Abstract
DNA double-strand breaks occur daily in all human cells and must be repaired with high fidelity to minimize genomic instability. Deficiencies in high-fidelity DNA repair by homologous recombination lead to dependence on DNA polymerase θ, which identifies DNA microhomologies in 3′ single-stranded DNA overhangs and anneals them to initiate error-prone double-strand break repair. The resulting genomic instability is associated with numerous cancers, thereby making this polymerase an attractive therapeutic target. However, despite the biomedical importance of polymerase θ, the molecular details of how it initiates DNA break repair remain unclear. Here, we present cryo-electron microscopy structures of the polymerase θ helicase ___domain bound to microhomology-containing DNA, revealing DNA-induced rearrangements of the helicase that enable DNA repair. Our structures show that DNA-bound helicase dimers facilitate a microhomology search that positions 3′ single-stranded DNA ends in proximity to align complementary bases and anneal DNA microhomology. We characterize the molecular determinants that enable the helicase ___domain of polymerase θ to identify and pair DNA microhomologies to initiate mutagenic DNA repair, thereby providing insight into potentially targetable interactions for therapeutic interventions.
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Data availability
Cryo-EM maps and associated atomic models were deposited to the EMDB and the PDB, respectively, with the following accession codes: apo PolθH tetramer, EMD-44534, PDB 9BH6; apo PolθH dimer, EMD-44535, PDB 9BH7; PolθH–DNA microhomology searching, EMD-44536, PDB 9BH8; PolθH–DNA microhomology aligning, EMD-44537, PDB 9BH9; PolθH–DNA microhomology annealed, EMD-44538, PDB 9BHA. The D2 symmetric tetramer map and the locally refined tetramer protomer map are available as additional maps under EMD-44534. The C2 symmetric dimer map and the locally refined dimer protomer map are available as additional maps under EMD-44535. Source data are provided with this paper.
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Acknowledgements
We thank J. C. Ducom at Scripps Research high-performance computing and C. Bowman at Scripps Research for computational support, as well as W. Lessin at the Scripps Research EM facility for microscopy support. Research reported in this publication was supported by the National Cancer Institute of the National Institutes of Health (NIH) under award number F32CA288144 (C.J.Z.). G.C.L. is supported by NIH grant GM14305 and the work used equipment supported by NIH grant S10OD032467.
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Contributions
C.J.Z. prepared all the cryo-EM samples, collected the data, produced the high-resolution structures, built all the atomic models, performed all the biochemical experiments, and wrote the paper. Y.B., B.A.S.-A., and T.G. provided initial support on the PolθH–DNA binding studies and the native PAGE assay. C.J.Z. and G.C.L. designed all the experiments and performed all the mechanistic interpretation. G.C.L. provided guidance in the cryo-EM data collection and analyses and edited the paper.
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Y.B., B.A.S.-A. and T.G. are employees of MOMA Therapeutics. The remaining authors declare no competing interests.
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Extended data
Extended Data Fig. 1 Comparison of apo PolθH tetramer cryo-EM and crystal structures.
a, Coomassie-stained SDS-PAGE of proteins used in this study. This experiment was independently repeated three times with similar results. WT: PolθH amino acids 2–894. ΔLH: PolθH with amino acids 838–860 deleted. ΔD5: PolθH amino acids 2–789. 2 µg of protein was loaded in each well. b, In the PolθH tetramer cryo-EM structure, two dimeric PolθH subunits are rotated about 4º with respect to the crystal structure (PDB: 5A9J). Residues 35–66 are omitted from the crystal structure representation.
Extended Data Fig. 2 Native PAGE screening for suitable substrates for PolθH-DNA cryo-EM studies.
a, An example gel from Native PAGE screening for DNA substrates that bind PolθH, consecutively stained with Diamond DNA stain (top) and Coomassie protein stain (bottom). This experiment was independently repeated three times with similar results. Gel migration of each species is labeled on the left, and the key is on the right. MH = microhomology. b, The stem-loop DNA species pursued for structural studies, with 6 bases of self-complementary microhomology (green) at the end of the 3′ overhang.
Extended Data Fig. 3 3′ ssDNA encounters a negatively charged patch on the C-terminal helix of D5 at the PolθH DNA tunnel exit.
DNA from the PolθH-DNA searching model is merged with the apo PolθH dimer model. The D5 surface of one PolθH protomer is colored by electrostatic potential, and the ratchet helix is colored pink.
Extended Data Fig. 4 DNA binding to PolθH causes two protomers to hinge apart, destabilizing the PolθH tetramer.
a, Surface representations of the atomic models of the apo and DNA-bound PolθH dimers are shown after aligning the lower subunit (colored red) of each. The upper subunits are colored blue, with the apo subunit semi-transparent. Upon DNA-binding, the two PolθH protomers hinge 14° apart from each other. For clarity, DNA and D5 (residues 790–894) are omitted from each surface representation. b, On the left, the apo tetramer is shown as a gray tube representation, with one dimer colored as in a and highlighted with a semi-transparent molecular surface. On the right, a DNA-bound dimer (colored and highlighted with DNA omitted) is overlaid on an apo tetramer (gray), aligned to the lower red protomer. The overlay demonstrates that upon DNA binding, the hinging and the D5 conformational change introduce steric clashes with the adjacent and diagonal tetramer protomers (clashing residues colored magenta), destabilizing the PolθH tetramer. Atoms less than 2 Å apart are considered to be clashing.
