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PDBsum entry 2f1h
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Recombination
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PDB id
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2f1h
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References listed in PDB file
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Key reference
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Title
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Asp302 determines potassium dependence of a rada recombinase from methanococcus voltae.
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Authors
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X.Qian,
Y.He,
Y.Wu,
Y.Luo.
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Ref.
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J Mol Biol, 2006,
360,
537-547.
[DOI no: ]
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PubMed id
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Abstract
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Archaeal RadA/Rad51 are close homologues of eukaryal Rad51/DMC1. Such
recombinases, as well as their bacterial RecA orthologues, form helical
nucleoprotein filaments in which a hallmark strand exchange reaction occurs
between homologous DNA substrates. Our recent ATPase and structure studies on
RadA recombinase from Methanococcus voltae have suggested that not only
magnesium but also potassium ions are absorbed at the ATPase center. Potassium,
but not sodium, stimulates the ATP hydrolysis reaction with an apparent
dissociation constant of approximately 40 mM. The minimal inhibitory effect by
40 mM NaCl further suggests that the protein does not have adequate affinity for
sodium. The wild-type protein's strand exchange activity is also stimulated by
potassium with an apparent dissociation constant of approximately 35 mM. We made
site-directed mutations at the potassium-contacting residues Glu151 and Asp302.
The mutant proteins are expectedly defective in promoting ATP hydrolysis.
Similar potassium preference in strand exchange is observed for the E151D and
E151K proteins. The D302K protein, however, shows comparable strand exchange
efficiencies in the presence of either potassium or sodium. Crystallized E151D
filaments reveal a potassium-dependent conformational change similar to what has
previously been observed with the wild-type protein. We interpret these data as
suggesting that both ATP hydrolysis and DNA strand exchange requires
accessibility to an "active" conformation similar to the crystallized
ATPase-active form in the presence of ATP, Mg2+ and K+.
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Figure 5.
Figure 5. The ATPase site of the E151D mutant RadA in
stereo. Two MvRadA subunits are colored yellow and gray,
respectively. K^+, Mg^2+ and water molecules are colored purple,
red and green, respectively. (a) E151D RadA/AMP–PNP in a 0.5 M
KCl. (b) E151D RadA/AMP–PNP complex in 0.5 M NaCl. (c) E151D
RadA/ADP complex in 0.5 M KCl. Most residues in the L2 region
are ordered in (a), but similarly disordered in (b) and (c). In
addition to a nucleoside triphosphate and Mg^2+, K^+ appears to
be required for stabilizing the more ordered “active” L2
conformation.
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Figure 6.
Figure 6. Two conformations of the E151D monomer in stereo.
The E151D monomer has an N-terminal dsDNA-binding domain (top)
and a larger ATPase core domain (bottom). The ATPase domain has
two half-sites for binding ATP (gold). Two putative DNA-binding
regions L1 (green) and L2 (magenta and blue for the two observed
conformations, respectively) are located near the filament axis
(dotted vertical line). Both ends of the L2 region contact the
ATP-binding site. Two K^+ (in yellow) were observed to form
bridges between the γ–phosphate of AMP–PNP and the short
helix in the L2 region (in magenta). When KCl was substituted
for NaCl, the L2 region (in blue) became largely disordered.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2006,
360,
537-547)
copyright 2006.
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