet. 13, e1006818 (2017).
Mann, A. et al. POLtheta prevents MRE11-NBS1-CtIP-dependent fork breakage in the absence of BRCA2/RAD51 by filling lagging-strand gaps. Mol. Cell 82, 4218–4231 e4218 (2022).
Mengwasser, K. E. et al. Genetic screens reveal FEN1 and APEX2 as BRCA2 synthetic lethal targets. Mol. Cell 73, 885–899 e886 (2019).
Boboila, C. et al. Robust chromosomal DNA repair via alternative end-joining in the absence of X-ray repair cross-complementing protein 1 (XRCC1). Proc. Natl Acad. Sci. USA 109, 2473–2478 (2012).
Masani, S., Han, L., Meek, K. & Yu, K. Redundant function of DNA ligase 1 and 3 in alternative end-joining during immunoglobulin class switch recombination. Proc. Natl Acad Sci. USA 113, 1261–1266 (2016).
Mateos-Gomez, P. A. et al. Mammalian polymerase theta promotes alternative NHEJ and suppresses recombination. Nature 518, 254–257 (2015).
Burgers, P. M. Polymerase dynamics at the eukaryotic DNA replication fork. J. Biol. Chem. 284, 4041–4045 (2009).
Levin, D. S., McKenna, A. E., Motycka, T. A., Matsumoto, Y. & Tomkinson, A. E. Interaction between PCNA and DNA ligase I is critical for joining of Okazaki fragments and long-patch base-excision repair. Curr. Biol. 10, 919–922 (2000).
Fan, J., Otterlei, M., Wong, H. K., Tomkinson, A. E. & Wilson, D. M. 3rd XRCC1 co-localizes and physically interacts with PCNA. Nucleic Acids Res. 32, 2193–2201 (2004).
Deshpande, M. et al. Error-prone repair of stalled replication forks drives mutagenesis and loss of heterozygosity in haploinsufficient BRCA1 cells. Mol. Cell 82, 3781–3793 e3787 (2022).
Llorens-Agost, M. et al. POLtheta-mediated end joining is restricted by RAD52 and BRCA2 until the onset of mitosis. Nat. Cell Biol. 23, 1095–1104 (2021).
Roerink, S. F., van Schendel, R. & Tijsterman, M. Polymerase theta-mediated end joining of replication-associated DNA breaks in C. elegans. Genome Res. 24, 954–962 (2014).
van Schendel, R., Romeijn, R., Buijs, H. & Tijsterman, M. Preservation of lagging strand integrity at sites of stalled replication by Pol alpha-primase and 9-1-1 complex. Sci. Adv. 7, eabf2278 (2021).
Wang, Z. et al. DNA polymerase theta (POLQ) is important for repair of DNA double-strand breaks caused by fork collapse. J. Biol. Chem. 294, 3909–3919 (2019).
Belan, O. et al. POLQ seals post-replicative ssDNA gaps to maintain genome stability in BRCA-deficient cancer cells. Mol. Cell 82, 4664–4680 e4669 (2022).
Heijink, A. M. et al. Sister chromatid exchanges induced by perturbed replication can form independently of BRCA1, BRCA2 and RAD51. Nat. Commun. 13, 6722 (2022).
Schrempf, A. et al. POLtheta processes ssDNA gaps and promotes replication fork progression in BRCA1-deficient cells. Cell Rep. 41, 111716 (2022).
Donnianni, R. A. et al. DNA polymerase delta synthesizes both strands during break-induced replication. Mol. Cell 76, 371–381 e374 (2019).
Layer, J. V. et al. Polymerase delta promotes chromosomal rearrangements and imprecise double-strand break repair. Proc. Natl Acad. Sci. USA 117, 27566–27577 (2020).
Wood, R. D. & Burki, H. J. Repair capability and the cellular age response for killing and mutation induction after UV. Mutat. Res. 95, 505–514 (1982).
Lange, S. S., Tomida, J., Boulware, K. S., Bhetawal, S. & Wood, R. D. The polymerase activity of mammalian DNA Pol zeta is specifically required for cell and embryonic viability. PLoS Genet. 12, e1005759 (2016).
Zatreanu, D. et al. Poltheta inhibitors elicit BRCA-gene synthetic lethality and target PARP inhibitor resistance. Nat. Commun. 12, 3636 (2021).
