1 bypassing lesions in dpo4 (oxo-g:a mismatch) a quantum mechanical/molecular mechanics (qm/mm)...
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Bypassing Lesions in DPO4 (oxo-G:A mismatch) Bypassing Lesions in DPO4 (oxo-G:A mismatch)
A Quantum Mechanical/Molecular Mechanics (QM/MM) A Quantum Mechanical/Molecular Mechanics (QM/MM)
Investigation of the Chemical StepInvestigation of the Chemical Step
Mihaela D. Bojin and Tamar Schlick
Retreat 02/08/08
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a) Biological challenges:
- Understand mismatched DPO4’s chemical step
- Compare our results with those obtained for the correct insertion of various
dNTPs into DPO4
- Identify similarities to repair processes that also occur via two-ion pathways, in
other polymerases (pol , pol , pol x, T7)
b) Modeling approaches:
- Determine appropriate models of the active site
- Employ QM and QM/MM computations
c) Results/Open questions/Significance
- Propose a favored mechanism and relevant intermediates on the potential
surface (How does DPO4 reach an active state? Does protonation matter? How
important are the water molecules from the active site?)
- Discuss what could we bring new to the field: understanding lesions versus
correct insertions, and the role of open active sites
OutlineOutline
3J. Mol. Evol., 2002, 54, 763
Numerous DNA polymerase sequences have been determined from three domains
of life: Archaea, Bacteria, Eukarya. They have been classified by Ito and Braithwaite
into the following classes: A, B, C, D, X, Y.
These polymerases operate via a two ion mechanism, which is a general repair
tool.
This implies a myriad of similar itineraries have in common the so-called “chemical
step” (breaking of the triphosphate and nucleotide transfer).
BackgroundBackground
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341 AA
29 DNA
Ca2+
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(A) Superimposition of the simulated structure (light
green) in the trajectory after chemistry with metal
ions and PPi removed to the ternary crystal
structure (light red) according to the palm domains.
(B) Enlarged view of the DNA duplexes before
(red) and after (green) the simulation. 8-OxoG and
dCTP are labeled as OxoG and C, respectively.
Black arrow indicates the direction of their
movements.
(C) Comparison of the LF domains before (light
red) and after (light green) simulation to that of the
Dbh apo-structure (blue) by superimposing the
palm domains.
Y. Wang, K. Arora, and T.Schlick (2006). Protein Sci., 15, 135-151.
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stability (from high to low) - pol G:C > 8-oxoG:C > 8-oxoG:A > G:A
The base pairing possibilities of 8-oxoG (8oG).
In an anti conformation it forms a Watson-Crick base pair with dCTP (a); by assuming a syn conformation, it can form a Hoogsteen base pair with dATP (b).
Wang and Schlick BMC Structural Biology 2007 7:7
Wang, Y., Reddy, S., Beard, W. A, Wilson, S. H, Schlick, T., Biophysical Journal, May 1, 2007
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“Efficient and High Fidelity Incorporation of dCTP Opposite 7,8-Dihydro-8-oxodeoxyguanosine by Sulfolobus solfataricus DNA Polymerase Dpo4 Formula” Hong Zang, Adriana Irimia, Jeong-Yun Choi, Karen C. Angel, Lioudmila V. Loukachevitch, Martin Egli, and F. Peter Guengerich J. Biol. Chem., Vol. 281, Issue 4, 2358-2372, January 27, 2006
Steady-state kinetics with the Y-family Sulfolobus solfataricus DNA
polymerase IV (Dpo4) showed 90-fold higher incorporation efficiency of dCTP
> dATP opposite 8-oxoG and 4-fold higher efficiency of extension beyond an
8-oxoG:C pair than an 8-oxoG:A pair.
The catalytic efficiency for these events (with dCTP or C) was similar for G
and 8-oxoG templates.
The 8-oxoG:A pair was in the syn:anti conformation
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Base pairing modes of 8-
oxoG (8OG) at the active
sites of the
Dpo4-dGTP, -dATP, and -
dCTP complexes
Eoff, R., et al, J. Biol. Chem., Vol. 282, Issue 27, 19831Zang, H. et al. J. Biol. Chem. 2006;281:2358-2372
O. Rechkoblit, L. Malinina, Y. Cheng, V. Kurvavvi, S. Broyde, N. E. Geacintov, and D. J. Patel (2006). PLoS. Biol. 1, e11.
9L. Wang, X. Yu, P. Hu, S. Broyde, and Y. Zhang, J. Am. Chem. Soc., 129 (15), 4731 -4737, 2007
(A) Dpo4 ternary complex active site based on molecular modeling/dynamics and subsequently ab initio QM/MM minimizations. (PDB ID: 1S0M).
(B) Active site of the Pol crystal structure (PDB ID: 2FMS)
10L. Wang, X. Yu, P. Hu, S. Broyde, and Y. Zhang, J. Am. Chem. Soc., 129 (15), 4731 -4737, 2007
M. Bojin, and T. Schlick, J Phys Chem B. 2007;111(38):11244-52
DPO4 (G:C)
Pol (G:C)
H H
H
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Problems solved for pol Problems solved for pol ’s mechanism ’s mechanism (computationally)(computationally)
Molecular dynamics MD simulations revealed an induced-fit mechanism and
delineated specific conformational changes that occur in the closing pathway of pol .
Modeling (MD simulations) demonstrated how an incorrect basepair inserted in the
DNA primer or template site introduces geometric deformations in the active site that
hamper conformational closing before the chemical reaction.
Transition path sampling (TPS) simulations revealed the major transition states
present in closing of the DNA polymerase and the energies associated with each step.
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Large models which include the primer terminus and incoming dNTP may not converge
Two unbound water molecules (H2O) provide a strong hydrogen network
Proposed model/Mechanistic StepsProposed model/Mechanistic Steps
Replace Ca2+ with Mg2+cat
Replace D 105, D7(Asp), E7 (Glu) by HCOO,
ddNTP and primer with CH3— groups
3-
P
O1
O2
O5'O3 P
O2
O1 O
Mgnuc PO
O2O1
Mgcat
O3«
Oc
Od O
O
Ob
H
H
Oa
HH2O
OH2
E106
D7
D105
H2O H2O
HH3C
CH3
Model for primer
Model for dNTP
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Mechanistic stepsMechanistic steps
1. Rearrangement.
2. Proton migration:
a) via direct transfer to O2(P)
b) indirectly, via a water molecule to triphosphate (P, or P)
c) directly to E106
d) indirectly (through water) to D7, or D105
3. Release of the pyrophosphate
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Work on an equilibrated active structure of DPO4 (MD)
Computations using B3LYP or MP2 functionals, and 6-31G or 6-
311+G(d,p), on the active site, with and without crystallographic water
molecules.
Improve the starting active site for QM/MM computations and explore
several potential mechanisms (direct proton transfer, indirect - to a water or
an aminoacid).
Consider protonation of the active site.
Summary of current results/plansSummary of current results/plans
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Acknowledgements
• Dr. Tamar Schlick
• Ms. Meredith Foley
• Dr. Yanli Wang