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

Nature Structural & Molecular Biology 24, 1116–1123 (2017) | Download Citation

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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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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.

Author information

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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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.

Competing interests

Agnel Sfeir is a cofounder and shareholder in Repare Therapeutics.

Corresponding author

Correspondence to Agnel Sfeir.

Integrated supplementary information

Supplementary figures

  1. 1.

    Polθ structure-function analysis

  2. 2.

    The function of Polθ-helicase and polymerase during homologous recombination

  3. 3.

    In vitro analysis of Polθ-helicase function

  4. 4.

    In vitro analysis of Polθ end-joining activity

  5. 5.

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

  6. 6.

    Investigating the interplay between Polθ-helicase and RPA during alt-NHEJ

Supplementary information

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  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–6 and Supplementary Tables 1–2

  2. 2.

    Life Sciences Reporting Summary

  3. 3.

    Supplementary Data Set 1

    Supplementary Data Set 1.

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DOI

https://doi.org/10.1038/nsmb.3494

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