Kaminski, A. M. et al. Analysis of diverse double-strand break synapsis with Pollambda reveals basis for unique substrate specificity in nonhomologous end-joining. Nat. Commun. 13, 3806 (2022).
Masuda, Y. et al. Dynamics of human replication factors in the elongation phase of DNA replication. Nucleic Acids Res. 35, 6904–6916 (2007).
Luedeman, M. E. et al. Poly(ADP) ribose polymerase promotes DNA polymerase theta-mediated end joining by activation of end resection. Nat. Commun. 13, 4547 (2022).
Holden, S. J., Uphoff, S. & Kapanidis, A. N. DAOSTORM: an algorithm for high-density super-resolution microscopy. Nat. Methods 8, 279–280 (2011).
Huang, F. et al. Video-rate nanoscopy using sCMOS camera-specific single-molecule localization algorithms. Nat. Methods 10, 653–658 (2013).
Huang, F., Schwartz, S. L., Byars, J. M. & Lidke, K. A. Simultaneous multiple-emitter fitting for single molecule super-resolution imaging. Biomed. Opt. Express 2, 1377–1393 (2011).
Yin, Y., Lee, W. T. C. & Rothenberg, E. Ultrafast data mining of molecular assemblies in multiplexed high-density super-resolution images. Nat. Commun. 10, 119 (2019).
Yin, Y. et al. A basal-level activity of ATR links replication fork surveillance and stress response. Mol. Cell 81, 4243–4257.e4246 (2021).
Sengupta, P. et al. Probing protein heterogeneity in the plasma membrane using PALM and pair correlation analysis. Nat. Methods 8, 969–975 (2011).
Veatch, S. L. et al. Correlation functions quantify super-resolution images and estimate apparent clustering due to over-counting. PLoS ONE 7, e31457 (2012).
Lee, W. T. C. et al. Single-molecule imaging reveals replication fork coupled formation of G-quadruplex structures hinders local replication stress signaling. Nat. Commun. 12, 2525 (2021).
Yin, Y. & Rothenberg, E. Probing the spatial organization of molecular complexes using triple-pair-correlation. Sci. Rep. 6, 30819 (2016).
Acknowledgements
We thank K. Kayama for wild-type Polδ E. coli expression constructs, A. Guerin and Y. Li (MD Anderson) for generation of mutant cell lines, A. Averill (University of Vermont) for expression and purification of Polδ, A. Kaminski (National Institute of Environmental Health Sciences) for purification of Polλ and 1P01CA247773 Cores B and C for supporting this work. We thank K. Takata for generation of U2OS POLQ−/− cells and initiating construction of the Halo-POLQ plasmid and R. Mouery (UNC) for generating Apex2−/− cells. We thank A. Tripathy and the UNC macromolecular interactions core for help with MST experiments. We appreciate A. Pharma Limited for supplying ART558. We thank members of the Ramsden laboratory for helpful discussion and critical review of the manuscript. All trainees and investigators were supported by grant no. 1P01CA247773 (except for T.A.K.), with extra support for D.A.R. from grant no. 5U01CA097096, for S.S. from grant nos. F32CA264891 and T32CA009156, and for R.D.W. from the J. Ralph Meadows Chair in Carcinogenesis Research.
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Conceptualization of the study was developed by S.S. and D.A.R. Experiments assessing the contribution of Polδ to cellular TMEJ were performed by S.S. The assays used for specific steps in cellular TMEJ developed in part by J.C-G., A.L. and D.W.W. Super resolution microscopy was performed by S.S. and D.G. Reagents and advice were provided by D.G., E.R., W.F. and G.P.G. Assays using purified proteins were performed by A.E. and S.S. Reagents and guidance came from R.W., S.D., R.L.D., M.H. and T.A.K. The initial draft of the manuscript was written by S.S and D.A.R. Editing of the manuscript and final drafting was done by all authors.
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D.A.R. has a materials transfer agreement with Artios Pharma and used an Artios Polθ inhibitor for research purposes in this work with no financial compensation. R.D.W. owns shares in Repare Therapeutics, Inc., which also has a Polθ inhibitor.
