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PDBsum entry 1q9y
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Transferase, replication/DNA
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PDB id
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1q9y
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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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Lesion (in)tolerance reveals insights into DNA replication fidelity.
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Authors
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E.Freisinger,
A.P.Grollman,
H.Miller,
C.Kisker.
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Ref.
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EMBO J, 2004,
23,
1494-1505.
[DOI no: ]
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PubMed id
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Abstract
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The initial encounter of an unrepaired DNA lesion is likely to be with a
replicative DNA polymerase, and the outcome of this event determines whether an
error-prone or error-free damage avoidance pathway is taken. To understand the
atomic details of this critical encounter, we have determined the crystal
structures of the pol alpha family RB69 DNA polymerase with DNA containing the
two most prevalent, spontaneously generated premutagenic lesions, an abasic site
and 2'-deoxy-7,8-dihydro-8-oxoguanosine (8-oxodG). Identification of the
interactions between these damaged nucleotides and the active site provides
insight into the capacity of the polymerase to incorporate a base opposite the
lesion. A novel open, catalytically inactive conformation of the DNA polymerase
has been identified in the complex with a primed abasic site template. This
structure provides the first molecular characterization of the DNA synthesis
barrier caused by an abasic site and suggests a general mechanism for polymerase
fidelity. In contrast, the structure of the ternary 8-oxodG:dCTP complex is
almost identical to the replicating complex containing unmodified DNA,
explaining the relative ease and fidelity by which this lesion is bypassed.
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Figure 1.
Figure 1 Oligonucleotide sequences and DNA adducts. (A) The 14
nt primers are identical in all trials and terminated by ddC
(indicated by C^*). Template strands are 18 nt long with a 3'-dG
overhang. X denotes the position of the lesion for
primer/template combinations (1) and (2), and the arrow the
position of dNTP incorporation. (B) Structures of the lesions at
position X in the templates 8-oxodG and tetrahydrofuran (abasic
site model).
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Figure 7.
Figure 7 Influence of Gly 568 on DNA binding to the polymerase
active site. Column (I) shows the event of nucleotide insertion
opposite an unmodified template strand with adenine in the
active site, while column (II) depicts the case of a template
containing an abasic site as in the AP:dG complex. Vertical
arrows specify the strained (red) or the relaxed state (green),
respectively. Diagonal arrows indicate whether the polymerase is
in the closed (red) or open conformation (green). The template
strand is depicted in magenta and the incoming nucleotide in
green. The yellow box indicates the position of Gly 568. (IA)
and (IIA) show the polymerase in the strained state and the open
conformation. Transition into the relaxed state presumably
causes the adenine base of the unmodified template to be pushed
back (IB), while the AP-containing template is unaffected (IIB).
(IC) and (IIC) depict an incoming dNTP bound to the base of the
fingers domains. Transition to the closed and strained
conformation ensures the correct positioning of all residues to
enable the catalytic phosphodiester bond formation (ID). In the
case of AP, a closed and relaxed conformation is feasible (IID).
The missing complementary base causes the dNTP to be held in
place less tightly and phosphodiester bond formation is less
efficient (IIE).
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The above figures are
reprinted
from an Open Access publication published by Macmillan Publishers Ltd:
EMBO J
(2004,
23,
1494-1505)
copyright 2004.
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