SUMMARY Linker histones associate with nucleosomes to promote the formation of higher-order chromatin structure, but the underlying molecular details are unclear. We investigated the structure of a 197 base-pair nucleosome bearing symmetric 25 base-pair linker DNA arms in complex with vertebrate linker histone H1. We determined electron cryo-microscopy (cryo-EM) and crystal structures of unbound and H1-bound nucleosomes and validated these structures by site-directed protein cross-linking and hydroxyl radical footprinting experiments. Histone H1 shifts the conformational landscape of the nucleosome by drawing the two linkers together and reducing their flexibility. The H1 C-terminal domain (CTD) localizes primarily to a single linker, while the H1 globular domain contacts the nucleosome dyad and both linkers, associating more closely with the CTD-distal linker. These findings reveal that H1 imparts a strong degree of asymmetry to the nucleosome, which is likely to influence the assembly and architecture of higher-order structures.
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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