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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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References listed in PDB file
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Key reference
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Title
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Alteration of sequence specificity of the type ii restriction endonuclease hincii through an indirect readout mechanism.
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Authors
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H.K.Joshi,
C.Etzkorn,
L.Chatwell,
J.Bitinaite,
N.C.Horton.
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Ref.
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J Biol Chem, 2006,
281,
23852-23869.
[DOI no: ]
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PubMed id
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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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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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