Extended Data Fig. 5 Representative cryo-EM density of PolθH.
Cryo-EM map (DNA microhomology annealed state) and model of one DNA-bound PolθH protomer with domains colored, surrounded by representative map density from alpha helices in each PolθH ___domain. Domain 1 (D1): residues 147–161. Domain 2 (D2): residues 347–360. Domain 3 (D3): residues 529–540. Domain 4 (D4): residues 745–769. Domain 5 (D5): residues 872–890. ssDNA: bases -2 to +8. dsDNA is in the same pose as in the central protomer.
Extended Data Fig. 6 Comparison of DNA-bound structures of PolθH and A. fulgidus Hel308.
a, PolθH-DNA structure (red and yellow) overlaid with A. fulgidus Hel308-DNA structure (PDB: 2P6R, purple and green). b, The unwinding loop of A. fulgidus Hel308 (purple, with residues shown) is larger than the equivalent PolθH loop (red). In the A. fulgidus Hel308 unwinding loop, F351 and Y354 are positioned to form pi-stacking interactions with DNA bases. Some bases in the DNA duplex have been hidden for clarity. c, R592 and W599 in the A. fulgidus Hel308 ratchet helix are positioned to stack with DNA. d, V757 and M761 in the PolθH ratchet helix wedge between DNA bases +5 and +6.
Extended Data Fig. 7 Apo cryo-EM data collection and initial processing.
Data collection and initial processing described for apo PolθH. CTF correction was performed in cryoSPARC live, and micrographs were exported to cryoSPARC for particle picking, extraction, and further processing based on particle presence in 2D and map homogeneity in 3D as described in the methods. One representative micrograph is shown with picked particles in red. For 2D sorting jobs, representative classes are shown. Initial models were created Ab initio from particles.
Extended Data Fig. 8 Apo cryo-EM image analysis workflow.
Representative processing workflow described for apo PolθH. For 3D sorting jobs, representative classes are shown. In this scheme, Ab initio models were utilized as volume inputs for iterative heterogeneous refinement, which separated tetrameric and dimeric PolθH particles. Non-uniform refinement was performed with symmetry imposed for both species to produce symmetric reconstructions. In addition, one protomer from each species was masked and symmetry expanded, subject to 3D variability analysis and/or 3D classification without alignment, and a consensus protomer of both species was locally refined. This yielded final protomer reconstructions both shown adjacent to an angular distribution plot and a 3DFSC histogram with the map-to-model FSC overlaid. To generate each final ‘combined’ map, the locally refined protomer was multiplied by its protomer mask, copied, and fit to the appropriate symmetric map.
Extended Data Fig. 9 PolθH-DNA cryo-EM data collection and initial processing.
Data collection and initial processing described for DNA-bound PolθH. CTF correction was performed in cryoSPARC live, and micrographs were exported to cryoSPARC for particle picking, extraction, and further processing based on particle presence in 2D and map homogeneity in 3D as described in the methods. One representative micrograph is shown with picked particles in red. For 2D sorting jobs, representative classes are shown. The initial model was created Ab initio from particles.
Extended Data Fig. 10 PolθH-DNA cryo-EM image analysis workflow.
Representative processing workflow described for DNA-bound PolθH. For 3D sorting jobs, representative classes are shown. In this scheme, the Ab initio model was utilized as a volume input for iterative heterogeneous refinement. Particles with apparent DNA density were subject to iterative 3D variability analysis with a mask encompassing the most heterogeneous regions to eliminate particles with no D5 density and poor DNA density. Particles were sorted into three groups based on DNA conformation (colored) and subject to non-uniform refinement to produce final reconstructions of PolθH in three different DNA pairing states, each above an angular distribution plot and a 3DFSC histogram with the map-to-model FSC overlaid. As some DNA density disappears upon map sharpening, a locally filtered map is also shown for each reconstruction.
Supplementary information
Supplementary Information
Supplementary Table 1 and Figs. 1 and 2.
Supplementary Video 1
PolθH undergoes conformational rearrangements to facilitate microhomology annealing.
Source data
Source Data Fig. 3
Statistical source data.
Source Data Fig. 3
Unprocessed native PAGE gels.
Source Data Extended Data Fig. 1
Unprocessed SDS–PAGE protein gel.
Source Data Extended Data Fig. 2
Unprocessed native PAGE gels.
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Zerio, C.J., Bai, Y., Sosa-Alvarado, B.A. et al. Human polymerase θ helicase positions DNA microhomologies for double-strand break repair. Nat Struct Mol Biol 32, 1061–1068 (2025). https://doi.org/10.1038/s41594-025-01514-8
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DOI: https://doi.org/10.1038/s41594-025-01514-8
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