1. 1 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

2. 2 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 Outline

3. 3 J. 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). Background

4. 4 341 AA 29 DNA Ca 2+

5. 5 (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.

6. 6 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

7. 7 “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

8. 8 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, 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 (2006). PLoS. Biol. 1, e11.

9. 9 L. 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)

10. 10 L. 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

11. 11 Problems solved for pol  ’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 .  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.

12. 12  Large models which include the primer terminus and incoming dNTP may not converge  Two unbound water molecules (H 2 O) provide a strong hydrogen network Proposed model/Mechanistic Steps  Replace Ca 2+ with Mg 2+ cat  Replace D 105, D7(Asp), E7 (Glu) by HCOO,  ddNTP and primer with CH 3 — groups 3-

13. 13 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

14. 14  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/plans

15. 15 Acknowledgements Dr. Tamar Schlick Ms. Meredith Foley Dr. Yanli Wang