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

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

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.

Mentions: At a low concentration of salt, RecA-like DNA strand exchange proteins are well known to be DNA-dependent ATPases. The single-stranded DNA overhang generated at the site of lesion is believed to be the in vivo site in most organisms [43] for the formation of a recombinase / ssDNA filament which is active in ATP hydrolysis and homologous strand exchange with a homologous dsDNA. We first examined the ssDNA-dependent activity of MvRadA in a pH range between 6.0 and 8.0. In this assay, 3 µM MvRadA and 18 µM poly-(dT)36 (in nucleotides) were used. RecA-like recombinases are known to bind approximately 3 nucleotides per subunit. A 2-fold overdose of poly-(dT)36 was used to ensure maximal ATPase activity. The initial turnover rates were derived (Fig. 1) by monitoring the time courses of phosphate release. The turnover rate was negligible at pH 6.0 (0.013 ± 0.005 min-1). The rate abruptly rose to 13.9 ± 0.3 min-1 at pH 6.4, and stayed above 13.9 min-1 at pH 6.8 - 8.0. The peak activity was observed at pH 7.2 (21.8 ± 0.4 min-1). By setting up hanging drops over pH 5 to 9, we observed that the concentrated protein (~30 mg / ml) was soluble at pH higher than 5.2. Therefore, inactivation at pH 6.0 by protein precipitation could be ruled out.


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

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

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.
Mentions: At a low concentration of salt, RecA-like DNA strand exchange proteins are well known to be DNA-dependent ATPases. The single-stranded DNA overhang generated at the site of lesion is believed to be the in vivo site in most organisms [43] for the formation of a recombinase / ssDNA filament which is active in ATP hydrolysis and homologous strand exchange with a homologous dsDNA. We first examined the ssDNA-dependent activity of MvRadA in a pH range between 6.0 and 8.0. In this assay, 3 µM MvRadA and 18 µM poly-(dT)36 (in nucleotides) were used. RecA-like recombinases are known to bind approximately 3 nucleotides per subunit. A 2-fold overdose of poly-(dT)36 was used to ensure maximal ATPase activity. The initial turnover rates were derived (Fig. 1) by monitoring the time courses of phosphate release. The turnover rate was negligible at pH 6.0 (0.013 ± 0.005 min-1). The rate abruptly rose to 13.9 ± 0.3 min-1 at pH 6.4, and stayed above 13.9 min-1 at pH 6.8 - 8.0. The peak activity was observed at pH 7.2 (21.8 ± 0.4 min-1). By setting up hanging drops over pH 5 to 9, we observed that the concentrated protein (~30 mg / ml) was soluble at pH higher than 5.2. Therefore, inactivation at pH 6.0 by protein precipitation could be ruled out.

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