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DOI no:
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J Biol Chem
275:2705-2712
(2000)
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PubMed id:
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Probing the structure of the PI-SceI-DNA complex by affinity cleavage and affinity photocross-linking.
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D.Hu,
M.Crist,
X.Duan,
F.A.Quiocho,
F.S.Gimble.
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ABSTRACT
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The PI-SceI protein is an intein-encoded homing endonuclease that initiates the
mobility of its gene by making a double strand break at a single site in the
yeast genome. The PI-SceI protein splicing and endonucleolytic active sites are
separately located in each of two domains in the PI-SceI structure. To determine
the spatial relationship between bases in the PI-SceI recognition sequence and
selected PI-SceI amino acids, the PI-SceI-DNA complex was probed by
photocross-linking and affinity cleavage methods. Unique solvent-accessible
cysteine residues were introduced into the two PI-SceI domains at positions 91,
97, 170, 230, 376, and 378, and the mutant proteins were modified with either
4-azidophenacyl bromide or iron
(S)-1-(p-bromoacetamidobenzyl)-ethylenediaminetetraacetate (FeBABE). The phenyl
azide-coupled proteins cross-linked to the PI-SceI target sequence, and the
FeBABE-modified proteins cleaved the DNA proximal to the derivatized amino acid.
The results suggest that an extended beta-hairpin loop in the endonuclease
domain that contains residues 376 and 378 contacts the major groove near the
PI-SceI cleavage site. Conversely, residues 91, 97, and 170 in the protein
splicing domain are in close proximity to a distant region of the substrate. To
interpret our results, we used a new PI-SceI structure that is ordered in
regions of the protein that bind DNA. The data strongly support a model of the
PI-SceI-DNA complex derived from this structure.
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Selected figure(s)
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Figure 2.
Fig. 2. Location of cysteine-substituted residues in
PI-SceI. The structure of PI-SceI showing the position of the
side chains of Arg-91, Lys-97, His-170, Ser-230, Lys-376, and
Lys-378 that have been individually mutated to cysteine and
conjugated to FeBABE and/or phenyl azide moieties.
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Figure 6.
Fig. 6. Affinity photocross-linking of the top and bottom
strands of the 219-bp fragment containing a single PI-SceI
recognition site by phenyl azide-conjugated PI-SceI proteins.
The DNA strand that is end-labeled with 32P is indicated at the
top of each set of lanes. The locations of photocross-linking
products generated by azidophenacyl-PI-SceI (  5Cys) are
indicated by open arrows, and those produced by
azidophenacyl-PI-SceI[97], azidophenacyl-PI-SceI[170], and
azidophenacyl-PI-SceI[376] are indicated by solid arrows. The
location of the PI-SceI cleavage site and the extent of the
PI-SceI recognition sequence are indicated as in Fig. 5.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2000,
275,
2705-2712)
copyright 2000.
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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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M.A.Johnson,
M.W.Southworth,
T.Herrmann,
L.Brace,
F.B.Perler,
and
K.Wüthrich
(2007).
NMR structure of a KlbA intein precursor from Methanococcus jannaschii.
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Protein Sci, 16,
1316-1328.
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PDB codes:
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S.Bräse,
C.Gil,
K.Knepper,
and
V.Zimmermann
(2005).
Organic azides: an exploding diversity of a unique class of compounds.
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Angew Chem Int Ed Engl, 44,
5188-5240.
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A.Bakhrat,
M.S.Jurica,
B.L.Stoddard,
and
D.Raveh
(2004).
Homology modeling and mutational analysis of Ho endonuclease of yeast.
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Genetics, 166,
721-728.
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R.David,
M.P.Richter,
and
A.G.Beck-Sickinger
(2004).
Expressed protein ligation. Method and applications.
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Eur J Biochem, 271,
663-677.
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J.C.Epinat,
S.Arnould,
P.Chames,
P.Rochaix,
D.Desfontaines,
C.Puzin,
A.Patin,
A.Zanghellini,
F.Pâques,
and
E.Lacroix
(2003).
A novel engineered meganuclease induces homologous recombination in yeast and mammalian cells.
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| |
Nucleic Acids Res, 31,
2952-2962.
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T.Fukuda,
S.Nogami,
and
Y.Ohya
(2003).
VDE-initiated intein homing in Saccharomyces cerevisiae proceeds in a meiotic recombination-like manner.
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Genes Cells, 8,
587-602.
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C.M.Moure,
F.S.Gimble,
and
F.A.Quiocho
(2002).
Crystal structure of the intein homing endonuclease PI-SceI bound to its recognition sequence.
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Nat Struct Biol, 9,
764-770.
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PDB codes:
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E.Werner,
W.Wende,
A.Pingoud,
and
U.Heinemann
(2002).
High resolution crystal structure of domain I of the Saccharomyces cerevisiae homing endonuclease PI-SceI.
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Nucleic Acids Res, 30,
3962-3971.
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PDB code:
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J.P.Gogarten,
A.G.Senejani,
O.Zhaxybayeva,
L.Olendzenski,
and
E.Hilario
(2002).
Inteins: structure, function, and evolution.
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Annu Rev Microbiol, 56,
263-287.
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K.L.Posey,
and
F.S.Gimble
(2002).
Insertion of a reversible redox switch into a rare-cutting DNA endonuclease.
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Biochemistry, 41,
2184-2190.
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S.Nogami,
T.Fukuda,
Y.Nagai,
S.Yabe,
M.Sugiura,
R.Mizutani,
Y.Satow,
Y.Anraku,
and
Y.Ohya
(2002).
Homing at an extragenic locus mediated by VDE (PI-SceI) in Saccharomyces cerevisiae.
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Yeast, 19,
773-782.
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B.S.Chevalier,
and
B.L.Stoddard
(2001).
Homing endonucleases: structural and functional insight into the catalysts of intron/intein mobility.
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Nucleic Acids Res, 29,
3757-3774.
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F.S.Gimble
(2001).
Degeneration of a homing endonuclease and its target sequence in a wild yeast strain.
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Nucleic Acids Res, 29,
4215-4223.
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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.
|
405 a.a.
