Two distinct conformational states define the interaction of human RAD51-ATP with single-stranded DNA

Ineke Brouwer; Tommaso Moschetti; Andrea Candelli; Edwige B. Garcin; Mauro Modesti; Luca Pellegrini; Gijs J.L. Wuite; Erwin J.G. Peterman

doi:10.15252/embj.201798162

Two distinct conformational states define the interaction of human RAD51-ATP with single-stranded DNA

Ineke Brouwer
, Tommaso Moschetti
, Andrea Candelli
, Edwige B. Garcin
, Mauro Modesti
, Luca Pellegrini^*
, Gijs J.L. Wuite
, Erwin J.G. Peterman

^*Corresponding author for this work

Research output: Contribution to Journal › Article › Academic › peer-review

Abstract

An essential mechanism for repairing DNA double-strand breaks is homologous recombination (HR). One of its core catalysts is human RAD51 (hRAD51), which assembles as a helical nucleoprotein filament on single-stranded DNA, promoting DNA-strand exchange. Here, we study the interaction of hRAD51 with single-stranded DNA using a single-molecule approach. We show that ATP-bound hRAD51 filaments can exist in two different states with different contour lengths and with a free-energy difference of ~4 k_BT per hRAD51 monomer. Upon ATP hydrolysis, the filaments convert into a disassembly-competent ADP-bound configuration. In agreement with the single-molecule analysis, we demonstrate the presence of two distinct protomer interfaces in the crystal structure of a hRAD51-ATP filament, providing a structural basis for the two conformational states of the filament. Together, our findings provide evidence that hRAD51-ATP filaments can exist in two interconvertible conformational states, which might be functionally relevant for DNA homology recognition and strand exchange.

Original language	English
Article number	e98162
Pages (from-to)	1-26
Number of pages	26
Journal	The EMBO Journal
Volume	37
Issue number	7
Early online date	5 Mar 2018
DOIs	https://doi.org/10.15252/embj.201798162
Publication status	Published - Apr 2018

Funding

We would like to thank Michael A. Longo for developing the hRAD51 purification protocol, based on RAD51's affinity for BRCA2 BRC4. We would like to thank Joseph Maman for helpful discussions and critical reading of the manuscript. This work was supported by VICI grants (G.J.L.W. and E.J.G.P.) from the Nederlandse Organisatie voor Wetenschappelijk Onderzoek, an European Research Council starting grant (G.J.L.W.), the French National Cancer Institute (Grant PLBIO13-103) (M.M.), the ARC Foundation for Cancer Research (M.M.), the A*MIDEX Project (no. ANR-11-IDEX-0001-02) and the “Investissements d'Avenir” French Government programme (M.M.); a postdoctoral fellowship of the Fondazione Cenci-Bolognetti to T.M.; a Wellcome Trust investigator award (104641/Z/14/Z) to L.P.; and a postdoctoral fellowship of the Fondation Aix-Marseille Université to E.B.G. We would like to thank Michael A. Longo for developing the hRAD51 purification protocol, based on RAD51’s affinity for BRCA2 BRC4. We would like to thank Joseph Maman for helpful discussions and critical reading of the manuscript. This work was supported by VICI grants (G.J.L.W. and E.J.G.P.) from the Nederlandse Organisatie voor Wetenschappelijk Onderzoek, an European Research Council starting grant (G.J.L.W.), the French National Cancer Institute (Grant PLBIO13-103) (M.M.), the ARC Foundation for Cancer Research (M.M.), the A*MIDEX Project (no. ANR-11-IDEX-0001-02) and the “Investissements d’Avenir” French Government programme (M.M.); a postdoctoral fellowship of the Fondazione Cenci-Bolognetti to T.M.; a Wellcome Trust investigator award (104641/Z/14/Z) to L.P.; and a postdoctoral fellowship of the Fondation Aix-Marseille Université to E.B.G.

