Research Article: Structure of a hexameric form of RadA recombinase from Methanococcus voltae

Date Published: May 1, 2012

Publisher: International Union of Crystallography

Author(s): Liqin Du, Yu Luo.


Hexameric rings of RadA recombinase from M. voltae have been crystallized. Structural comparisons suggest that homologues of RadA tend to form double-ringed assemblies.

Partial Text

Bacterial RecA (Clark & Margulies, 1965 ▶), archaeal RadA (Sandler et al., 1996 ▶) and eukaryal Rad51 (Shinohara et al., 1992 ▶) and DMC1 (Bishop et al., 1992 ▶) proteins form a superfamily of recombinases (also called DNA strand-exchange proteins; Seitz & Kowalczykowski, 2000 ▶). Homologous recombination appears to be essential in the repair of double-stranded DNA breaks and the restarting of stalled replication forks (Cox, 1998 ▶; Cox et al., 2000 ▶; Courcelle et al., 2001 ▶; Lusetti & Cox, 2002 ▶; Kowalczykowski, 2000 ▶). These proteins facilitate a pivotal DNA strand-exchange process between a single-stranded DNA (ssDNA) and a homologous double-stranded DNA (dsDNA) in homologous recombination. Electron-microscopic and crystallo­graphic results have revealed strikingly similar ‘active’ recombinase assemblies in the form of right-handed helical filaments with approximately six monomers per turn (VanLoock et al., 2003 ▶; Conway et al., 2004 ▶; Wu et al., 2004 ▶; Chen et al., 2008 ▶; Sheridan et al., 2008 ▶; Li et al., 2009a ▶). The milestone structures of Escherichia coli RecA (EcRecA) in complex with a series of DNA molecules have shed light on the exact mechanism of homologous DNA strand exchange (Chen et al., 2008 ▶). Crystallized ‘inactive’ filaments with shorter helical pitches have also been observed (Story et al., 1992 ▶). Ring-shaped forms with 6–8 protomers have also been commonly observed by electron microscopy (Heuser & Griffith, 1989 ▶; Yu & Egelman, 1997 ▶; Passy et al., 1999 ▶; Yang et al., 2001 ▶; Galkin et al., 2006 ▶; McIlwraith et al., 2001 ▶; Masson et al., 1999 ▶). Only heptameric rings of Pyrococcus furiosus RadA (PfRadA) and octameric Homo sapiens DMC1 (HsDMC1) have previously been crystallized (Shin et al., 2003 ▶; Kinebuchi et al., 2004 ▶). A reconstructed hexameric EcRecA model has been derived from electron microscopy (Yu & Egelman, 1997 ▶). In addition to the three commonly found forms, crystal structures of overwound three-monomer-per-turn filaments (Ariza et al., 2005 ▶) and left-handed filaments of Sulfolobus solfataricus RadA (SsRadA; Chen et al., 2007 ▶; Chang et al., 2009 ▶) have also been observed. Here, we report the first crystal structure of hexameric RadA from Methanococcus voltae devoid of its first 60 amino-acid residues (Δ60MvRadA). Crystal-packing analysis and comparison with the heptameric PfRadA structure and the octameric HsDMC1 structure indicated that these proteins can form two-ringed assemblies.

Unlike the structures of filamentous MvRadA, the hexameric Δ60MvRadA structure revealed a different conformation of Arg74 which is no longer capable of retaining the salt bridge to Glu96. As such, the crystal structure of Δ60MvRadA further supports the notion that the residue equivalent to Arg74 of MvRadA modulates the conformational changes which give rise to flexibility in the protein assemblies of orthologous proteins (Chen et al., 2007 ▶).