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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