Crystal structure of a Rad51 filament

Abstract

Rad51, the major eukaryotic homologous recombinase, is important for the repair of DNA damage and the maintenance of genomic diversity and stability. The active form of this DNA-dependent ATPase is a helical filament within which the search for homology and strand exchange occurs. Here we present the crystal structure of a Saccharomyces cerevisiae Rad51 filament formed by a gain-of-function mutant. This filament has a longer pitch than that seen in crystals of Rad51's prokaryotic homolog RecA, and places the ATPase site directly at a new interface between protomers. Although the filament exhibits approximate six-fold symmetry, alternate protein-protein interfaces are slightly different, implying that the functional unit of Rad51 within the filament may be a dimer. Additionally, we show that mutation of His352, which lies at this new interface, markedly disrupts DNA binding.

Two distinct conformational states define the interaction of human RAD 51‐ ATP with single‐stranded DNA

“…As hRAD51 exists in a range of oligomeric species in solution that depend on concentration for their relative abundance, it is likely that crystal growth was seeded by heptameric hRAD51 present in the crystallization buffer. The arrangement of the hRAD51 protomers within the filament and their mode of self‐association is similar to what was observed for the filament structure of yeast Rad51 (Conway et al , 2004). The heptameric repeat of the helical hRAD51‐ATP filament has a pitch of 128.0 Å and a protomer rise of 18.3 Å.…”

“…Previous crystallographic analysis of the yeast RAD51 filament had revealed the presence of two slightly different interfaces in the asymmetric unit, implying that the functional unit of the filament might be a dimer (Conway et al , 2004). Inspection of the 12 independent dimer interfaces in the two heptameric turns of the hRAD51‐ATP filament structure showed the presence of two distinct dimer conformations, alternating along the filament (Fig 5A), in a qualitatively similar arrangement to what had been observed for yeast Rad51 (Conway et al , 2004).…”

“…In addition, although the NPF can readily form in the presence of both ATP (van Mameren et al , 2009b) and ADP (Hilario et al , 2009), hRAD51 disassembly from dsDNA is critically dependent on ATP hydrolysis (van Mameren et al , 2009b). X‐ray crystallography has identified the location of the ATP‐binding pocket between adjacent monomers in the NPF (Conway et al , 2004). Therefore, it is likely that ATP binding and hydrolysis play a significant role in the conformation and stability of hRAD51 NPFs.…”

“…Although large‐scale differences in NPF structure, such as variations in pitch, have been widely reported, local changes in filament structure at the single protomer level have not been described yet. The only exception comes from a crystallographic model of yeast Rad51 in filament form, which showed the presence of different protomer interfaces that were proposed to be functionally relevant (Conway et al , 2004). …”

BRCA2: a universal recombinase regulator

“…Recombinant RAD51 and DMC1 proteins form oligomeric ring structures of seven or eight monomers, respectively (Shin et al, 2003;Kinebuchi et al, 2004). However, they are functionally active when associated with DNA in the form of a highly ordered right-handed helical nucleoprotein filament (Figure 3b) (Benson et al, 1994;Conway et al, 2004;Sehorn et al, 2004). It is within this nucleoprotein filament that DNA interactions take place between homologous sequences.…”