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Probing Rad51-DNA interactions by changing DNA twist.

Atwell S, Disseau L, Stasiak AZ, Stasiak A, Renodon-Cornière A, Takahashi M, Viovy JL, Cappello G - Nucleic Acids Res. (2012)

Bottom Line: The stretching mode is observed when DNA is unwound towards a helical repeat of 18.6 bp/turn, whereas a non-stretching mode is observed when DNA molecules are not permitted to change their native helical repeat.We also show that the two forms of complexes are interconvertible and that by enforcing changes of DNA twist one can induce transitions between the two forms.Our observations permit a better understanding of the role of ATP hydrolysis in Rad51-mediated homologous pairing and strand exchange.

View Article: PubMed Central - PubMed

Affiliation: Institut Curie, Centre de Recherche-Physico-Chimie-Curie, CNRS UMR168, Université Pierre et Marie Curie, Paris F-75231, France.

ABSTRACT
In eukaryotes, Rad51 protein is responsible for the recombinational repair of double-strand DNA breaks. Rad51 monomers cooperatively assemble on exonuclease-processed broken ends forming helical nucleo-protein filaments that can pair with homologous regions of sister chromatids. Homologous pairing allows the broken ends to be reunited in a complex but error-free repair process. Rad51 protein has ATPase activity but its role is poorly understood, as homologous pairing is independent of adenosine triphosphate (ATP) hydrolysis. Here we use magnetic tweezers and electron microscopy to investigate how changes of DNA twist affect the structure of Rad51-DNA complexes and how ATP hydrolysis participates in this process. We show that Rad51 protein can bind to double-stranded DNA in two different modes depending on the enforced DNA twist. The stretching mode is observed when DNA is unwound towards a helical repeat of 18.6 bp/turn, whereas a non-stretching mode is observed when DNA molecules are not permitted to change their native helical repeat. We also show that the two forms of complexes are interconvertible and that by enforcing changes of DNA twist one can induce transitions between the two forms. Our observations permit a better understanding of the role of ATP hydrolysis in Rad51-mediated homologous pairing and strand exchange.

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Electron micrographs of Rad51-dsDNA complexes in stretched (A, B) and non-stretched form (C–E).
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gks1131-F3: Electron micrographs of Rad51-dsDNA complexes in stretched (A, B) and non-stretched form (C–E).

Mentions: In principle, the perceived extension and shrinking as reported in Figure 1F could be also explained by plectonemic coiling of entire Rad51-DNA filaments. Because these filaments reach torsionally relaxed state at σ of ∼−0.43 they would not form plectonemes at this σ, but as the DNA is wound towards σ = 0 the entire filaments could fold on themselves and form plectonemes. However, such a plectonemic coiling of the entire filaments is expected to give a much higher slope of perceived length change than this is the case of protein-free DNA. This expectation results from the fact that the stiffness of Rad51-dsDNA filaments is ca. 10 times higher than that of protein-free DNA and the physical diameter of filaments is ca. 5 times bigger than that of protein-free DNA (5). The observed slope of the extension change is, however, less steep than that of overwound DNA forming plectonemes, where a much smaller number of DNA rotations resulted in bringing together two ends of plectonemically coiled DNA molecules (see inset in Figure 1F). In addition, electron micrographs of Rad51-dsDNA complexes formed under comparable conditions on torsionally constrained plasmids (see Figure 3) revealed them to be essentially free from plectonemes, thus suggesting that the torque induced by Rad51 binding to DNA is weak.


Probing Rad51-DNA interactions by changing DNA twist.

Atwell S, Disseau L, Stasiak AZ, Stasiak A, Renodon-Cornière A, Takahashi M, Viovy JL, Cappello G - Nucleic Acids Res. (2012)

Electron micrographs of Rad51-dsDNA complexes in stretched (A, B) and non-stretched form (C–E).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3526263&req=5

gks1131-F3: Electron micrographs of Rad51-dsDNA complexes in stretched (A, B) and non-stretched form (C–E).
Mentions: In principle, the perceived extension and shrinking as reported in Figure 1F could be also explained by plectonemic coiling of entire Rad51-DNA filaments. Because these filaments reach torsionally relaxed state at σ of ∼−0.43 they would not form plectonemes at this σ, but as the DNA is wound towards σ = 0 the entire filaments could fold on themselves and form plectonemes. However, such a plectonemic coiling of the entire filaments is expected to give a much higher slope of perceived length change than this is the case of protein-free DNA. This expectation results from the fact that the stiffness of Rad51-dsDNA filaments is ca. 10 times higher than that of protein-free DNA and the physical diameter of filaments is ca. 5 times bigger than that of protein-free DNA (5). The observed slope of the extension change is, however, less steep than that of overwound DNA forming plectonemes, where a much smaller number of DNA rotations resulted in bringing together two ends of plectonemically coiled DNA molecules (see inset in Figure 1F). In addition, electron micrographs of Rad51-dsDNA complexes formed under comparable conditions on torsionally constrained plasmids (see Figure 3) revealed them to be essentially free from plectonemes, thus suggesting that the torque induced by Rad51 binding to DNA is weak.

Bottom Line: The stretching mode is observed when DNA is unwound towards a helical repeat of 18.6 bp/turn, whereas a non-stretching mode is observed when DNA molecules are not permitted to change their native helical repeat.We also show that the two forms of complexes are interconvertible and that by enforcing changes of DNA twist one can induce transitions between the two forms.Our observations permit a better understanding of the role of ATP hydrolysis in Rad51-mediated homologous pairing and strand exchange.

View Article: PubMed Central - PubMed

Affiliation: Institut Curie, Centre de Recherche-Physico-Chimie-Curie, CNRS UMR168, Université Pierre et Marie Curie, Paris F-75231, France.

ABSTRACT
In eukaryotes, Rad51 protein is responsible for the recombinational repair of double-strand DNA breaks. Rad51 monomers cooperatively assemble on exonuclease-processed broken ends forming helical nucleo-protein filaments that can pair with homologous regions of sister chromatids. Homologous pairing allows the broken ends to be reunited in a complex but error-free repair process. Rad51 protein has ATPase activity but its role is poorly understood, as homologous pairing is independent of adenosine triphosphate (ATP) hydrolysis. Here we use magnetic tweezers and electron microscopy to investigate how changes of DNA twist affect the structure of Rad51-DNA complexes and how ATP hydrolysis participates in this process. We show that Rad51 protein can bind to double-stranded DNA in two different modes depending on the enforced DNA twist. The stretching mode is observed when DNA is unwound towards a helical repeat of 18.6 bp/turn, whereas a non-stretching mode is observed when DNA molecules are not permitted to change their native helical repeat. We also show that the two forms of complexes are interconvertible and that by enforcing changes of DNA twist one can induce transitions between the two forms. Our observations permit a better understanding of the role of ATP hydrolysis in Rad51-mediated homologous pairing and strand exchange.

Show MeSH