Structure of human Rad51 protein filament from molecular modeling and site-specific linear dichroism spectroscopy
Artikel i vetenskaplig tidskrift, 2009

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.

Författare

Anna Reymer

Chalmers, Kemi- och bioteknik, Fysikalisk kemi

Karolin Frykholm

Chalmers, Kemi- och bioteknik, Fysikalisk kemi

K. Morimatsu

UC Davis

Chalmers University of Technology

M. Takahashi

Universite de Nantes

Bengt Nordén

Chalmers, Kemi- och bioteknik, Fysikalisk kemi

Proceedings of the National Academy of Sciences of the United States of America

0027-8424 (ISSN) 1091-6490 (eISSN)

Vol. 106 13248-13253

Ämneskategorier

Fysikalisk kemi

DOI

10.1073/pnas.0902723106