Structures of Mismatch Replication
Errors Observed in a DNA Polymerase
Sean J. Johnson and Lorena S. Beese
Biochemistry 4000
Tracy Wang
High-fidelity DNA polymerases
maintain replication accuracy through:
• Select for correct base pairing, while strongly
discriminating against mismatched bases ( prior to
covalent incorporation)
• Stalling of the polymerases, thereby favoring
subsequent mismatch excision (after incorporation)
Klenow Fragment
• DNA polymerase I
• Highly processive
• Fingers, Thumb
and Palm
Taq polymerase
• Closed conformation
• dNTP bound
• Open conformation
• No incoming nt.
Model system
• Use the thermophilic Bacillus DNA
polymerase I fragment ( BF).
• BF is a high-fidelity Family A polymerase.
• Structural homology to the Klenow
Fragement of E.coli (KF) and T. aquaticus
(Taq) polymerases.
Model system
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Insertion site (n position )
Catalytic site
Preinsertion site
Postinsertion site (n-1 position )
DNA duplex binding region (n-2,…, n-5 positions )
Replication mechanism
• Transfer of the n template base from the pre-insertion site
to the insertion site:
- open to closed conformation
- hinge-bending motion
• Base pairs with an incoming base, covalent incorporation
takes place.
• Newly synthesized base moves into the post-insertion site.
• The DNA in the duplex binding region translocates by one
base pair.
• The next template base moves into the pre-insertion site.
Cognate G•C base pair bound at the postinsertion
(n-1) site.
• Hydrogen bond between
Asp830 and the 3’ primer
terminus.
• Arg615 and Gln797
interact with hydrogen
bond acceptor atom
located in the DNA minor
groove.
Objective
• To obtain and solve the structure of all 12
possible mismatches captured at the active site
of a DNA polymerase.
• To understand the mechanisms that lead to
mismatch-induced stalling of the polymerase.
Crystallographic capture of
mismatches
• Crystals with mismatches were obtained either
by enzymatic incorporation in the crystals (E), or
by cocrystallization of DNA duplexes that contain
a mismatch at the primer terminus (C).
• In the presence of Mg2+, accurate DNA
replication is obtained.
• In the presence of Mn2+, enzymatic
incorporation of mismatches is allowed.
• E: use mutagenic reaction conditions (MgSO4 
MnSO4 )
• C: BF cocrystallized with DNA substrates
Results
Results
• 12 possible covalently incorporated DNA
mismatches.
• Six are placed at the post-insertion site and are
well ordered (G•T, T•G, T•T, C•T, A•G, G•G).
• Three are placed at the post-insertion site but
are too disordered for interpretation (T•C, A•C,
C•A).
• Three are frayed (A•A, C•C, G•A).
Primer strand is at the insertion site
Template strand is at the pre-insertion site
Results
Results
• Structure of each mismatch is distinct.
• Some structures had been previously observed,
others do not.
• The type and degree of disruptions vary
depending on the identity of the mismatch.
Mismatch-induced disruptions at the active site
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Displacement of the template strand
Repositioning of Tyr714
Block the template pre-insertion site due to the rearrangement
B form DNA at the active site rather than A form
The catalytic site is undisturbed
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G adopts a conformation similar to
that of a cognate base  intact
catalytic site
Wobble conformation, T is positioned
toward DNA major groove
Minor groove interaction with Gln797
is lost  displacement of template
strand
Primer base G rotates 180 into a syn
conformation, template base remains
in an anti conformation  helical
width is closer to a Watson-Crick base
pair
More closely resembles the G•T
complex
Template strand is displaced
N3 is moved from the minor groove 
interaction between Arg615 and N3 is
lost
• T•C, A•C : disordered mismatches
• The disorder is confined primarily to the template strand
• Disorder on the primer strand is localized to the primer
base
• A mixture of A and B forms of DNA can be discerned in
the DBR.
• Protein adopts a distorted open conformation in which
the pre-insertion site is blocked.
• The primer terminus is displaced
• Interaction between Asp830 and the primer 3’ hydroxyl
at the catalytic site is disrupted
• The DNA backbone of the template strand is undisturbed
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wobble conformation.
