Identification of the Subunit–Subunit Interface of Xenopus Rad51.1 Protein: Similarity to RecA

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

“…20 To verify this hypothesis, we monitored the changes in fluorescence of HsRad51-H294W upon addition of 3 M urea, which dissociates the Rad51 filament without unfolding the protein. 25,26 Supporting the hypothesis, HsRad51-H294W became fluorescent in the presence of 3 M urea (Fig. 7a), which reduced only slightly the CD signal at 222 nm (data not shown).…”

“…22 For human Rad51 (HsRad51), the 3-D structure of the central part has been revealed by X-ray crystallography and that of the N-terminal part by NMR. 23,24 To determine the structure of the HsRad51-DNA complex filament, we previously identified its subunit/subunit contact interface 25,26 and DNA-binding sites 27 by monitoring the fluorescence of a tryptophan residue inserted, as a probe, at the putative sites. In this report, we have investigated, using a similar but more systematic approach, the ATPinduced conformational changes of the HsRad51 filament to understand how Rad51 interacts with ATP and DNA to form the active nucleofilament, the first step of the strand exchange reaction.…”

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

“…This idea also provides a possible explanation for the stretched DNA conformation reported for the RecA nucleo-protein filament (15), where exactly 3 nucleotides correspond to a protein monomer and are followed by a spatial gap that in RecA is filled by Ile-199 of the L2 loop, and in HsRad51 is filled by Tyr-232 of the L1 loop. That Tyr-232 is indeed intercalated is supported by the observation that its replacement by a tryptophan, which is a more bulky residue, strongly reduces the binding affinity (26). Arg-235, also sitting on L1 and shown to facilitate the strand-exchange reaction, occupies a position enabling direct binding to the DNA phosphate backbone of the other strand.…”

“…Orientation of the monomers in the filamentous structure was set to be consistent with the results from a tryptophan-scanning mutagenesis of informatively positioned aromatic residues across HsRad51: H294, Y315, and Y191 at the subunit-subunit interface (18,26), Y232 and Y301 in the DNA binding regions and thus located in the nearest proximity of the filament center (18,24,25). Loop modeling was performed according to a protocol described by Simmerling et al (30) for a loop of similar length as the L2 loop, using the molecular dynamics software package AMBER10.…”

“…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).…”