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PDBsum entry 2z4s
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DNA binding protein
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PDB id
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2z4s
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References listed in PDB file
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Key reference
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Title
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A common mechanism for the ATP-Dnaa-Dependent formation of open complexes at the replication origin.
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Authors
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S.Ozaki,
H.Kawakami,
K.Nakamura,
N.Fujikawa,
W.Kagawa,
S.Y.Park,
S.Yokoyama,
H.Kurumizaka,
T.Katayama.
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Ref.
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J Biol Chem, 2008,
283,
8351-8362.
[DOI no: ]
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PubMed id
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Abstract
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Initiation of chromosomal replication and its cell cycle-coordinated regulation
bear crucial and fundamental mechanisms in most cellular organisms. Escherichia
coli DnaA protein forms a homomultimeric complex with the replication origin
(oriC). ATP-DnaA multimers unwind the duplex within the oriC unwinding element
(DUE). In this study, structural analyses suggested that several residues
exposed in the central pore of the putative structure of DnaA multimers could be
important for unwinding. Using mutation analyses, we found that, of these
candidate residues, DnaA Val-211 and Arg-245 are prerequisites for initiation in
vivo and in vitro. Whereas DnaA V211A and R245A proteins retained normal
affinities for ATP/ADP and DNA and activity for the ATP-specific conformational
change of the initiation complex in vitro, oriC complexes of these mutant
proteins were inactive in DUE unwinding and in binding to the single-stranded
DUE. Unlike oriC complexes including ADP-DnaA or the mutant DnaA, ATP-DnaA-oriC
complexes specifically bound the upper strand of single-stranded DUE. Specific
T-rich sequences within the strand were required for binding. The corresponding
conserved residues of the DnaA ortholog in Thermotoga maritima, an ancient
eubacterium, were also required for DUE unwinding, consistent with the idea that
the mechanism and regulation for DUE unwinding can be evolutionarily conserved.
These findings provide novel insights into mechanisms for pore-mediated origin
unwinding, ATP/ADP-dependent regulation, and helicase loading of the initiation
complex.
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Figure 1.
FIGURE 1. Structural models of DnaA oligomers and tmaDnaA
mutants. A, crystal structure of tmaDnaA AAA^+ domain. The
crystal structure of tmaDnaA AAA^+ domain bound to ADP was
solved by molecular replacement. The A. aeolicus DnaA AAA^+
domain structure (22) was used as a search model. The subdomains
IIIa and IIIb within AAA^+ domain are depicted as a ribbon model
in different colors. B, structure comparison. Structures of
AAA^+ domain of tmaDnaA and A. aeolicus DnaA, colored purple and
cyan, respectively, were superimposed for subdomain IIIa.
Structures are shown in a frame model. C, a ring model structure
of tmaDnaA. A hexameric ring model was constructed using the
determined structure of tmaDnaA AAA^+ domain. Each protomer is
colored differently. The amino acid residues of Val-176,
Met-179, Lys-180, Lys-209, and Gly-211 are exposed on the pore
surface of the ring and are colored in red. For i–vi, see
panel F. D, a spiral filament model structure of tmaDnaA. A
spiral filament model was constructed using the structure of
tmaDnaA AAA^+ domain, based on the spiral filament structure of
the A. aeolicus DnaA AAA^+ domain (37). The model filament is
shown viewed down the helical axis (top), as well as
perpendicularly to the axis (bottom). Each protomer is colored
differently. The amino acid residues described above are also
exposed on the pore surface of the spiral filament and are
colored red. E, the pore surfaces of the tmaDnaA ring and spiral
filament. Three consecutive molecules of tmaDnaA within the ring
(C) and spiral (D) models are shown. The amino acid residues
described above and analyzed in this study are colored red and
indicated. F, amino acid sequences, including the putative pore
region of representative DnaA orthologs. i–vi correspond to E.
coli DnaA residues of Val-211, Leu-214, Gln-215, Lys-223,
Lys-243, and Arg-245, respectively. Residues corresponding to
these are highlighted in yellow for hydrophobic residues or in
purple for basic residues. The secondary structures of A.
aeolicus DnaA (helix  3- 6) are also shown (22).
Ecoli, Escherichia coli; Thema, Thermotoga maritima; Aquae,
Aquifex aeolicus; Helph, Helicobacter pylori; Bacsu, Bacillus
subtilis; Myctu, Mycobacterium tuberculosis; Chlmu, and
Chlamydia muridarum. G, tma-oriC unwinding assay. The indicated
amounts of wild-type (WT) or mutant tmaDnaA proteins were
incubated at 48 °C for 10 min in the presence of pOZ14 (200
fmol), which bears the tma-oriC, followed by digestion with P1
nuclease and AlwNI.
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Figure 10.
FIGURE 10. A model for open complex formation. A, a model
of DnaA complexes and ATP-dependent conformational change. Light
blue, ADP; light pink, ATP; yellow, H-motif; blue, B-motif; and
red, arginine finger. B, possible mixed complexes. The
initiation activity of the mixed complexes is indicated by
± (moderate) or + (active). Mixed complexes of wild-type
DnaA with mutant DnaA of the B-motif (i), H-motif (ii), or
arginine finger (iii) are shown. Light gray color indicates
mutant DnaA protomers.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2008,
283,
8351-8362)
copyright 2008.
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