The primer base lifts up into the
DNA major groove, and the
template base rotates slightly
toward the minor groove.
Shift of the 3’ hydroxyl 
breaking the hydrogen bond
with Asp830  disrupting
assembly of the catalytic site.
Not a wobble conformation.
Base pair directly opposite each
other
Significant opening in the minor
groove
Bridging water molecule
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Displacement of the template strand.
Blocking of the pre-insertion site.
Displacement of the primer strand.
Disruption of the catalytic site.
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Both bases maintain an anti
conformation  increase of the
helical width  extensive
movement of both template and
primer strands.
Altering the interaction with Asp830,
but without breaking it.
Wobble conformation.
An additional water-mediated Hbond.
Template is displaced
Interaction between Arg615 and the
primer base is lost
Sugar ring of T is disordered 
primer terminus is displaced, but
interaction between Asp830 and
primer 3’ hydroxyl may be retained
to some degree.
• Structure is frayed.
• The primer base is bound at the insertion site.
• The template base is bound at the pre-insertion site.
• A•A, C•C, G•A: frayed mismatches.
• Do not bind in the post-insertion site.
• Tyr714 blocks access of the template base to the
insertion site.
• Tyr714 also prevents the primer base from stacking to
the DNA helix. Primer base stacks against Phe710.
• Catalytic site is completely disrupted.
Mismatch extensions
• By transferring BF.DNA cocrystals containing a
mismatch at the 3’ primer terminus into reaction buffers
containing nucleotides complementary to the template
strand.
• By soaking in different combinations of nucleotides in the
presence of Mg2+ to control the final location of the
mismatch
Results
• The A•G, T•T, T•G, and C•C mismatches failed to extend.
• The G•T, C•T, and G•G mismatches were all
successfully extended.
• For the T•C mismatch, DNA is too disordered to permit
interpretation.
G•T Extension
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At n-2 position: wobble
At n-2 and n-3: disrupt A to B form
transition, similar to the disruptions
observed when G•T is at n-1
 disrupted active site
At n-4: partially restores the normal
DNA structure
 mixture of both a
disrupted and an
undisrupted active site
At n-3 and n-4 : the wobble inverts
inconsistent with the interbase Hbonding geometry associated with
the major tautomeric form of nt.
 tautomeric shift or
ionization of the mismatch
At n-6: fully restores the DNA
conformation
C•T Extension
• Extended in a single round of replication
• At n-2 position: fully restores the catalytic site.
Summary
• The high-resolution structures of all 12 possible
covalently incorporated DNA mismatches at the active
site of the BF polymerase were obtained.
• The mismatch-induced disruptions can be divided into
four broad categories.
1. Disruption of template strand and pre-insertion site.
2. Disruption of primer strand and assembly of catalytic site.
3. Disruption of template and primer strands.
4. Fraying of DNA at insertion site.
• The polymerase retains a short-term memory of the
mismatch incorporation event.
Discussion
• Category of mismatch-induced disruptions  differences in
mismatch extension efficiencies ?
• G•T: disruption may account for the reduced extension efficiency
ten-fold to a thousand-fold.
G•G: more than 100-fold slower than that of G•T.
• C•T: can be extended readily due to the flexibility of DNA.
• A•G: efficiencies are reduced by up to million-fold.
Discussion
• A heteroduplex can adjust its size in order to
translocating through the DBR.
• The structural adaptations are confined primarily to the
DNA, with some local protein side chain motions.
• Exonucleolytic excision of mismatches requires
dissociation of the heteroduplex from the polymerase:
A number of the structures show diminished interactions.
• Some mismatch conformations have not been previously
described in a DNA heteroduplex.
- Structural adaptations are not confined to simple
conformational rearrangements.
• Each equivalent mismatch interacts in a unique manner
with the protein. (e.g. G•T, T•G)
Future work
• Basis for future biochemical and structural
studies.
• Study the structural changes of a given
mismatch under different conditions (pH,
sequence context).
• To see if these mechanisms can be
applied to other DNA polymerases and
even RNA polymerases.
References
• Johnson, S. J. and Beese, L. B. Cell 2004, 116, 803-816.