Structure of human Rad51 protein filament from molecular modeling and site-specific linear dichroism spectroscopy
KAUST Grant NumberKUK-11-008-23
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AbstractTo 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 nucleo-protein 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.
CitationReymer A, Frykholm K, Morimatsu K, Takahashi M, Norden B (2009) Structure of human Rad51 protein filament from molecular modeling and site-specific linear dichroism spectroscopy. Proceedings of the National Academy of Sciences 106: 13248–13253. Available: http://dx.doi.org/10.1073/pnas.0902723106.
SponsorsWe thank Professor Edward H. Egelman (University of Virginia, Charlottesville, VA) for kindly providing us with the surface map of a 3D reconstruction of a HsRad51 filament electron micrograph and Dr. Axelle Renodon-Cornière for performing the recombinase activity measurements. This work was supported by King Abdullah University of Science and Technology Grant KUK-11-008-23 and the Association pour la Recherche sur le Cancer (M.T.).
PubMed Central IDPMC2726390
CollectionsPublications Acknowledging KAUST Support
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- Authors: Fornander LH, Frykholm K, Reymer A, Renodon-Cornière A, Takahashi M, Nordén B
- Issue date: 2012 Jun
- Visualizing the assembly of human Rad51 filaments on double-stranded DNA.
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- Issue date: 2006 Oct 27
- 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.
- Authors: Renodon-Cornière A, Takizawa Y, Conilleau S, Tran V, Iwai S, Kurumizaka H, Takahashi M
- Issue date: 2008 Nov 14
- Roles of the human Rad51 L1 and L2 loops in DNA binding.
- Authors: Matsuo Y, Sakane I, Takizawa Y, Takahashi M, Kurumizaka H
- Issue date: 2006 Jul
- Real-time assembly and disassembly of human RAD51 filaments on individual DNA molecules.
- Authors: van der Heijden T, Seidel R, Modesti M, Kanaar R, Wyman C, Dekker C
- Issue date: 2007