Characterization of the active site of DNA polymerase ? by molecular dynamics and quantum chemical calculation
It is well established that the fully formed polymerase active site of the DNA repair enzyme, polymerase b (pol b), including two bound Mg2+ cations and the nucleoside triphosphate (dNTP) substrate, exists at only one point in the catalytic cycle just prior to the chemical nucleotidyl transfer step. The structure of the active conformation has been the subject of much interest as it relates to the mechanism of the chemical step and also to the question of fidelity assurance. While crystal structures of ternary pol b - (primer-template) DNA - dNTP complexes have provided the main structural features of the active site, they are necessarily incomplete due to intentional alterations, e.g. removal of the 3?OH groups from primer and substrate, that were needed to obtain a structure from mid-cycle. Working from the crystal structure closest to the fully formed active site, (pdb: 1bpy), two MD simulations of the solvated ternary complex were performed; one with the missing 3?OH?s restored, via modeling, to the primer and substrate, and the other without restoration of the 3?OH?s. The results of the simulations, taken together with ab initio optimizations on simplified active site models, indicate that the missing primer 3?OH in the crystal structure is responsible for a significant perturbation in the coordination sphere of the catalytic cation and allow us to suggest several corrections and additions to the active site structure as observed by crystallography. In addition, the calculations help to resolve questions that have been raised regarding the protonation states of coordinating ligands.
- Research Organization:
- Pacific Northwest National Laboratory (PNNL), Richland, WA (US), Environmental Molecular Sciences Laboratory (EMSL)
- Sponsoring Organization:
- USDOE
- DOE Contract Number:
- AC06-76RL01830
- OSTI ID:
- 15006222
- Report Number(s):
- PNNL-SA-37442; 2270; 2401; KP1101010
- Journal Information:
- Proteins, Vol. 53, Issue 3; ISSN 0887-3585
- Publisher:
- Wiley
- Country of Publication:
- United States
- Language:
- English
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