1. 1 Exploring the Possible Pathways of DNA Polymerase λ’s Nucleotidyl Transfer Reaction Meredith Foley Schlick Lab Retreat -- February 9, 2008 Chemistry

2. 2 Available Data on the Reaction Pathway Using k pol values, the activation energy of the reaction can be estimated from transition state theory (pol λ: 16- 17 kcal/mol; pol β: 16-18 kcal/mol) Many computational studies have focused on pol β’s reaction using both QM and QM/MM methods Among the QM/MM-determined reaction mechanisms for pol β, initial O3′ proton transfer to a water or a catalytic aspartate are the most favorable For pol λ, I have considered 4 different initial proton transfer pathways as well as the case when O3′ attacks Pα without forcing O3′ proton transfer

3. 3 Methodology Model built from ternary complex with misaligned DNA (2.00 Å; pdb: 2bcv) Solvated complex in a box with 150 mM ionic strength Equilibrated system in CHARMM Reduced model for QM/MM calculations and added link atoms 3 movement areas defined (free, semi-fixed, and fixed) MM region treated with CHARMM ff QM region treated with HF/3-21G basis set QM/MM equilibration performed using CHARMM/Gamess-UK Reaction pathways followed using a constrained minimization approach Fixed Semi- Fixed Free 7 Å 13 Å

4. 4 Active Site Model 75 atoms including 6 link atoms in the QM region −3 charge in QM region O3′H (H3T atom) points toward O5′ (not a viable pathway) Used this structure as a starting point for all reaction mechanisms explored

5. 5 O3′ Attack on Pα Reaction Coordinate: O3’-Pa-O3A Energy (kcal/mol) Start End As O3′ attacks Pα, the cat Mg—dTTP:O1A distance decreases while the O3′--cat Mg distance increases (Mg doesn’t need to stabilize oxyanion) Pα-O3A breaks H3T is closer to D490:OD1 than O5′ D490

6. 6 Proton Transfer to Asp490 Energy (kcal/mol) Reaction Coordinate: O3′-H3T-Asp490:OD1 Start End O3′-Pα increases H3T breaks from O5′ O3′-Pα ↑ cat Mg-O1A ↓ O3′-cat Mg ↑ O3′-cat Mg ↓ cat Mg-O1A ↓ Cat Mg helps to stabilize oxyanion O3′ as H3T is transferred to Asp490:OD1 O3′-Pα decreases as H3T is transferred to Asp490:OD1

7. 7 Proton Transfer to dTTP:O2A Energy (kcal/mol) Reaction Coordinate: O3’-H3T-dTTP:O2A Start End H3T moving away from O5′ H3T equidistant from O3′ and O2A O3′-Pα distance decreases during the proton transfer O3′-cat Mg distance increases until H3T is transferred to O2A. Then, it decreases

8. 8 Proton Transfer to Asp429 Energy (kcal/mol) Reaction Coordinate: O3′-H3T-Asp429:OD1 Start End H3T is equidistant from O3′ and Asp:OD1 H3T rotates to Asp:OD1; O3′-Pα increases O3′-cat Mg decreases O3′-Pα distance increases as H3T rotates toward Asp429, but then decreases as proton is transferred

9. 9 Proton Transfer to Water Energy (kcal/mol) Reaction Coordinate: O3′-H3T-Water1:OH2 Start End O3′-Pα distance increases until H3T rotates away from O5′ toward Water1 H3T breaks away from O5′ Cat Mg – O3′ distance decreases

10. 10 Future Work – Build New Models Continue following reaction pathways following proton transfer and O3′ attack Improve starting geometry (e.g., using models at left) Refine favored pathways with a larger basis set and smaller step size Consider simultaneous proton transfer and O3’ attack Proton transfer alone causes rearrangement of the catalytic ion D490 Wat1 2.83.0 1.6 1.65 2.22.1 H3T toward Wat1H3T toward Asp490

11. 11

12. 12 Possible Step 2 – to Another Water Molecule Energy (kcal/mol) Rxn Coordinate: Wat1:OH2-Wat1:H1-Wat2:OH2 Start End

13. 13 Possible Step 2 – O3′ Attack on Pα Energy (kcal/mol) Reaction Coordinate: O3′-Pα-O3A Start End

14. 14 Pol β -- 2006 Rotated O3′H toward Asp256 to obtain initial geometry γ-phosphate oxygen protonated 64 atoms in QM region with −2 charge ONIOM method (QM region: B3LYP and 6-31G*; MM region: Amber ff) Followed O3’-Pα-O3A reaction coordinate with 0.10 Å step size Estimate that TS occurs at O3′- Pα = 2.2 Å and Pα-O3A = 1.9 Å with 21.5 kcal/mol higher energy than the reactant Lin, Pedersen, Batra, Beard, Wilson, Pedersen, 2006, PNAS 103:13294-13299

15. 15 Radhakrishnan & Schlick 2006 G:C and G:A systems (1bpy) equilibrated in CHARMM; aspartates and dNTPs are unprotonated QM region has 86 atoms (includes S180 and R183); -1 charge Reduced waters to 3 solvation shells in QM/MM model; added SLA QM region:B3LYP and 6-311G; MM region: CHARMM ff QM/MM equilibration followed by five 1 ps trajectories along O3’-Pa-O3A coordinate; O3’-cat Mg restrained to 2 A From trajectories, 50 snapshots were chosen and minimized without constraints; in both systems 6 different states were obtained; G:C free energy of activation at least 17 kcal/mol, G:A free energy of activation at least 21 kcal/mol

16. 16 Alberts & Schlick 2006

17. 17 Transition State Theory Insertion rate constant of reaction = k pol k pol = vexp[−ΔG ‡ /RT] At 25°C, v = 6.212x10 12 s −1 (v = kT/h)