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Extended data figures and tables
Extended Data Fig. 1 Fundamental mechanisms and validation of TMEJ.
a, Quantification of minimal and flapped TMEJ repair normalized to NHEJ with and without 2 µM Pol θi (ART558). Data are from 3 biological replicates. Data are mean ± SD. Values represent the maximum fraction of detected repair, comparing Pol θi to untreated cells. b, Quantification of TMEJ with locked nucleic acids at the indicated positions. Data are from 3 biological replicates. Data are mean ± SD. c, Schematic of TMEJ mismatched BamHI substrate to measure strand displacement of 20 and 50 bp. d, Gel of DNA substrates in a. e, Quantification of strand displacement (BamHI sensitivity) of 20 bp or 50 bp. Data is from 3 independent experiments. Data are mean ± SEM. f, Standard curve of qPCR CT values of a unflapped and flapped TMEJ product where the amount of flapped product is constant and unflapped is varied. g, Identical to f, but flapped is varied and unflapped is constant. h, Standard curve of qPCR CT values of a 45 bp and 25 bp TMEJ synthesis products where the 25 bp product is constant and the 45 bp product is varied. I, Identical to h, but the 45 bp product is varied and the 25 bp product is constant. j, Standard curve of qPCR CT values of a 70 bp and 25 bp TMEJ synthesis products where the 25 bp product is constant and the 70 bp product is varied. k, Identical to j, but the 25 bp product is varied and the 70 bp product is constant. l, Standard curve of qPCR CT values of the LBR repair signature and reference where the signature product is varied and the reference product is constant. m, Identical to l, but the reference is varied and the signature is constant.
Extended Data Fig. 2 Polymerase δ is essential for TMEJ.
a, Schematic of viral timeline to generate cell lines. b, Validation of POLD2 siRNA in RPE1 cells. Actin was used as a loading control. c, Validation of POLD3 siRNA in RPE1 cells. Actin was used as a loading control. d, Quantification of repair of a 70 bp, 5 nt flapped substrate relative to the minimal TMEJ substrate in cells depleted of POLD2 or POLD3, relative to WT. Data are from 3 biological replicates analysed with a one-way ANOVA and Dunnet’s method. Data are mean ± SD. e, Quantification of repair of the minimal TMEJ substrate in POLD1 depletion and mutant backgrounds. Data is from 5 (WT) or 6 biological replicates. Data are mean ± SD. f, Western blot validation of PolM2 expression, siScramble and shPOLD1 were included as controls. g, Western blot validation of PIPM expression, siScramble and shPOLD1 were included as controls. h, 25 nM WT human Polδ, PolM2, and ExoM was incubated with 25 nM of a 10 nt 3′ recessed 5′ labelled substrates for 10 min. I, 25 nM WT human Polδ, PolM2, and ExoM were incubated with 25 nM of 5′ labeled forked substrate with a 5 nt 3′ ssDNA overhang for 10 min. j, Polθ is inhibited (Pol θi) with 2 µM ART558. POLD1 in RPE1 cells is endogenously expressed (+) or depleted (−) by shPOLD1, or depleted by shPOLD1 and complemented by expression of ExoM, PolM, or WT POLD1. Quantification of repair of a 70 bp, 5 nt flapped substrate relative to the minimal TMEJ substrate, normalized to parental cells. Data are from 3 biological replicates analyzed with a one-way ANOVA and Dunnet’s method. Data are mean ± SD. Part of these data are also displayed in Fig. 2a.
Extended Data Fig. 3 TMEJ flap cleavage depends on Polδ.
a, Polθ is inhibited (Pol θi) with 2 µM ART558. POLD1 in RPE1 cells is endogenously expressed (+) or depleted (−) by shPOLD1 treatment, or depleted by shPOLD1 treatment and complemented by expression of PolM2. Quantification of repair of a 5 nt flapped substrate relative to the minimal TMEJ substrate, normalized to WT. Data are from 3 biological replicates analyzed with a one-way ANOVA and Dunnet’s method. Data are mean ± SD. b, Quantification of repair of a 2 nt flapped substrate performed as in a. c, Quantification of repair of a 10 nt flapped substrate performed as in a. d, Western blot validation of APEX2−/− cells, Vinculin was used as a loading control. e, Quantification of repair of a 5 nt flapped substrate performed as in a, with WT and two independently generated APEX2−/− clones. f, Quantification of repair of a 5 nt flapped substrate performed as in a, with WT and ERCC1−/− cells. g, Quantification of a templated insertion product at the chromosomal rosa26e locus in MEFs from a Cas9-induced DSB. Experiments were performed with Polθ inhibited (Pol θi), APEX2−/−, and WT MEF cells. Data are from 3 biological replicates and normalized to a chromosomal reference. Data are mean ± SD. The dashed line indicates an independent experiment.
