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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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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. Anenlarged view of B. Both His-276 and His-280 are located in a short helix within the mobile L2 region.
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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. Anenlarged view of B. Both His-276 and His-280 are located in a short helix within the mobile L2 region.

Mentions: RecA recombinase and its orthologs promote DNA strand exchange in the presence of ATP and magnesium ion. The DNA bound along the axis of such recombinase filaments is in an extended state competent for DNA strand exchange [26, 49]. The formation of such nucleoprotein filaments possibly requires the participation of both L1 and L2 regions. The ATPase-active conformation appears to have well dispositioned L1 and L2 regions along the filament axis (Fig. 5A), a feature suitable for optimal interaction with DNA. We have previously observed that the ATPase and strand exchange activities of MvRadA are both K+-dependent and correlated with accessibility to the ATPase-active conformation [28]. Our structural and functional studies on the pH dependence of MvRadA reveal a similar correlation. At pH 6.0, activities of MvRadA are much lower and the crystallized filament is in an ATPase-inactive conformation with a disordered L2 loop. Consistent with known requirements for cofactors but not ATP hydrolysis per se, the ordered L2 region is likely required in both polarizing ATP and promoting DNA strand exchange. The correlation between pH-dependent activities and conformational differences further supports the notion that L2 play an important role in binding DNA and / or promoting strand exchange.


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)

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. Anenlarged view of B. Both His-276 and His-280 are located in a short helix within the mobile L2 region.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

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. Anenlarged view of B. Both His-276 and His-280 are located in a short helix within the mobile L2 region.
Mentions: RecA recombinase and its orthologs promote DNA strand exchange in the presence of ATP and magnesium ion. The DNA bound along the axis of such recombinase filaments is in an extended state competent for DNA strand exchange [26, 49]. The formation of such nucleoprotein filaments possibly requires the participation of both L1 and L2 regions. The ATPase-active conformation appears to have well dispositioned L1 and L2 regions along the filament axis (Fig. 5A), a feature suitable for optimal interaction with DNA. We have previously observed that the ATPase and strand exchange activities of MvRadA are both K+-dependent and correlated with accessibility to the ATPase-active conformation [28]. Our structural and functional studies on the pH dependence of MvRadA reveal a similar correlation. At pH 6.0, activities of MvRadA are much lower and the crystallized filament is in an ATPase-inactive conformation with a disordered L2 loop. Consistent with known requirements for cofactors but not ATP hydrolysis per se, the ordered L2 region is likely required in both polarizing ATP and promoting DNA strand exchange. The correlation between pH-dependent activities and conformational differences further supports the notion that L2 play an important role in binding DNA and / or promoting strand exchange.

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