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Distinct RPA domains promote recruitment and the helicase-nuclease activities of Dna2

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  • Ananya Acharya

    (Università della Svizzera italiana (USI), Faculty of Biomedical Sciences
    Eidgenössische Technische Hochschule (ETH))

  • Kristina Kasaciunaite

    (Universität Leipzig)

  • Martin Göse

    (Universität Leipzig)

  • Vera Kissling

    (Eidgenössische Technische Hochschule (ETH))

  • Raphaël Guérois

    (Institute for Integrative Biology of the Cell (I2BC), Commissariat à l’Energie Atomique, CNRS, Université Paris-Sud, Université Paris-Saclay)

  • Ralf Seidel

    (Universität Leipzig)

  • Petr Cejka

    (Università della Svizzera italiana (USI), Faculty of Biomedical Sciences
    Eidgenössische Technische Hochschule (ETH))

Abstract

The Dna2 helicase-nuclease functions in concert with the replication protein A (RPA) in DNA double-strand break repair. Using ensemble and single-molecule biochemistry, coupled with structure modeling, we demonstrate that the stimulation of S. cerevisiae Dna2 by RPA is not a simple consequence of Dna2 recruitment to single-stranded DNA. The large RPA subunit Rfa1 alone can promote the Dna2 nuclease activity, and we identified mutations in a helix embedded in the N-terminal domain of Rfa1 that specifically disrupt this capacity. The same RPA mutant is instead fully functional to recruit Dna2 and promote its helicase activity. Furthermore, we found residues located on the outside of the central DNA-binding OB-fold domain Rfa1-A, which are required to promote the Dna2 motor activity. Our experiments thus unexpectedly demonstrate that different domains of Rfa1 regulate Dna2 recruitment, and its nuclease and helicase activities. Consequently, the identified separation-of-function RPA variants are compromised to stimulate Dna2 in the processing of DNA breaks. The results explain phenotypes of replication-proficient but radiation-sensitive RPA mutants and illustrate the unprecedented functional interplay of RPA and Dna2.

Suggested Citation

  • Ananya Acharya & Kristina Kasaciunaite & Martin Göse & Vera Kissling & Raphaël Guérois & Ralf Seidel & Petr Cejka, 2021. "Distinct RPA domains promote recruitment and the helicase-nuclease activities of Dna2," Nature Communications, Nature, vol. 12(1), pages 1-15, December.
  • Handle: RePEc:nat:natcom:v:12:y:2021:i:1:d:10.1038_s41467-021-26863-y
    DOI: 10.1038/s41467-021-26863-y
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    References listed on IDEAS

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    Cited by:

    1. Ananya Acharya & Hélène Bret & Jen-Wei Huang & Martin Mütze & Martin Göse & Vera Maria Kissling & Ralf Seidel & Alberto Ciccia & Raphaël Guérois & Petr Cejka, 2024. "Mechanism of DNA unwinding by MCM8-9 in complex with HROB," Nature Communications, Nature, vol. 15(1), pages 1-18, December.

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