Extended Data Fig. 4 Polδ performs processive synthesis in TMEJ.
a, Polθ is inhibited (Pol θi) with 2 µM ART558. POLD1 in RPE1 cells is endogenously expressed (+) or depleted (−) by shPOLD1, or depleted by shPOLD1 and complemented by expression of PolM2. Quantification of repair of a 70 nt synthesis substrate relative to the minimal TMEJ substrate, normalized to parental cells, with PolM2 complemented cells. Data are from 3 biological replicates analyzed with a one-way ANOVA and Dunnet’s method. Data are mean ± SD. b, Quantification of repair of a 45 bp synthesis substrate relative to the minimal TMEJ substrate, normalized to WT. Data are from 3 biological replicates analyzed with a one-way ANOVA and Dunnet’s method. Data are mean ± SD. c, Quantification of strand displacement in TMEJ repair substrates requiring 45 bp of synthesis. Relative joining efficiency for each background assessed was also measured. d, Western blot of POLD1 knockdown in WT MEF cells. siScramble was used as a technical control, Actin was used as a loading control. e, Western blot validation of siPCNA in RPE1 cells, siScramble was used as a technical control and Actin as a loading control. f, Quantification of repair of a 70 bp, 5 nt flapped substrate relative to the minimal TMEJ substrate, normalized to WT, with PolN−/− cells. Data are from 3 biological replicates analyzed with a one-way ANOVA and Dunnet’s method. Data are mean ± SD. g, Quantification of repair of a 70 nt, 5 bp flapped substrate relative to the minimal TMEJ substrate, normalized to REV3L−/+, with REV3L−/− cells. REV3L is the gene name for Pol zeta. Data are from 3 biological replicates analyzed with a one-way ANOVA and Dunnet’s method. Data are mean ± SD.
Extended Data Fig. 5 Chromosomal LBR reporter characterization and controls.
a, Predicted microhomology-mediated deletion repair products at the LBR locus. b, Sequence alignments of predicted microhomology-mediated deletion repair intermediates. c, Quantification of TMEJ at a terminal, chromosomal MH by qPCR. RPE1 cells were untreated (−) or treated with Pol θi (+). POLD1 in RPE1 cells is endogenously expressed (+) or depleted (−) by shPOLD1 treatment. Data are from 3 biological replicates analyzed with a one-way ANOVA and Dunnet’s method. Data are mean ± SD. d, Quantification of TMEJ at a terminal, chromosomal MH by qPCR. RPE1 cells were either depleted of Polθ (Pol θi) or POLD2/3 by siRNA. e, NHEJ repair quantification performed as in c. f, NHEJ repair quantification performed as in d. g, Western blot of shPOLE treated RPE1 cells and an untreated control. Actin was used as a loading control. h, Quantification of the terminal TMEJ repair product at LBR relative to the signature NHEJ product for WT, shPOLD1, and shPOLE treated RPE1 cells. Bars represent data mean and SD. i, Representative flow cytometry cell cycle plot of shPOLD1 transduced RPE1 cells with EdU and DAPI staining. j, Cell cycle profiles of shPOLD1 and shPOLE transduced cells, relative to RPE1 WT.
