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PDBsum entry 1l1o
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DNA binding protein
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PDB id
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1l1o
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Contents |
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115 a.a.
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123 a.a.
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173 a.a.
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* Residue conservation analysis
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References listed in PDB file
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Key reference
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Title
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Structure of the rpa trimerization core and its role in the multistep DNA-Binding mechanism of rpa.
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Authors
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E.Bochkareva,
S.Korolev,
S.P.Lees-Miller,
A.Bochkarev.
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Ref.
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EMBO J, 2002,
21,
1855-1863.
[DOI no: ]
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PubMed id
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Abstract
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The human single-stranded DNA-binding protein, replication protein A (RPA) binds
and stable
( approximately 30 nt). Switching from 8 to 30 nt mode is associated with a
large conformational change. Here we report the 2.8 A structure of the RPA
trimerization core comprising the C-terminal DNA-binding domain of subunit RPA70
(DBD-C), the central DNA-binding domain of subunit RPA32 (DBD-D) and the entire
RPA14 subunit. All three domains are built around a central
oligonucleotide/oligosaccharide binding (OB)-fold and flanked by a helix at the
C-terminus. Trimerization is mediated by three C-terminal helices arranged in
parallel. The OB-fold of DBD-C possesses unique structural features; embedded
zinc ribbon and helix-turn-helix motifs. Using time-resolved proteolysis with
trypsin, we demonstrate that the trimerization core does not contribute to the
binding with substrates of 10 nt, but interacts with oligonucleotides of 24 nt.
Taken together, our data indicate that switching from 8-10 to 30 nt mode is
mediated by DNA binding with the trimerization core.
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Figure 3.
Figure 3 DBD-C modeled on a ssDNA molecule derived from the
DBD-AB/ssDNA co-crystal structure. The DBD-C was aligned with
DBD-B as shown in Figure 2C and then docked onto the 3 nt DBD-B
binding site (shown in red). Side chains in DBD-C (the position
for Y581 is putative) that are orientated to interact with the
sugar-phosphate backbone are colored in green and blue (for
carbon and nitrogen atoms, respectively), and those in position
to hydrogen bond, or stack with, DNA bases are yellow. Putative
hydrogen bonds with the phosphate groups are shown as dashed
lines. The position of conserved C486 is shown as a reference.
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Figure 6.
Figure 6 Suggested model of the apo RPA trimer. Cartoon
representing RPA as a hexamer of OB-folds centered around a
multi-helical bundle. Helices are represented by circles, and
the six OB-folds by palms, which are labeled as 14, 70-NTD
(N-terminal domain), A, B, C and D (for RPA14, RPA70N, DBD-A,
-B, -C and -D, respectively). The trimerization core is outlined
with a triangle. Putative elements, an interaction between
RPA70N and RPA14, and the transient helix of RPA70N, are shown
with question mark. See text for more details.
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The above figures are
reprinted
from an Open Access publication published by Macmillan Publishers Ltd:
EMBO J
(2002,
21,
1855-1863)
copyright 2002.
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Secondary reference #1
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Title
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The role for zinc in replication protein a.
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Authors
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E.Bochkareva,
S.Korolev,
A.Bochkarev.
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Ref.
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J Biol Chem, 2000,
275,
27332-27338.
[DOI no: ]
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PubMed id
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Figure 3.
Fig. 3. Metal ion modulates ssDNA-binding activity of the
RPA14·32-(43-171)·70-(436-616) trimer. The trimer
was incubated with 20 fmol of 32P-labeled (dN)[31] in the
absence/presence of 10 mM EDTA (A) or 1 mM OP ( B). Complexes
were resolved by nondenaturing gel electrophoresis and
visualized by autoradiography. The numbers above each line refer
to the molecular excess of the protein over the DNA probe. The
mobility of the free ssDNA is shown.
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Figure 4.
Fig. 4. A comparison of ssDNA binding activity of
RPA14·32-(43-171)·70-(436-616) and
RPA70-(181-432). The electrophoretic mobilities of the
designated subcomplexes were estimated the same way as indicated
in the legend for Fig. 3. In this experiment, 0.5 mM DTT was
added to the running buffer (0.5× TB).
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The above figures are
reproduced from the cited reference
with permission from the ASBMB
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