Binding of a Second Magnesium Is Required for ATPase Activity of RadA from Methanococcus voltae

Abstract

RecA-like strand exchange proteins, which include closely related archaeal Rad51/RadA and eukaryal Rad51 and DMC1, play a key role in DNA repair by forming helical nucleoprotein filaments which promote a hallmark strand exchange reaction between homologous DNA substrates. Our recent crystallographic studies on a RadA recombinase from Methanococcus voltae (MvRadA) have unexpectedly revealed a secondary magnesium at the subunit interface approximately 11 A from the primary one coordinated by ATP and the canonical P-loop. The DNA-dependent ATPase activity of MvRadA appears to be dependent on the concentration of free Mg2+, while the strand exchange activity does not. We also made site-directed mutagenesis at the Mg2+-liganding residue Asp-246. The mutant proteins exhibited approximately 20-fold reduced ATPase activity but normal strand exchange activity. Structurally, the main chain carbonyl of the conserved catalytic residue Glu-151 is hydrogen bonded with one of the magnesium-liganding water molecules. Changes in the secondary magnesium site may therefore induce conformational changes around this catalytic glutamate and affect the ATPase activity without significantly altering the stability of the extended recombinase filament. Asp-246 is somewhat conserved among archaeal and eukaryal homologues, implying some homologues may share this allosteric site for ATPase function.

Structure of a hexameric form of RadA recombinase fromMethanococcus voltae

“…This region also contains a conserved Arg74 residue which has been shown to be important for the conformational flexibility of RadA and Rad51 (Chen et al, 2007). In all previously determined filamentous structures of RadA from M. voltae (Wu et al, 2004(Wu et al, , 2005Qian et al, 2005Qian et al, , 2006Qian et al, , 2007Galkin et al, 2006;Li et al, 2009b) and M. maripaludis (MmRadA; Li et al, 2009a), this Arg74 residue forms a salt bridge with Glu96 (yellow and cyan structures in Fig. 3).…”

Structural and functional analyses of the DMC1-M200V polymorphism found in the human population

“…In the crystal structure of the archaeal RadA protein, the α-helix corresponding to the α-13 helix of the human DMC1 protein contains the Asp246 residue that binds to Mg 2+ . This Mg 2+ ion is also bound by several residues from the neighboring protomer, and could be essential for the formation of the active filament structure (48). Therefore, mutations in the α-helix harboring these residues may alter the structure of the metal-binding site.…”

From meiosis to postmeiotic events: Uncovering the molecular roles of the meiosis‐specific recombinase Dmc1

“…DMC1‐M200V requires a higher Mg 2+ concentration for optimal D‐loop formation activity. The crystal structure of the M. voltae RadA revealed a second Mg 2+ ‐binding site (apart from the ATP‐binding site) that is probably well conserved within the RecA/Rad51/Dmc1 recombinase superfamily, and is essential for active helical filament formation [40]. The amino acids constituting this binding site are somewhat conserved between RadA and Dmc1/Rad51.…”