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Mechanism of microhomology-mediated end-joining promoted by human DNA polymerase θ

Nature Structural & Molecular Biology volume 22, pages 230–237 (2015)Cite this article

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Abstract

Microhomology-mediated end-joining (MMEJ) is an error-prone alternative double-strand break–repair pathway that uses sequence microhomology to recombine broken DNA. Although MMEJ has been implicated in cancer development, the mechanism of this pathway is unknown. We demonstrate that purified human DNA polymerase θ (Polθ) performs MMEJ of DNA containing 3′ single-strand DNA overhangs with ≥2 bp of homology, including DNA modeled after telomeres, and show that MMEJ is dependent on Polθ in human cells. Our data support a mechanism whereby Polθ facilitates end-joining and microhomology annealing, then uses the opposing overhang as a template in trans to stabilize the DNA synapse. Polθ exhibits a preference for DNA containing a 5′-terminal phosphate, similarly to polymerases involved in nonhomologous end-joining. Finally, we identify a conserved loop domain that is essential for MMEJ and higher-order structures of Polθ that probably promote DNA synapse formation.

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Figure 1: Polθ promotes microhomology-mediated end-joining in vitro and in vivo.
Figure 2: Polθ uses the opposing overhang as a template in trans to stabilize the DNA synapse.
Figure 3: Template preferences for Polθ MMEJ.
Figure 4: Polθ promotes MMEJ of DNA containing internal microhomology.
Figure 5: Polθ promotes DNA synapse formation and strand annealing separately from its replication function.
Figure 6: Insertion loop 2 promotes microhomology-mediated end-joining, DNA binding and formation of polymerase complexes.
Figure 7: Models of Polθ MMEJ.

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Acknowledgements

Research was funded by US National Institutes of Health grant (4R00CA160648-03) awarded to R.T.P. and Temple University School of Medicine start-up funds to R.T.P. We thank S. Wallace (University of Vermont) for wild-type Polθ and Polθ L2 expression vectors, J. Stark (Beckman Research Institute, City of Hope) for the U2OS cell line EJ2-GFP24 and A.K. Aggarwal (Mount Sinai Hospital) for Polκ.

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Authors and Affiliations

  1. Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, Pennsylvania, USA

    Tatiana Kent, Gurushankar Chandramouly, Shane Michael McDevitt, Ahmet Y Ozdemir & Richard T Pomerantz

  2. Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania, USA

    Tatiana Kent, Gurushankar Chandramouly, Shane Michael McDevitt, Ahmet Y Ozdemir & Richard T Pomerantz

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  1. Tatiana Kent
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  2. Gurushankar Chandramouly
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  3. Shane Michael McDevitt
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  4. Ahmet Y Ozdemir
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Contributions

R.T.P., T.K., G.C., S.M.M. and A.Y.O. performed the experiments. R.T.P. designed the experiments and wrote the paper.

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Correspondence to Richard T Pomerantz.

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Integrated supplementary information

Supplementary Figure 1 Further analysis of Polθ MMEJ products.

a, Time course of Polθ MMEJ. (Left panels) Non-denaturing gels showing MMEJ time course with indicated pssDNA. (Right) Plot of MMEJ extension products. % MMEJ = intensity of MMEJ products (upper band (*))/total intensity in each lane. b, Restriction analysis of Polθ MMEJ products. pssDNA with half EcoRI site (left). Schematic of assay (middle). Non-denaturing gel showing MMEJ products following incubation with (lane 3) or without (lane 2) EcoRI (right). c, Length analysis of Polθ MMEJ products. pssDNA-4 substrates of different lengths (left). Schematic of assay (middle). Non-denaturing gel showing MMEJ in the presence of 100 nM pssDNA-4A and indicated concentrations of pssDNA-4B.

Supplementary Figure 2 Polθ performs efficient snap-back replication.

