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

Rao DE, Luo Y - Protein Pept. Lett. (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Saskatchewan, 2D01 Health Sciences Building, 107 Wiggins Road, Saskatoon, Saskatchewan, Canada S7N 5E5. yu.luo@usask.ca.

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.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4150490&req=5

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.

Mentions: The other His residue in the L2 region of MvRadA, His-276, (Fig. 3) is conserved in approximately three-fourths of known RadA / Rad51 / DMC1 sequences. The remaining ~1/4 sequences have an Asn at the equivalent positions. We therefore made an H276N mutant RadA. This mutant protein’s peak ssDNA-dependent ATPase activity (Fig. 4A) was observed to be 19.9 ± 0.1 min-1, approximately 90% of the wild-type MvRadA (21.8 ± 0.4 min-1). Interestingly, the ssDNA-dependent ATPase activity of the H276N protein appeared less dependent on pH. Even at pH 6.0, it recorded a turnover rate (8.22 ± 0.08 min-1) ~40% of its peak rate at pH 7.2. In contrast, the wild-type protein exhibited a negligible turnover rate. The salt-stimulated ATPase activity in the presence of 1.0 M KCl (Fig. 4A) also peaked at pH 7.2 (8.3 ± 0.2 min-1), again ~90% of the wild-type protein (9.1 ± 0.3 min-1). At pH 6.0, the wild-type and the H276N proteins both exhibited turnover rates less than 0.2 min-1, a minimum level for our assay to precisely quantify. At pH 6.4, however, the salt-stimulated ATPase activity of the mutant protein was ~3 fold as active as the wild-type protein (3.0 versus 0.9 min-1). It appears that the salt-stimulated ATPase activity of the H276N protein is also less dependent on pH than the wild-type protein. This mutant protein was also active in promoting strand exchange in the presence of either ATP or the non-hydrolysable AMP-PNP (Fig. 4B). In the presence of AMP-PNP, however, the existence of multiple bands on the agarose gel appears to indicate that the DNA strand exchange reaction was partially stalled before resolving various ssDNA-dsDNA joint molecules. Meaningful quantification was therefore infeasible for reactions in the presence of AMP-PNP. As observed for the ATPase activity of this mutant protein, the strand exchange yields in the presence of ATP (Fig. 4C and 4D) also exhibited less pronounced pH dependence. Though only about half as high as the wild-type RadA on average, the strand exchange yields promoted by the H276N protein at pH 6.0 and 6.4 were nearly 50% higher than those at pH 6.8 to 8.0. In contrast, the wild-type protein was largely inefficient at pH 6.0. Collectively, we interpret the attenuated pH dependence of the H276N protein as suggesting ionization at the His-276 side chain is partially responsible for causing the pH dependence of the wild-type MvRadA.


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

Rao DE, Luo Y - Protein Pept. Lett. (2014)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4150490&req=5

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.
Mentions: The other His residue in the L2 region of MvRadA, His-276, (Fig. 3) is conserved in approximately three-fourths of known RadA / Rad51 / DMC1 sequences. The remaining ~1/4 sequences have an Asn at the equivalent positions. We therefore made an H276N mutant RadA. This mutant protein’s peak ssDNA-dependent ATPase activity (Fig. 4A) was observed to be 19.9 ± 0.1 min-1, approximately 90% of the wild-type MvRadA (21.8 ± 0.4 min-1). Interestingly, the ssDNA-dependent ATPase activity of the H276N protein appeared less dependent on pH. Even at pH 6.0, it recorded a turnover rate (8.22 ± 0.08 min-1) ~40% of its peak rate at pH 7.2. In contrast, the wild-type protein exhibited a negligible turnover rate. The salt-stimulated ATPase activity in the presence of 1.0 M KCl (Fig. 4A) also peaked at pH 7.2 (8.3 ± 0.2 min-1), again ~90% of the wild-type protein (9.1 ± 0.3 min-1). At pH 6.0, the wild-type and the H276N proteins both exhibited turnover rates less than 0.2 min-1, a minimum level for our assay to precisely quantify. At pH 6.4, however, the salt-stimulated ATPase activity of the mutant protein was ~3 fold as active as the wild-type protein (3.0 versus 0.9 min-1). It appears that the salt-stimulated ATPase activity of the H276N protein is also less dependent on pH than the wild-type protein. This mutant protein was also active in promoting strand exchange in the presence of either ATP or the non-hydrolysable AMP-PNP (Fig. 4B). In the presence of AMP-PNP, however, the existence of multiple bands on the agarose gel appears to indicate that the DNA strand exchange reaction was partially stalled before resolving various ssDNA-dsDNA joint molecules. Meaningful quantification was therefore infeasible for reactions in the presence of AMP-PNP. As observed for the ATPase activity of this mutant protein, the strand exchange yields in the presence of ATP (Fig. 4C and 4D) also exhibited less pronounced pH dependence. Though only about half as high as the wild-type RadA on average, the strand exchange yields promoted by the H276N protein at pH 6.0 and 6.4 were nearly 50% higher than those at pH 6.8 to 8.0. In contrast, the wild-type protein was largely inefficient at pH 6.0. Collectively, we interpret the attenuated pH dependence of the H276N protein as suggesting ionization at the His-276 side chain is partially responsible for causing the pH dependence of the wild-type MvRadA.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Saskatchewan, 2D01 Health Sciences Building, 107 Wiggins Road, Saskatoon, Saskatchewan, Canada S7N 5E5. yu.luo@usask.ca.

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.

Show MeSH
Related in: MedlinePlus