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PDBsum entry 2ewf
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* Residue conservation analysis
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DOI no:
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J Mol Biol
384:489-502
(2008)
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PubMed id:
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Structural analysis of the heterodimeric type IIS restriction endonuclease R.BspD6I acting as a complex between a monomeric site-specific nickase and a catalytic subunit.
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G.S.Kachalova,
E.A.Rogulin,
A.K.Yunusova,
R.I.Artyukh,
T.A.Perevyazova,
N.I.Matvienko,
L.A.Zheleznaya,
H.D.Bartunik.
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ABSTRACT
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The heterodimeric restriction endonuclease R.BspD6I from Bacillus species D6
recognizes a pseudosymmetric sequence and cuts both DNA strands outside the
recognition sequence. The large subunit, Nt.BspD6I, acts as a type IIS
site-specific monomeric nicking endonuclease. The isolated small subunit,
ss.BspD6I, does not bind DNA and is not catalytically active. We solved the
crystal structures of Nt.BspD6I and ss.BspD6I at high resolution. Nt.BspD6I
consists of three domains, two of which exhibit structural similarity to the
recognition and cleavage domains of FokI. ss.BspD6I has a fold similar to that
of the cleavage domain of Nt.BspD6I, each containing a PD-(D/E)XK motif and a
histidine as an additional putative catalytic residue. In contrast to the
DNA-bound FokI structure, in which the cleavage domain is rotated away from the
DNA, the crystal structure of Nt.BspD6I shows the recognition and cleavage
domains in favorable orientations for interactions with DNA. Docking models of
complexes of Nt.BspD6I and R.BspD6I with cognate DNA were constructed on the
basis of structural similarity to individual domains of FokI, R.BpuJI and
HindIII. A three-helix bundle forming an interdomain linker in Nt.BspD6I acts as
a rigid spacer adjusting the orientations of the spatially separated domains to
match the distance between the recognition and cleavage sites accurately.
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Selected figure(s)
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Figure 5.
Fig. 5. (a) Structural alignment of the active sites of
Nt.BspD6I (red), ss.BspD6I (green), FokI (marine) and BamHI
(grey). The segments 418–489 (Nt.BspD6I), 22–93 (ss.BspD6I),
425–488 (1FOK), 61–134 (2BAM) are depicted as cartoons,
omitting for each structure the loop between the first and the
second β-strand. Putative catalytic residues are shown as
sticks, the calcium ion sites A and B of 2BAM as spheres. (b)
Hydrogen bonding network in the catalytic center of the
Nt.BspD6I cleavage domain. The residues E418, D456, E469 and
E482 are essential for DNA cleavage activity.
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Figure 8.
Fig. 8. Superposition of the D1 and D2 subdomains of
DNA-bound FokI (1FOK), BpuJI (2VLA) and the binary docking
complex of Nt.BspD6I. FokI (brown) interacts with the antisense
strand, BpuJI (magenta) and Nt.BspD6I (cyan) with the sense
strand of DNA.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2008,
384,
489-502)
copyright 2008.
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Figures were
selected
by an automated process.
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Literature references that cite this PDB file's key reference
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PubMed id
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Reference
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S.E.Halford,
L.E.Catto,
C.Pernstich,
D.A.Rusling,
and
K.L.Sanders
(2011).
The reaction mechanism of FokI excludes the possibility of targeting zinc finger nucleases to unique DNA sites.
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Biochem Soc Trans,
39,
584-588.
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S.H.Chan,
B.L.Stoddard,
and
S.Y.Xu
(2011).
Natural and engineered nicking endonucleases--from cleavage mechanism to engineering of strand-specificity.
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Nucleic Acids Res,
39,
1.
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M.Lelke,
L.Brunotte,
C.Busch,
and
S.Günther
(2010).
An N-terminal region of Lassa virus L protein plays a critical role in transcription but not replication of the virus genome.
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J Virol,
84,
1934-1944.
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P.H.Too,
Z.Zhu,
S.H.Chan,
and
S.Y.Xu
(2010).
Engineering Nt.BtsCI and Nb.BtsCI nicking enzymes and applications in generating long overhangs.
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Nucleic Acids Res,
38,
1294-1303.
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K.L.Sanders,
L.E.Catto,
S.R.Bellamy,
and
S.E.Halford
(2009).
Targeting individual subunits of the FokI restriction endonuclease to specific DNA strands.
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Nucleic Acids Res,
37,
2105-2115.
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The most recent references are shown first.
Citation data come partly from CiteXplore and partly
from an automated harvesting procedure. Note that this is likely to be
only a partial list as not all journals are covered by
either method. However, we are continually building up the citation data
so more and more references will be included with time.
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Links