a, Models of Polθ extension of ssDNA. (Left) Polθ template independent extension of ssDNA via terminal transferase activity would allow for the incorporation of any dNTP. (Middle) Polθ template dependent extension in cis via ‘snap-back’ replication would limit incorporation to a dNTP (dGTP) complementary to the template sequence (poly-dC). (Right) Polθ template dependent extension in trans via non-homologous end-joining would allow incorporation of a dNTP (dATP) complementary to the opposing template sequence (poly-dT). b, Poly-dC and poly-dT ssDNA templates (top). Denaturing gels showing Polθ extension products in the presence of indicated dNTPs and DNA substrates (bottom). Efficient ssDNA extension exclusively in the presence of dGTP demonstrates Polθ ‘snap-back’ replication activity (right) while ruling out terminal transferase and non-homologous end-joining activities. c, Evidence for ‘snap-back’ replication on non-homo-polynucleotide ssDNA. (Left) Non-denaturing gel showing MMEJ with the indicated substrate and all 4 dNTPs. (Middle) Non-denaturing gel showing Polθ extension in the presence of indicated dNTPs for the indicated times. Preferential incorporation of dCTP and dTTP confirm ‘snap-back’ replication. Models of Polθ ‘snap-back’ replication (right). d, Evidence for ‘snap-back’ replication on telomere-like pssDNA. Non-denaturing gel showing MMEJ with all 4 dNTPs and the indicated pssDNA (left). The major small molecular weight byproduct is generated by ‘snap-back’ replication as indicated by displacement of the short 25 nt strand by Polθ (right). (Right) Non-denaturing gel showing MMEJ on the indicated pssDNA radio-labeled on the 25 nt strand. Displacement of the 25 nt strand (lane 2) and the small size of the majority of Polθ products produced in the left panel (lane 2) indicates ‘snap-back’ replication. Limited extension of the 25 nt strand also indicates ‘snap-back’ replication (lane 4).

Supplementary Figure 3 Control for polymerase activity on a primer template.

Non-denaturing gel showing primer extension in the presence of indicated Pols and primer-template.

Supplementary Figure 4 Polθ promotes MMEJ of pssDNA-containing internal microhomology relatively far from the 3′ ends of overhangs.

pssDNA substrates (left). Non-denaturing gel showing MMEJ in the presence of the indicated pssDNA substrates (middle). Model of MMEJ (right). Orange boxes highlight microhomology. * = 32P, * = MMEJ products.

Supplementary Figure 5 Structural and functional analysis of insertion loop 2.

a, Polθ L2 binds ssDNA. Non-denaturing gels showing EMSA with Polθ WT (left) and Polθ L2 (right) on Cy3 conjugated ssDNA (top). b, Polθ L2 fails to extend ssDNA. Denaturing gel showing a time course of ssDNA extension on the indicated substrate in the presence of Polθ WT (lanes 2–5) and Polθ L2 (lanes 6–9). c, Loop 2 promotes strand annealing. Schematic of annealing assay (left). Non-denaturing gel showing ssDNA annealing in the presence of Polθ WT (lane 4) and Polθ L2 (lane 3). Error bars, s.d. (n = 3 independent experiments). % annealing = (intensity of upper band)/(sum of the intensities of upper and lower bands). d, Loop 2 promotes Polθ dimers and multimers. (Left) Gel filtration profile of Polθ WT (90 kDa). The presence of monomers, dimers and multimers are indicated. (Middle panels) Native gel of Polθ WT (90 kDa; left) and Polθ L2 (83 kDa; right). The presence of a single band demonstrates that Polθ L2 behaves as a monomer (right); Polθ WT migrates as multiple bands, demonstrating complexes (left). (Right panel) SDS gel of Polθ WT (90 kDa; lane 1) and Polθ L2 (83 kDa; lane 2).

Supplementary Figure 6 Structural model of Polθ superimposed on Bacillus Pol I–DNA complex.

a, Superposition of Bacillus Pol I structure (blue; PDB code 4DQQ)31 in complex with primer-template (orange) and Polθ model (grey; residues 1944-2590) assembled by Swiss Model server (http://swissmodel.expasy.org)30 using Bacillus Pol I:DNA structure (PDB code 4DQQ)31 as a template. Loop 2 lies in between palm and thumb domains and is in close proximity to the 3’ terminus of the primer. b, Critical conserved residues involved in polymerase activity (Asp 539, 749) and fidelity (Tyr 600) are closely aligned with homologous residues in Bacillus Pol I, demonstrating the validity of the model. Bacillus Pol I residues indicated in parentheses. c, Loop 2 lies in close proximity to the 3’ terminus of the primer. Orange, DNA. Blue, positively charged amino acids (K, R). Green, hydrophobic amino acids. Red, negatively charged amino acids (D, E)

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Kent, T., Chandramouly, G., McDevitt, S. et al. Mechanism of microhomology-mediated end-joining promoted by human DNA polymerase θ. Nat Struct Mol Biol 22, 230–237 (2015). https://doi.org/10.1038/nsmb.2961

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