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. 2008 Nov 1;7(11):1824-34.
doi: 10.1016/j.dnarep.2008.07.007. Epub 2008 Aug 30.

Catalytic mechanism of human DNA polymerase lambda with Mg2+ and Mn2+ from ab initio quantum mechanical/molecular mechanical studies

G Andrés Cisneros  1 , Lalith Perera, Miguel García-Díaz, Katarzyna Bebenek, Thomas A Kunkel, Lee G Pedersen
Affiliations

Catalytic mechanism of human DNA polymerase lambda with Mg2+ and Mn2+ from ab initio quantum mechanical/molecular mechanical studies

G Andrés Cisneros et al. DNA Repair (Amst). .

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 (Pollambda) have been reported both for pre- and post-catalytic complexes [García-Díaz 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 Pollambda using ab initio quantum mechanical/molecular mechanical methods. The reaction path has been calculated using Mg(2+) and Mn(2+) 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 Polbeta. Furthermore, Pollambda residues R420 and E529 found to be important from the energy decomposition analysis, are homologous to residues R183 and E295 in Polbeta, 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.

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Conflict of interest statement

Conflict of Interest statement

The authors declare that there are no conflicts of interest.

Figures

Figure 1
Figure 1
Tested reaction schemes for the Polλ catalyzed reaction. Scheme 1 (left): proton transfer to D490, scheme 2 (middle): proton transfer to D429, scheme 3 (right): proton transfer to ordered water.
Figure 2
Figure 2
Superposition of active sites for the calculated reactants with the X-ray crystal structure (pdb id 2PFO). X-ray structure is shown in green, Mn2+ structure is shown in purple and Mg2+ structure is shown in orange. Hydrogen atoms except for the O3′-H have been omitted for clarity. The metals, O3′, Pα, waters that complete the metal coordination and the H atom on O3′ are highlighted as spheres. Catalytic metal is on the left and nucleotide binding metal is on the right. Metal ligand distances are reported in table S1 (supplementary materials).
Figure 3
Figure 3
Superposition of active sites for the calculated products with the X-ray crystal structure (pdb id 2PFQ). X-ray structure is shown in green, Mn2+ structure is shown in purple and Mg2+ structure is shown in orange. Hydrogen atoms except for the transferred proton have been omitted for clarity. The metals, O3′, Pα, waters that complete the metal coordination and the transferred proton are highlighted as spheres. Catalytic metal is on the left and nucleotide binding metal is on the right. Metal ligand distances are reported in table S1 (supplementary materials).
Figure 4
Figure 4
Calculated reaction paths for the Mg2+(left) and Mn2+ (right) catalyzed reactions.
Figure 5
Figure 5
Superposition of active sites for the calculated transition states (TS1 top, TS2 bottom). Mg2+ structures are shown in orange and Mn2+ structures are shown in purple. Hydrogen atoms except for the transferred proton have been omitted for clarity. The metals, O3′, Pα, waters that complete the metal coordination and transferred proton are highlighted as spheres. Catalytic metal is on the left and nucleotide binding metal is on the right. Metal ligand distances are reported in table S1 (supplementary materials).
Figure 6
Figure 6
Selected distance changes along the reaction coordinate for the Mg2+(left) and Mn2+ (right) catalyzed reactions. Circles: O3′-Pα; squares: Pα-O; rhombus: O3′-H; triangles: H-O(D490).
Figure 7
Figure 7
Electrostatic stabilization energy per residue for the Mg2+ and Mn2+ catalyzed reactions (protein only).
Figure 8
Figure 8
Active site positions of selected residues in the Mg2+ system (top) that contribute to the (de)stabilization of the TS during catalysis. The residues shown in red correspond to those which are common to all TSs or have a large contribution (see text). Bottom: Sequence alignment of the X family polymerases for the selected residues (highlighted in yellow). Key: “*”indicates residues conserved in all sequences,“:” indicates residues conserved in at least three of the four sequences, “.” indicates homologous residues present in at least three of the four sequences.
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