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PDBsum entry 1v8o
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Structural genomics, unknown function
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PDB id
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1v8o
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Contents |
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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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Distant structural homology leads to the functional characterization of an archaeal pin domain as an exonuclease.
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Authors
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V.L.Arcus,
K.Bäckbro,
A.Roos,
E.L.Daniel,
E.N.Baker.
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Ref.
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J Biol Chem, 2004,
279,
16471-16478.
[DOI no: ]
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PubMed id
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Abstract
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Genome sequencing projects have focused attention on the problem of discovering
the functions of protein domains that are widely distributed throughout living
species but which are, as yet, largely uncharacterized. One such example is the
PIN domain, found in eukaryotes, bacteria, and Archaea, and with suggested roles
in signaling, RNase editing, and/or nucleotide binding. The first reported
crystal structure of a PIN domain (open reading frame PAE2754, derived from the
crenarchaeon, Pyrobaculum aerophilum) has been determined to 2.5 A resolution
and is presented here. Mapping conserved residues from a multiple sequence
alignment onto the structure identifies a putative active site. The discovery of
distant structural homology with several exonucleases, including T4 phage RNase
H and flap endonuclease (FEN1), further suggests a likely function for PIN
domains as Mg2+-dependent exonucleases, a hypothesis that we have confirmed in
vitro. The tetrameric structure of PAE2754, with the active sites inside a
tunnel, suggests a mechanism for selective cleavage of single-stranded overhangs
or flap structures. These results indicate likely DNA or RNA editing roles for
prokaryotic PIN domains, which are strikingly numerous in thermophiles, and in
organisms such as Mycobacterium tuberculosis. They also support previous
hypotheses that eukaryotic PIN domains participate in RNAi and nonsense-mediated
RNA degradation.
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Figure 2.
FIG. 2. Oligomeric state for PAE2754 showing conserved
residues. A, view of the dimer showing the residues that are
conserved across COG4113 (also shown in the alignment in Fig.
3). For chain A, three of the residues are labeled with their
one-letter amino acid code. B, surface depiction (in the same
orientation as A) of the PAE2754 dimer showing electrostatic
charges at the surface using GRASP. C, orthogonal views of the
tetramer surface showing the tunnel inside which the putative
active site resides. This figure was drawn using PyMol (38) and
GRASP (39).
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Figure 5.
FIG. 5. In vitro exonuclease activity of PAE2754. A
polyacrylamide/urea denaturing gel showing DNA stained by
ethidium bromide (see text). Lane A shows the 54-bp
oligonucleotide alone. Lanes B-G show 1-5- and 19-h incubations,
respectively, of annealed oligonucleotides (54 + 18 bp) with
PAE2754 and MgCl[2] at 37 °C. Lane H shows a 19-h incubation
of annealed oligonucleotides (54 + 18 bp) with PAE2754 at 37
°C in the absence of MgCl[2].
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2004,
279,
16471-16478)
copyright 2004.
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