Extended Data Fig. 6 Polδ physically associates with Polθ by Co-IP.
a, Extracts of RPE1 cells expressing Halo-Polθ were immunoprecipitated with an antibody to Halo (αHalo) or without antibody (−), and recovery probed with an antibody to POLE (αPOLE). b, Co-IP of FLAG-tagged domains of Polθ in POLQ−/− U2OS cells. Polθ domains were pulled down with an antibody to FLAG, and recovery probed with a POLD1 antibody. Negative controls include parallel experiments using cells not expressing FLAG tagged constructs or cells expressing FLAG-tagged constructs but with αFLAG omitted. c, Co-IP of FLAG-tagged domains of Polθ in POLQ−/− U2OS cells. Polθ domains were pulled down with an antibody to FLAG, and recovery probed with a POLE antibody. Cells expressing FLAG-tagged constructs but with αFLAG omitted were used as negative controls. d, Protein lysates and DNA ladder incubated with and without benzonase. e, Extracts of RPE1 cells expressing FLAG-mNeonGreen or no construct were immunoprecipitated with a FLAG antibody and recovery was probed with a POLD1 antibody (αPOLD1). f, Western blot of FLAG-tagged Polθ domains in RPE1 cells expressing Myc-tagged POLD1 constructs. Actin was used as a loading control. g, Polθ is inhibited (Pol θi) with 2 µM ART558. POLD1 in RPE1 cells is endogenously expressed (+) or depleted (−) by shPOLD1 treatment, or depleted by shPOLD1 treatment and complemented by expression of POLD1 cDNA. Quantification of repair of a 5 nt flapped, 70 nt ssDNA substrate relative to the minimal TMEJ substrate, normalized to WT. Data are from 3 biological replicates analyzed with a one-way ANOVA and Dunnet’s method. Data are mean ± SD. h, Western blot of Myc-tagged POLD1 constructs used in RPE1 cells for functional assays in g. Actin was used as a loading control.
Extended Data Fig. 7 Polδ physically associates with Polθ by Microscale thermophoresis.
a, Protein gel of Polδ holoenzyme purification showing every second fraction which were all collectively pooled for downstream applications. Dark lines indicate cropped lanes. b, Extrachromosomal TMEJ repair frequency relative to NHEJ repair in Polθ-null cells complemented with either full length Halo-tagged or Polθ ΔCen. Data represents 3 biological replicates. Bars represent data means and SDs. c, Protein gel of Polθ ΔCen and Polλ purifications used for downstream applications. Dark lines indicate cropped lanes. d, Microscale thermophoresis of labeled Polθ ΔCen with varied amounts of Polλ. Fnorm was plotted as binding was not concluded. Data are mean ± SEM.
Extended Data Fig. 8 Polδ associates with Polθ at sites of damage.
a, Representative STORM images of 53BP1, POLD1, and Polθ in cells untreated (−NCS) or treated with Neocarzinostatin (+NCS). Scale bar = 1500 nm. b, Images from a, analyzed after ROIs were randomized or experimentally acquired. Data are from 3 biological replicates analyzed with an unpaired Mann Whitney test. All violin plots display data median (solid line) and quartiles (dashed lined). N = 55 nuclei. c, Images from a, analyzed after ROIs were randomized or experimentally acquired. Data are from 3 biological replicates analyzed with an unpaired Mann Whitney test. N = 191 (−NCS) and 210 (+NCS) ROIs. (d) Nuclear density of POLD1, Polθ, and 53BP1 with and without damage (+/− NCS). Data are from 3 biological replicates and analyzed with an unpaired Mann Whitney test. N = 96 (-NCS) and 105 ( + NCS) nuclei. e, Uncropped western blot of POLD1 from Fig. 2b showing antibody specificity. Endogenous RPE1 POLD1 expression (Lane 1-2), shPOLD1 in RPE1 (Lane 3), and exogenous expression of shRNA-resistant POLD1 constructs (Lane 4–6). f, Representative STORM images of POLD1 staining in an S phase (Edu positive) and non-S phase cell (EdU negative). g, Representative STORM images of Halo ligand staining in RPE1 Polθ and RPE1 Halo-Polθ both −/+ NCS treatment in an S phase nuclei. h, Quantification of 53BP1 nuclear density detected by antibody with STORM in U2OS WT and 53BP1−/− cells with and without NCS damage induction. Data was analyzed with an unpaired Mann Whitney test. N = 52 (WT), 57, (WT + NCS), 47 (53BP1−/−), 47 (53BP1−/− + NCS) ROIs.
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Stroik, S., Carvajal-Garcia, J., Gupta, D. et al. Stepwise requirements for polymerases δ and θ in theta-mediated end joining. Nature 623, 836–841 (2023). https://doi.org/10.1038/s41586-023-06729-7
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DOI: https://doi.org/10.1038/s41586-023-06729-7
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