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PDBsum entry 2dtu
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Transferase/DNA
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PDB id
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2dtu
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References listed in PDB file
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Key reference
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Title
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Structural and biochemical investigation of the role in proofreading of a beta hairpin loop found in the exonuclease domain of a replicative DNA polymerase of the b family.
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Authors
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M.Hogg,
P.Aller,
W.Konigsberg,
S.S.Wallace,
S.Doublié.
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Ref.
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J Biol Chem, 2007,
282,
1432-1444.
[DOI no: ]
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PubMed id
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Abstract
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Replicative DNA polymerases, as exemplified by the B family polymerases from
bacteriophages T4 and RB69, not only replicate DNA but also have the ability to
proofread misincorporated nucleotides. Because the two activities reside in
separate protein domains, polymerases must employ a mechanism that allows for
efficient switching of the primer strand between the two active sites to achieve
fast and accurate replication. Prior mutational and structural studies suggested
that a beta hairpin structure located in the exonuclease domain of family B
polymerases might play an important role in active site switching in the event
of a nucleotide misincorporation. We show that deleting the beta hairpin loop in
RB69 gp43 affects neither polymerase nor exonuclease activities. Single binding
event studies with mismatched primer termini, however, show that the beta
hairpin plays a role in maintaining the stability of the polymerase/DNA
interactions during the binding of the primer DNA in the exonuclease active site
but not on the return of the corrected primer to the polymerase active site. In
addition, the deletion variant showed a more stable incorporation of a
nucleotide opposite an abasic site. Moreover, in the 2.4 A crystal structure of
the beta hairpin deletion variant incorporating an A opposite a templating
furan, all four molecules in the crystal asymmetric unit have DNA in the
polymerase active site, despite the presence of DNA distortions because of the
misincorporation, confirming that the primer strand is not stably bound within
the exonuclease active site in the absence of the beta hairpin loop.
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Figure 1.
FIGURE 1. Switching of the  hairpin loop in response
to nucleotide incorporation opposite a lesion. A, the closed
ternary complex of RB69 gp43 trapped with an incoming dTTP
opposite a templating A (PDB code 1IG9 (17)). The 5'-end of the
template DNA (gray) stacks against Trp-574 (gold), and the hairpin
is in an up position. B, an open binary complex of RB69 gp43
after successful incorporation of an A opposite a furan (PDB
code 1RV2, chain C (15)). In this structure the 5'-end of the
template is sandwiched between Phe-359 (purple) and the hairpin,
which is in a down position.
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Figure 7.
FIGURE 7. In the crystal structure of the hairpin
deletion variant, all four molecules have DNA in the polymerase
active site. A and B, two of the four complexes found in the
asymmetric unit of the previously solved furan-dAMP binary
complex (PDB code 1RV2 (15)). One complex has DNA in the
exonuclease active center (molecule B) (A); the other has DNA in
the polymerase active site (molecule C) (B). C and D, the
equivalent complexes from the structure of the -variant
inserting an A opposite furan. In this structure none of the
four complexes within the asymmetric unit has DNA in the
exonuclease (exo) domain. The polymerase domains are colored red
for the palm, green for the thumb, blue for the fingers, cyan
for the exonuclease (with the hairpin in black), and
orange for the N-terminal domain. The primer strand is shown in
magenta and the template in dark blue. The tip of the hairpin
loop was disordered in A.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2007,
282,
1432-1444)
copyright 2007.
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