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)
A Quantum Mechanical/Molecular Mechanics (QM/MM)
Investigation of the Chemical Step
Mihaela D. Bojin and Tamar Schlick
Retreat 02/08/08
1
Outline
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 b, pol l, 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
2
Background
 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).
3
J. Mol. Evol., 2002, 54, 763
341 AA
29 DNA
Ca2+
4
(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.
5
Y. Wang, K. Arora, and T.Schlick (2006). Protein Sci., 15, 135-151.
stability (from high to low) - pol b
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
6
 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 8oxoG: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
“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
7
Base pairing modes of 8oxoG (8OG) at the active
sites of the
Dpo4-dGTP, -dATP, and dCTP complexes
Eoff, R., et al, J. Biol. Chem., Vol. 282,
Issue 27, 19831
Zang, 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
8 1, e11.
(2006). PLoS. Biol.
(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 b crystal
structure (PDB ID: 2FMS)
L. Wang, X. Yu, P. Hu, S. Broyde, and Y. Zhang, J. Am.
Chem. Soc., 129 (15), 4731 -4737, 2007
9
DPO4 (G:C)
Pol b (G:C)
H
H
H
M. Bojin, and T. Schlick, J Phys Chem B. 2007;111(38):11244-52
L. Wang, X. Yu, P. Hu, S. Broyde, and Y. Zhang, J. Am. Chem. Soc., 129 (15), 4731 -4737, 2007
10
Problems solved for pol b’s mechanism (computationally)
 Molecular dynamics MD simulations revealed an induced-fit mechanism and
delineated specific conformational changes that occur in the closing pathway of pol b.
 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 b and the energies associated with each step.
11
Proposed model/Mechanistic Steps
 Large models which include the primer terminus and incoming dNTP may not converge
 Two unbound water molecules (H2O) provide a strong hydrogen network
Model for
dNTP
Model for
primer
CH3
O5'
O2
O3b
H
P
H3C
3-
O2b
Pb
O1b
O
O3«
O1
Oa
E106
Mgcat H O
2
Ob
H
H2O
Od
Oc
 Replace Ca2+ with Mg2+cat
D7
OH2
H2O
O
H
Mgnuc
O1
P
O
O2
O
D105
H
 Replace D 105, D7(Asp), E7 (Glu) by HCOO,
 ddNTP and primer with CH3— groups
12
Mechanistic 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
13
Summary of current results/plans
 Work on an equilibrated active structure of DPO4 (MD)
 Computations using B3LYP or MP2 functionals, and 6-31G or 6311+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.
14
Acknowledgements
• Dr. Tamar Schlick
• Ms. Meredith Foley
• Dr. Yanli Wang
15
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