Identification of the Subunit–Subunit Interface of Xenopus Rad51.1 Protein: Similarity to RecA

Selmane, Tassadite; Camadro, Jean‐Michel; Conilleau, Sébastien; Fleury, Fabrice; Tran, V.H.; Prévost, Chantal; Takahashi, Masayuki

doi:10.1016/j.jmb.2003.11.045

Cited by 13 publications

(20 citation statements)

References 44 publications

(40 reference statements)

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“…20 To verify this hypothesis, we monitored the changes in fluorescence of HsRad51-H294W upon addition of 3 M urea, which dissociates the Rad51 filament without unfolding the protein. 25,26 Supporting the hypothesis, HsRad51-H294W became fluorescent in the presence of 3 M urea (Fig. 7a), which reduced only slightly the CD signal at 222 nm (data not shown).…”

Section: Fluorescence Of Engineered Hsrad51supporting

confidence: 61%

“…22 For human Rad51 (HsRad51), the 3-D structure of the central part has been revealed by X-ray crystallography and that of the N-terminal part by NMR. 23,24 To determine the structure of the HsRad51-DNA complex filament, we previously identified its subunit/subunit contact interface 25,26 and DNA-binding sites 27 by monitoring the fluorescence of a tryptophan residue inserted, as a probe, at the putative sites. In this report, we have investigated, using a similar but more systematic approach, the ATPinduced conformational changes of the HsRad51 filament to understand how Rad51 interacts with ATP and DNA to form the active nucleofilament, the first step of the strand exchange reaction.…”

Section: Introductionmentioning

confidence: 99%

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Structural Analysis of the Human Rad51 Protein–DNA Complex Filament by Tryptophan Fluorescence Scanning Analysis: Transmission of Allosteric Effects between ATP Binding and DNA Binding

Renodon-Cornière¹,

Takizawa²,

Conilleau³

et al. 2008

Journal of Molecular Biology

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Section: Fluorescence Of Engineered Hsrad51supporting

confidence: 61%

Section: Introductionmentioning

confidence: 99%

Structural Analysis of the Human Rad51 Protein–DNA Complex Filament by Tryptophan Fluorescence Scanning Analysis: Transmission of Allosteric Effects between ATP Binding and DNA Binding

Renodon-Cornière¹,

Takizawa²,

Conilleau³

et al. 2008

Journal of Molecular Biology

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“…This idea also provides a possible explanation for the stretched DNA conformation reported for the RecA nucleo-protein filament (15), where exactly 3 nucleotides correspond to a protein monomer and are followed by a spatial gap that in RecA is filled by Ile-199 of the L2 loop, and in HsRad51 is filled by Tyr-232 of the L1 loop. That Tyr-232 is indeed intercalated is supported by the observation that its replacement by a tryptophan, which is a more bulky residue, strongly reduces the binding affinity (26). Arg-235, also sitting on L1 and shown to facilitate the strand-exchange reaction, occupies a position enabling direct binding to the DNA phosphate backbone of the other strand.…”

Section: Discussionmentioning

confidence: 92%

“…Orientation of the monomers in the filamentous structure was set to be consistent with the results from a tryptophan-scanning mutagenesis of informatively positioned aromatic residues across HsRad51: H294, Y315, and Y191 at the subunit-subunit interface (18,26), Y232 and Y301 in the DNA binding regions and thus located in the nearest proximity of the filament center (18,24,25). Loop modeling was performed according to a protocol described by Simmerling et al (30) for a loop of similar length as the L2 loop, using the molecular dynamics software package AMBER10.…”

Section: Methodsmentioning

confidence: 99%

“…The trial axis giving the best fit with the experimental angles was set as the filament axis. To define the inner orientations of the monomers in the filament relative to each other and the filament axis, additional information from tryptophan-scanning mutagenesis of strategically positioned amino acid residues was used (18,(24)(25)(26). The filament was constructed to have a 99 Å pitch and 6.39 monomers per turn (10,27).…”

Section: Hsrad51 Monomer and Filament Structuresmentioning

confidence: 99%

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Structure of human Rad51 protein filament from molecular modeling and site-specific linear dichroism spectroscopy

Reymer

Frykholm

Morimatsu

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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To get mechanistic insight into the DNA strand-exchange reaction of homologous recombination, we solved a filament structure of a human Rad51 protein, combining molecular modeling with experimental data. We build our structure on reported structures for central and N-terminal parts of pure (uncomplexed) Rad51 protein by aid of linear dichroism spectroscopy, providing angular orientations of substituted tyrosine residues of Rad51-dsDNA filaments in solution. The structure, validated by comparison with an electron microscopy density map and results from mutation analysis, is proposed to represent an active solution structure of the nucleoprotein complex. An inhomogeneously stretched double-stranded DNA fitted into the filament emphasizes the strategic positioning of 2 putative DNA-binding loops in a way that allows us speculate about their possibly distinct roles in nucleo-protein filament assembly and DNA strand-exchange reaction. The model suggests that the extension of a single-stranded DNA molecule upon binding of Rad51 is ensured by intercalation of Tyr-232 of the L1 loop, which might act as a docking tool, aligning protein monomers along the DNA strand upon filament assembly. Arg-235, also sitting on L1, is in the right position to make electrostatic contact with the phosphate backbone of the other DNA strand. The L2 loop position and its more ordered compact conformation makes us propose that this loop has another role, as a binding site for an incoming double-stranded DNA. Our filament structure and spectroscopic approach open the possibility of analyzing details along the multistep path of the strand-exchange reaction.homologous recombination ͉ HsRad51 H omologous recombination, the process of exchanging DNA strands of homologous sequences, is important for both the repair of damaged DNA and the maintenance of genomic diversity. In eukaryotes, from yeast to human, Rad51 is involved in homology search and DNA strand-exchange, central processes for recombination (1, 2). The mechanism of DNA strand-exchange appears evolutionarily conserved (3), and has been extensively studied with the bacterial RecA as a model protein (4-8). The nucleo-protein filament structures appear similar among the RecA and RecA-like proteins, including Rad51 (3, 9, 10). The first structure of a RecA filament was solved Ͼ15 years ago (11) and more recently structures of Rad51 proteins have been reported (12)(13)(14). Despite these efforts, though, no detailed filamentous structure of the human Rad51 protein has been reported and only the principal steps of the recombination process are known.RecA and Rad51 protein filaments formed in presence of DNA and ATP, or a nonhydrolysable ATP analogue, all show an extended conformation with a helical pitch of Ϸ90-100 Å. The RecA and Rad51 filament structures vary significantly depending on the presence of DNA and type of nucleotide cofactor (10,(15)(16)(17)(18)(19) suggesting that the filament structure may change during the strand-exchange reaction, possibly related to its function. Thus, a st...

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RecA Superfamily Proteins

McGrew

Knight

2006

Encyclopedia of Molecular Cell Biology and Molecular Medicine

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Identification of the Subunit–Subunit Interface of Xenopus Rad51.1 Protein: Similarity to RecA

Cited by 13 publications

References 44 publications

Structural Analysis of the Human Rad51 Protein–DNA Complex Filament by Tryptophan Fluorescence Scanning Analysis: Transmission of Allosteric Effects between ATP Binding and DNA Binding

Structural Analysis of the Human Rad51 Protein–DNA Complex Filament by Tryptophan Fluorescence Scanning Analysis: Transmission of Allosteric Effects between ATP Binding and DNA Binding

Structure of human Rad51 protein filament from molecular modeling and site-specific linear dichroism spectroscopy

RecA Superfamily Proteins

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