Cisneros, G. Andres, Perera, Lalith, Schaaper, Roel M, Pedersen, Lars C., London, Robert E., Pedersen, Lee G, Darden, Thomas A. JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 131: 1550-1556, 2009
Abstract
The 28 kDa epsilon subunit of Escherichia coli DNA polymerase III is the exonucleotidic proofreader responsible for editing polymerase insertion errors. Here, we study the mechanism by which E carries out the exonuclease activity. We performed quantum mechanics/molecular mechanics calculations on the N-terminal domain containing the exonuclease activity. Both the free-E and a complex E bound to a 0 homologue (HOT) were studied. For the epsilon-HOT complex Mg2+ or Mn2+ were investigated as the essential divalent metal cofactors, while only Mg2+ was used for free-e. In all calculations a water molecule bound to the catalytic metal acts as the nucleophile for hydrolysis of the phosphate bond. Initially, a direct proton transfer to H162 is observed.
Subsequently, the nucleophilic attack takes place followed by a second proton transfer to E14. Our results show that the reaction catalyzed with Mn2+ is faster than that with Mg2+, in agreement with experiment. In addition, the E-HOT complex shows a slightly lower energy barrier compared to free-E. In all cases the catalytic metal is observed to be pentacoordinated. Charge and frontier orbital analyses suggest that charge transfer may stabilize the pentacoordination. Energy decomposition analysis to study the contribution of each residue to catalysis suggests that there are several important residues. Among these, H98, D103, D129, and D146 have been implicated in catalysis by mutagenesis studies. Some of these residues were found to be structurally conserved on human TREX1, the exonuclease domains from E. coli DNA-Pol 1, and the DNA polymerase of bacteriophage RB69.
Cisneros, G. Andres, Perera, Lalith, Garcia-Diaz, Miguel, Bebenek, Katarzyna, Kunkel, Thomas A., Pedersen, Lee G. DNA REPAIR 7: 1824-1834 2008
Abstract
DNA polymerases play a crucial role in the cell cycle due to their involvement in genome replication and repair. Understanding the reaction mechanism by which these polymerases carry out their function can provide insights into these processes, Recently, the crystal structures of human DNA polymerase lambda (Pol lambda) have been reported both for pre- and post-catalytic complexes [Garcia-Diaz et al., DNA Repair 3 (2007), 1333]. Here we employ the pre-catalytic complex as a starting structure for the determination of the catalytic mechanism of Pol lambda using ab initio quantum mechanical/molecular mechanical methods. The reaction path has been calculated using Mg2+ and Mn2+ as the catalytic metals. In both cases the reaction proceeds through a two-step mechanism where the 3'-OH of the primer sugar ring is deprotonated by one of the conserved Asp residues (D490) in the active site before the incorporation of the nucleotide to the nascent DNA chain.
A significant charge transfer is observed between both metals and some residues in the active site as the reaction proceeds. The optimized reactant and product structures agree with the reported crystal structures. In addition, the calculated reaction barriers for both metals are close to experimentally estimated barriers. Energy decomposition analysis to explain individual residue contributions suggests that several amino acids surrounding the active site are important for catalysis. Some of these residues, including R420, R488 and E529, have been implicated in catalysis by previous mutagenesis experiments on the homologous residues on Pol beta. Furthermore, Pol lambda residues R420 and E529 found to be important from the energy decomposition analysis, are homologous to residues R183 and E295 in Pol beta, both of which are linked to cancer. In addition, residues R386, E391, K422 and K472 appear to have an important role in catalysis and could be a potential target for mutagenesis experiments. There is partial conservation of these residues across the Pol X family of DNA polymerases.
Liu, Shubin, Govind, Niranjan, Pedersen, Lee G. JOURNAL OF CHEMICAL PHYSICS 129 094104 2008
Abstract
Continuing our recent endeavor, we systematically investigate in this work the origin of internal rotational barriers for small molecules using the new energy partition scheme proposed recently by one of the authors [S. B. Liu, J. Chem. Phys. 126, 244103 (2007)], where the total electronic energy is decomposed into three independent components, steric, electrostatic, and fermionic quantum. Specifically, we focus in this work on six carbon, nitrogen, and oxygen containing hydrides, CH3CH3, CH3NH2, CH3OH, NH2NH2, NH2OH, and H2O2, with only one rotatable dihedral angle angle H-X-Y-H (X,Y=C,N,O). The relative contributions of the different energy components to the total energy difference as a function of the internal dihedral rotation will be considered. Both optimized-geometry (adiabatic) and fixed-geometry (vertical) differences are examined, as are the results from the conventional energy partition and natural bond orbital analysis.
A wealth of strong linear relationships among the total energy difference and energy component differences for different systems have been observed but no universal relationship applicable to all systems for both cases has been discovered, indicating that even for simple systems such as these, there exists no omnipresent, unique interpretation on the nature and origin of the internal rotation barrier. Different energy components can be employed for different systems in the rationalization of the barrier height. Confirming that the two differences, adiabatic and vertical, are disparate in nature, we find that for the vertical case there is a unique linear relationship applicable to all the six molecules between the total energy difference and the sum of the kinetic and electrostatic energy differences. For the adiabatic case, it is the total potential energy difference that has been found to correlate well with the total energy difference except for ethane whose rotation barrier is dominated by the quantum effect.