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PDBsum entry 3bep
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Transferase, transcription/DNA
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PDB id
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3bep
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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 a sliding clamp on DNA.
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Authors
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R.E.Georgescu,
S.S.Kim,
O.Yurieva,
J.Kuriyan,
X.P.Kong,
M.O'Donnell.
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Ref.
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Cell, 2008,
132,
43-54.
[DOI no: ]
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PubMed id
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Abstract
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The structure of the E. coli beta clamp polymerase processivity factor has been
solved in complex with primed DNA. Interestingly, the clamp directly binds the
DNA duplex and also forms a crystal contact with the ssDNA template strand,
which binds into the protein-binding pocket of the clamp. We demonstrate that
these clamp-DNA interactions function in clamp loading, perhaps by inducing the
ring to close around DNA. Clamp binding to template ssDNA may also serve to hold
the clamp at a primed site after loading or during switching of multiple factors
on the clamp. Remarkably, the DNA is highly tilted as it passes through the beta
ring. The pronounced 22 degrees angle of DNA through beta may enable DNA to
switch between multiple factors bound to a single clamp simply by alternating
from one protomer of the ring to the other.
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Figure 3.
Figure 3. Structure of the β-DNA Complex
(A) Ribbon
representation of the β-DNA complex: front and side views. DNA
is tilted  vert,
similar 22° from the C2 rotation axis of β. The Cy5 moiety
is not shown for clarity but is shown in Figure S4.
(B)
Detailed view of R24 (top) and Q149 (bottom) in the β-DNA
complex compared to the apo β structure (blue).
(C)
Replication assays using primed M13 ssDNA coated with SSB
contain Pol III^* and the indicated amount of either WT β (blue
diamonds), β[Q149A] (red squares), β[R24A] (orange triangles),
or β[R24A/Q149A] (green circles).
(D) Polymerase extension
rate was determined with primed M13 ssDNA to which β, or mutant
β, is first assembled on the DNA, followed by initiating
synchronous chain extension. Reactions were quenched at the
indicated times, and products were analyzed in a native agarose
gel.
(E) The scheme illustrates the bead conjugated primed
DNA in which SSB blocks ^32P-β from sliding off the end of the
DNA. Clamp loading rate was assessed in assays with either
β[WT] (blue), β[Q149A] (red), β[R24A] (orange), or
β[R24A,Q149A] (green).
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Figure 4.
Figure 4. Interaction of β with the ssDNA Region of the
Primed Site
(A) Crystal lattice showing two molecules of
the β-DNA complex. The ssDNA makes a crystal contact between
two molecules of β.
(B) Surface representation of the
charged residues that line the channel directing ssDNA to the
hydrophobic protein-binding site of β. Basic residues are
colored blue. The protein-binding site is shaded purple and
subsites I and II (defined in Burnouf et al., 2004) are
indicated.
(C) View of ssDNA (orange) positioned inside the
hydrophobic protein-binding pocket of β. Thy[11] and Thy[12]
occupy subsite I of the β hydrophobic pocket; the exposed R246,
R240 side chains interact with the DNA phosphate backbone. Y153
and Y154 (green) stack with Ade[15] and Thy[13], respectively.
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The above figures are
reprinted
from an Open Access publication published by Cell Press:
Cell
(2008,
132,
43-54)
copyright 2008.
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