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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.

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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.
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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.

Mentions: The strand exchange activity promoted by this archaeal protein was then examined in the same pH range in the presence of 3 mM ATP and 3 mM MgCl2 (Fig. 2A and 2C). A higher concentration of MvRadA (20 µM) and approximately 3 nucleotides / base pairs of DNA substrates per RadA subunit were used to enable facile fluorescence detection on the acrylamide gel. The trend was similar to that of the ATPase activity. At pH 6.0, only ~3% of the 63-nt ssDNA substrate was converted into the 63-nt / 36-nt hdDNA. This level of activity was comparable to basal level activities in the absence of ATP or in the presence of ADP (1-5%, data not shown). The yield abruptly rose to ~23% at pH 6.4 and remained steady around 45% at pH 6.8 - 8.0. In both the ATPase and strand exchange assays, activities close to optimal (within 2 fold) were observed at pH 6.4 - 8.0 while drastically reduced activities were observed at pH 6.0.


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

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

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.
Mentions: The strand exchange activity promoted by this archaeal protein was then examined in the same pH range in the presence of 3 mM ATP and 3 mM MgCl2 (Fig. 2A and 2C). A higher concentration of MvRadA (20 µM) and approximately 3 nucleotides / base pairs of DNA substrates per RadA subunit were used to enable facile fluorescence detection on the acrylamide gel. The trend was similar to that of the ATPase activity. At pH 6.0, only ~3% of the 63-nt ssDNA substrate was converted into the 63-nt / 36-nt hdDNA. This level of activity was comparable to basal level activities in the absence of ATP or in the presence of ADP (1-5%, data not shown). The yield abruptly rose to ~23% at pH 6.4 and remained steady around 45% at pH 6.8 - 8.0. In both the ATPase and strand exchange assays, activities close to optimal (within 2 fold) were observed at pH 6.4 - 8.0 while drastically reduced activities were observed at pH 6.0.

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