Funders	Funder number
Fondazione Cenci-Bolognetti
Nederlandse Organisatie voor Wetenschappelijk Onderzoek
Fondation Aix-Marseille Universite
VICI
European Research Council
???publication-publication-funding-organisation-not-added???	ANR-11-IDEX-0001
Wellcome Trust	104641, 104641/Z/14/Z
Fondation ARC pour la Recherche sur le Cancer	ANR-11-IDEX-0001-02
Institut National Du Cancer	PLBIO13-103

Keywords

DNA repair
homologous recombination
RAD51
single-stranded DNA

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10.15252/embj.201798162

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title = "Two distinct conformational states define the interaction of human RAD51-ATP with single-stranded DNA",

abstract = "An essential mechanism for repairing DNA double-strand breaks is homologous recombination (HR). One of its core catalysts is human RAD51 (hRAD51), which assembles as a helical nucleoprotein filament on single-stranded DNA, promoting DNA-strand exchange. Here, we study the interaction of hRAD51 with single-stranded DNA using a single-molecule approach. We show that ATP-bound hRAD51 filaments can exist in two different states with different contour lengths and with a free-energy difference of \textasciitilde{}4 kBT per hRAD51 monomer. Upon ATP hydrolysis, the filaments convert into a disassembly-competent ADP-bound configuration. In agreement with the single-molecule analysis, we demonstrate the presence of two distinct protomer interfaces in the crystal structure of a hRAD51-ATP filament, providing a structural basis for the two conformational states of the filament. Together, our findings provide evidence that hRAD51-ATP filaments can exist in two interconvertible conformational states, which might be functionally relevant for DNA homology recognition and strand exchange.",

keywords = "DNA repair, homologous recombination, RAD51, single-stranded DNA",

author = "Ineke Brouwer and Tommaso Moschetti and Andrea Candelli and Garcin, \{Edwige B.\} and Mauro Modesti and Luca Pellegrini and Wuite, \{Gijs J.L.\} and Peterman, \{Erwin J.G.\}",

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AU - Brouwer, Ineke

AU - Moschetti, Tommaso

AU - Candelli, Andrea

AU - Garcin, Edwige B.

AU - Modesti, Mauro

AU - Pellegrini, Luca

AU - Wuite, Gijs J.L.

AU - Peterman, Erwin J.G.

PY - 2018/4

Y1 - 2018/4

N2 - An essential mechanism for repairing DNA double-strand breaks is homologous recombination (HR). One of its core catalysts is human RAD51 (hRAD51), which assembles as a helical nucleoprotein filament on single-stranded DNA, promoting DNA-strand exchange. Here, we study the interaction of hRAD51 with single-stranded DNA using a single-molecule approach. We show that ATP-bound hRAD51 filaments can exist in two different states with different contour lengths and with a free-energy difference of ~4 kBT per hRAD51 monomer. Upon ATP hydrolysis, the filaments convert into a disassembly-competent ADP-bound configuration. In agreement with the single-molecule analysis, we demonstrate the presence of two distinct protomer interfaces in the crystal structure of a hRAD51-ATP filament, providing a structural basis for the two conformational states of the filament. Together, our findings provide evidence that hRAD51-ATP filaments can exist in two interconvertible conformational states, which might be functionally relevant for DNA homology recognition and strand exchange.

AB - An essential mechanism for repairing DNA double-strand breaks is homologous recombination (HR). One of its core catalysts is human RAD51 (hRAD51), which assembles as a helical nucleoprotein filament on single-stranded DNA, promoting DNA-strand exchange. Here, we study the interaction of hRAD51 with single-stranded DNA using a single-molecule approach. We show that ATP-bound hRAD51 filaments can exist in two different states with different contour lengths and with a free-energy difference of ~4 kBT per hRAD51 monomer. Upon ATP hydrolysis, the filaments convert into a disassembly-competent ADP-bound configuration. In agreement with the single-molecule analysis, we demonstrate the presence of two distinct protomer interfaces in the crystal structure of a hRAD51-ATP filament, providing a structural basis for the two conformational states of the filament. Together, our findings provide evidence that hRAD51-ATP filaments can exist in two interconvertible conformational states, which might be functionally relevant for DNA homology recognition and strand exchange.

KW - DNA repair

KW - homologous recombination

KW - RAD51

KW - single-stranded DNA

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Two distinct conformational states define the interaction of human RAD51-ATP with single-stranded DNA

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