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PDBsum entry 2gih
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Hydrolase/DNA
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PDB id
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2gih
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Enzyme class:
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E.C.3.1.21.4
- type Ii site-specific deoxyribonuclease.
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Reaction:
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Endonucleolytic cleavage of DNA to give specific double-stranded fragments with terminal 5'-phosphates.
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Cofactor:
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Mg(2+)
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DOI no:
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J Biol Chem
281:23852-23869
(2006)
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PubMed id:
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Alteration of sequence specificity of the type II restriction endonuclease HincII through an indirect readout mechanism.
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H.K.Joshi,
C.Etzkorn,
L.Chatwell,
J.Bitinaite,
N.C.Horton.
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ABSTRACT
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The functional and structural consequences of a mutation of the DNA
intercalating residue of HincII, Q138F, are presented. Modeling has suggested
that the DNA intercalation by Gln-138 results in DNA distortions potentially
used by HincII in indirect readout of its cognate DNA, GTYRAC (Y = C or T, R = A
or G) (Horton, N. C., Dorner, L. F., and Perona, J. J. (2002) Nat. Struct. Biol.
9, 42-47). Kinetic data presented here indicate that the mutation of glutamine
138 to phenylalanine (Q138F) results in a change in sequence specificity at the
center two base pairs of the cognate recognition site. We show that the
preference of HincII for cutting, but not binding, the three cognate sites
differing in the center two base pairs has been altered by the mutation Q138F.
Five new crystal structures are presented including Q138F HincII bound to GTTAAC
and GTCGAC both with and without Ca2+ as well as the structure of wild type
HincII bound to GTTAAC. The Q138F HincII/DNA structures show conformational
changes in the protein, bound DNA, and at the protein-DNA interface, consistent
with the formation of adaptive complexes. Analysis of these structures and the
effect of Ca2+ binding on the protein-DNA interface illuminates the origin of
the altered specificity by the mutation Q138F in the HincII enzyme.
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Selected figure(s)
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Figure 6.
FIGURE 6. Stereo diagrams of superpositions using base
atoms of purine 8 of wild type HincII/CG/Ca^2+ (black) showing
the central two base pairs of the recognition sequence GTYRAC
(underlined) of both strands. A, Q138F/TA (red). B,
Q138F/TA/Ca^2+ (pink). C, Q138F/CG (blue). D, Q138F/CG/Ca^2+
(cyan). E, Q138F/CG/Ca^2+ (cyan), Q138F/TA/Ca^2+ (pink), and
wild type HincII/CG/Ca^2+ (gray) showing the propagation of
differences in stacking with the center step purines to the
phosphate position of Ade 9. A residue of the active site,
Asp-127, as well as the adjacent residue implicated in potential
steric conflicts with the DNA, Thr-130, are also shown. Py, a
pyrimidine; Pur, a purine.
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Figure 9.
FIGURE 9. Stereo diagrams of active site superpositions,
wild type HincII/CG/Ca^2+ (gray) and Q138F/DNA structures
(colored). A, subunit A of Q138F/TA (red) and Q138F/CG (blue).
B, subunit B of Q138F/TA (red) and Q138F/CG (blue). C, subunit A
of Q138F/TA/Ca^2+ (pink) and Q138F/CG/Ca^2+ (cyan). D, subunit B
of Q138F/TA/Ca^2+ (pink) and Q138F/CG/Ca^2+ (cyan).
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2006,
281,
23852-23869)
copyright 2006.
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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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J.H.Chang,
X.Jiao,
K.Chiba,
C.Oh,
C.E.Martin,
M.Kiledjian,
and
L.Tong
(2012).
Dxo1 is a new type of eukaryotic enzyme with both decapping and 5'-3' exoribonuclease activity.
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Nat Struct Mol Biol,
19,
1011-1017.
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PDB codes:
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M.Firczuk,
M.Wojciechowski,
H.Czapinska,
and
M.Bochtler
(2011).
DNA intercalation without flipping in the specific ThaI-DNA complex.
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Nucleic Acids Res,
39,
744-754.
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PDB code:
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S.Guan,
A.Blanchard,
P.Zhang,
and
Z.Zhu
(2010).
Alteration of sequence specificity of the type IIS restriction endonuclease BtsI.
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PLoS One,
5,
e11787.
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S.K.Menon,
B.J.Eilers,
M.J.Young,
and
C.M.Lawrence
(2010).
The crystal structure of D212 from sulfolobus spindle-shaped virus ragged hills reveals a new member of the PD-(D/E)XK nuclease superfamily.
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J Virol,
84,
5890-5897.
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PDB code:
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S.R.Coffin,
and
N.O.Reich
(2009).
Escherichia coli DNA adenine methyltransferase: the structural basis of processive catalysis and indirect read-out.
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J Biol Chem,
284,
18390-18400.
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A.C.Babic,
E.J.Little,
V.M.Manohar,
J.Bitinaite,
and
N.C.Horton
(2008).
DNA distortion and specificity in a sequence-specific endonuclease.
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J Mol Biol,
383,
186-204.
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PDB codes:
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E.J.Little,
A.C.Babic,
and
N.C.Horton
(2008).
Early interrogation and recognition of DNA sequence by indirect readout.
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Structure,
16,
1828-1837.
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PDB code:
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J.Orlowski,
and
J.M.Bujnicki
(2008).
Structural and evolutionary classification of Type II restriction enzymes based on theoretical and experimental analyses.
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Nucleic Acids Res,
36,
3552-3569.
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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.
Where a reference describes a PDB structure, the PDB
codes are
shown on the right.
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Links
