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-Cornière, Axelle; Takizawa, Yoshimasa; Conilleau, Sébastien; Tran, V.H.; Iwai, Shigenori; Kurumizaka, Hitoshi; Takahashi, Masayuki

doi:10.1016/j.jmb.2008.08.030

Cited by 11 publications

(20 citation statements)

References 47 publications

(59 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…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%

“…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 structure of the human Rad51 protein filament in its complex with DNA and ATP may yield essential mechanistic information for the recombination reaction, in analogy with insight gained from crystal structures of RecA in its active filament with DNA and ATP␥S (15).…”

mentioning

confidence: 99%

See 1 more Smart Citation

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.

Self Cite

View full text Add to dashboard Cite

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...

show abstract

Section: Hsrad51 Monomer and Filament Structuresmentioning

confidence: 99%

mentioning

confidence: 99%

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.

Self Cite

View full text Add to dashboard Cite

show abstract

“…Flexible loops are involved in DNA binding and may also play a role in Rad51 oligomerization and in interaction with recombination mediators. The nucleotide binding site is located on the subunit-subunit interface between two monomers and strong allosteric coupling is found between ATP binding, DNA binding and ATP hydrolysis (18) (19). Binding of the nucleotide increases affinity for DNA while ATP hydrolysis reduces DNA affinity and triggers disassembly.…”

Section: Rad51 Nucleoprotein Filamentmentioning

confidence: 99%

Assembly and disassembly of Rad51 filaments on single-stranded DNA: A novel assay to study the dynamics of protein-ssDNA interactions at the single-molecule level

Franker¹

2016

JOSHA

View full text Add to dashboard Cite

JOSHA is a service that helps scholars, researchers, and students discover, use, and build upon a wide range of content SummaryEukaryotic recombinase protein Rad51 is the key player in homologous recombination, an essential DNA repair mechanism used for the repair of double-strand breaks. Double-strand breaks can lead to chromosome fragmentation and are particularly hazardous during and shortly after DNA replication. The mechanism of homologous recombination is highly conserved between species and recombinase proteins are expressed in a wide range of prokaryotic and eukaryotic cells. The primary event in homologous recombination is the formation of a helical nucleoprotein filament on singlestranded DNA overhangs around double-strand breaks. The nucleoprotein filament mediates all subsequent steps of homologous recombination and is capable of performing strand exchange reactions unassisted in vitro. Dynamic assembly and disassembly interactions between the nucleoprotein filament and its DNA substrate are essential for strand exchange. Investigating the Rad51 -single-stranded DNA complex presents numerous challenges and it has never been observed directly. This study presents a novel assay to investigate these interactions at the singlemolecule level. A combination of dual-beam optical trapping and high-resolution fluorescence microscopy allows in situ creation and manipulation of single-stranded DNA molecules, while at the same time binding of fluorescently labeled Rad51 proteins can be observed directly. Using this novel single-molecule approach, it is possible to distinguish between the different steps of filament formation: nucleation, extension and disassembly. We are able to simultaneously interrogate the molecular mechanisms of filament assembly and disassembly, and determine nucleation, extension and disassembly rates as well as binding unit sizes without any a priori assumptions. Furthermore, we are able to investigate the effects of tension on the various stages of filament formation and observe Rad51-induced forces in real time.

show abstract

“…Figure . Mutagenesis experiments have determined various functional regions in the core domain 15,16 . Figure …”

Section: Monomermentioning

confidence: 99%

“…The nucleoprotein filament elongates and partly unwinds DNA, extending it to ~150% of its RecA and Rad51 are capable of forming filaments on both ssDNA and dsDNA in the presence of ATP or an ATP analog and strong allosteric coupling between ATP binding, DNA binding and ATPase activity is observed 15 . Binding affinity of RecA is significantly higher for ssDNA, while…”

Section: Filament Assemblymentioning

confidence: 99%

Homologous recombination: Single-molecule experiments and their lessons for the in vivo situation

Franker¹

2016

JOSHA

View full text Add to dashboard Cite

show abstract

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

Cited by 11 publications

References 47 publications

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

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

Assembly and disassembly of Rad51 filaments on single-stranded DNA: A novel assay to study the dynamics of protein-ssDNA interactions at the single-molecule level

Homologous recombination: Single-molecule experiments and their lessons for the in vivo situation

Contact Info

Product

Resources

About