Conformational flexibility revealed by the crystal structure of a crenarchaeal RadA

Abstract

Homologous recombinational repair is an essential mechanism for repair of double-strand breaks in DNA. Recombinases of the RecA-fold family play a crucial role in this process, forming filaments that utilize ATP to mediate their interactions with single- and double-stranded DNA. The recombinase molecules present in the archaea (RadA) and eukaryota (Rad51) are more closely related to each other than to their bacterial counterpart (RecA) and, as a result, RadA makes a suitable model for the eukaryotic system. The crystal structure of Sulfolobus solfataricus RadA has been solved to a resolution of 3.2 A in the absence of nucleotide analogues or DNA, revealing a narrow filamentous assembly with three molecules per helical turn. As observed in other RecA-family recombinases, each RadA molecule in the filament is linked to its neighbour via interactions of a short beta-strand with the neighbouring ATPase domain. However, despite apparent flexibility between domains, comparison with other structures indicates conservation of a number of key interactions that introduce rigidity to the system, allowing allosteric control of the filament by interaction with ATP. Additional analysis reveals that the interaction specificity of the five human Rad51 paralogues can be predicted using a simple model based on the RadA structure.

Figure 1

The structure of SsRadA. (a) The RadA monomer (b) The RadA filament observed in the crystal viewed perpendicular to the 3₁ axis. [All molecular graphics composed with PYMOL (43)]. (c) A structure-based sequence alignment of SsRadA with other RadA/Rad51 structures. Italic font indicates residues not observed in the crystal structures. Numbering and secondary structure assignment [DSSP (44)] pertain to SsRadA. Black asterisks highlight residues explicitly mentioned in the text, composed using INDONESIA (38) and ALINE (available from the authors).

Figure 2

The oligomerization strand. (a) σ_A-weighted electron density (1.2 σ; purple mesh) of the region surrounding Phe73. Atoms from different monomers are coloured differently (grey/blue). (b) A cluster of salt bridges stabilizes the SsRadA oligomerization motif. (c) Superposition of the oligomerization strands of SsRadA (blue), EcRecA (green) and HsRad51/BRCA2 (magenta). The common ATPase domain of the interacting subunit is shown as a grey surface. (d and e) Conserved interactions between the N-terminal domain of SsRadA, MvRadA and ScRad51, with the neighbouring ATPase domain.

Figure 3

Interactions between ATPase subunits for SsRadA (blue), ScRad51 (red) and PfRadA (green). Structures were superimposed on one subunit (shown as backbone trace). The neighbouring subunits are shown as semi-transparent surfaces, with a solid cartoon representation of residues between helices α10 and α12. (a) Side view. (b) Top view.