Structure of human Rad51 protein filament from molecular modeling and site-specific linear dichroism spectroscopy.
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 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.
Reymer
Anna
A
Physical Chemistry, Department of Chemical and Biological Engineering, Chalmers University of Technology, SE-41296 Göteborg, Sweden.
Frykholm
Karolin
K
Morimatsu
Katsumi
K
Takahashi
Masayuki
M
Nordén
Bengt
B
D004247:DNA / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D051135:Rad51 Recombinase / Q000737:chemistry* / Q000378:metabolism; D013057:Spectrum Analysis; D014443:Tyrosine / Q000378:metabolism
D014443:Tyrosine; D004247:DNA; C495153:RAD51 protein, human; D051135:Rad51 Recombinase