pH-dependent activities and structural stability of loop-2-anchoring helix of RadA recombinase from Methanococcus voltae

Abstract

RadA is an archaeal orthologue of human recombinase Rad51. This superfamily of recombinases, which also includes eukaryal meiosis-specific DMC1 and remotely related bacterial RecA, form filaments on single-stranded DNA in the presence of ATP and promote a strand exchange reaction between the single-stranded DNA and a homologous double stranded DNA. Due to its feasibility of getting crystals and similarity (> 40% sequence identity) to eukaryal homologues, we have studied RadA from Methanococcus voltae (MvRadA) as a structural model for understanding the molecular mechanism of homologous strand exchange. Here we show this protein's ATPase and strand exchange activities are minimal at pH 6.0. Interestingly, MvRadA's pH dependence is similar to the properties of human Rad51 but dissimilar to that of the well-studied E. coli RecA. A structure subsequently determined at pH 6.0 reveals features indicative of an ATPase- inactive form with a disordered L2 loop. Comparison with a previously determined ATPase-active form at pH 7.5 implies that the stability of the ATPase-active conformation is reduced at the acidic pH. We interpret these results as further suggesting an ordered disposition of the DNA-binding L2 region, similar to what has been observed in the previously observed ATPase-active conformation, is required for promoting hydrolysis of ATP and strand exchange between singleand double-stranded DNA. His-276 in the mobile L2 region was observed to be partially responsible for the pH-dependent activities of MvRadA.

Figure. (1)

PH-dependent ATPase Activities. The turnover rates were derived by monitoring the time course of phosphate release. All reaction solutions contained 5 mM ATP, 10 mM MgCl2, and 50 mM of Hepes-Tris buffer at indicated pH. ThessDNA-dependent ATP hydrolysis were carried out in the presence of 3 mM MvRadA and 18 mM single-stranded poly-(dT)₃₆ (in nucleotides) and 0.1 M of KCl. The salt-stimulated ATP hydrolysis was carried out in the presence of 3 mM MvRadA and 1.0 M KCl. The activities at pH 6.0 are too low to show. Standard deviations from multiple experiments are shown as error bars.

Figure. (2)

PH-dependent Strand Exchange Activities. A scheme of the reaction is shown on the top right corner. The acrylamide gels were stained by ethidium bromide. Strand exchange yields were quantified by intensities of fluorescence at bands corresponding to the 36-nt / 63-nt heteroduplex DNA (hdDNA). The pH values are shown atop the gel lanes. A. Strand exchange activity in the presence of an ATP-regenerating system. B. Strand exchange activity in the presence of AMP-PNP. C. Quantified stand exchange yields from A and B.

Figure. (3)

ATPase Site in Stereo. Two recombinase subunits are coloured in yellow and grey, respectively. K⁺ ions, Mg²⁺ ions and water molecules are coloured in purple, red and green, respectively. The putative hydrolysis water in each structure is shown in a bigger sphere. Selected hydrogen bonds and metal-coordination bonds are shown in dashed lines in brown. A. ATPase site previously seen in a KCl-soaked crystal at pH 7.5. B. ATPase site seen in KCl-soaked crystals at pH 6.0. At the acidic pH, the direct contacts between the g-phosphate moiety of the ATP analogue and the electron-withdrawing potassium ions and His-280 side chain are lost.

Figure. (4)

PH-dependent Activities of H276N Protein. Activities were measured and shown for the H276N protein as in Figure 1 and 2. A. ATP hydrolysis rates in the presence of poly-(dT)₃₆ or high concentration of KCl. B. Strand exchange activities in the presence of ATP and AMP-PNP at pH 7.2. The reaction seems stalled in the presence of AMP-PNP. C. Strand exchange activities in the presence of an ATP-regenerating system. D. Quantified stand exchange yields from C as well as from Figure 2A. The H276N protein exhibited attenuated pH dependence.

Figure. (5)

DNA-Binding Site in Stereo. A. A crystallized MvRadA filament with subunits shown in alternating colours. The ATP analogues are shown in cyan. Three DNA strands (structure unknown) are depicted along the helical groove near the axis. B. A stereo view of one MvRadA protomer. The L1 region is highlighted in green. Ordered parts of the L2 regions are highlighted (magenta at pH 6.0 and blue at pH 7.5). The filament axis lies vertically. Two AMP-PNP molecules and the side chains of Arg-218, Arg-224, Arg-230, His-276 and His-280 are shown in ball-and-stick model. Residues 260 to 278 in the L2 region of the pH 6.0 structure were disordered in the crystal. C. An enlarged view of B. Both His-276 and His-280 are located in a short helix within the mobile L2 region.