Theoretical investigation of the effects of mutations of the charged amino acids on the dissociation and rate constants for the DNA replication by human DNA polymerase b
V. Martínek1,2, J. Florián2
1Department of Biochemistry, Faculty of Science, Charles University, Albertov 2030, 128 40 Prague 2, Czech Republic
2Department of Chemistry, Loyola University Chicago, Chicago, IL 60626, USA
DNA polymerase b (pol b), the most studied member of the X family of DNA polymerases, is known to be an important part of the eukaryotic base-excision repair system. Furthermore, mutations of this enzyme are associated with many colon and lung tumors and esophageal cancer (1). Pol b is a relatively small (39 kDa) template-dependent DNA polymerase without proofreading activity, which makes it an attractive system for in vitro and in silico studies of the replication fidelity.
We have performed correlation studies for human pol b (WT) and six pol b mutants (R183A, Y271A, D276V, K280G, R283A, E295A). The fidelity related experimental kinetic data, specifically the equilibrium dissociation constant (KD) and the rate of incorporation (kpol), are available for all mutants employed in this study. dNTP binding free energy (DGbind), reaction activation energy (Dg#cat) and an apparent reaction activation energy (Dg#enz) derived from KD, kpol and kpol/KD were correlated with several structural and energetic parameters calculated from molecular dynamics (MD) trajectories. For each pol b mutant, a 4 ns MD simulation was carried out in a solvent sphere of 25 Ǻ radius using Amber-95 force field. DGbind for the dCTP bound to polymerase-DNA complex containing the correct templating nucleotide (G) was computed using the linear interaction energy and linear response approximation methods. In addition, population analysis of the set of 12 distance parameters including the near-attack configuration of the phosphor-ester forming bond was correlated to the experimental data. The statistically significant correlation was observed between experimental Dg#enz, Dg#cat and DGbind versus the population ration of conformations forming H-bond (ND2 of Asn279 and O2 of the incoming dCTP base). This finding supports hypothesis that Asn279 residue play a significant role not only in the substrate binding stabilization (corr. coefficient 0.72), but is also as factor influencing the reaction activation energy (corr. coefficient 0.77). These two effects results in high correlation coefficient (0.92) between the H-bond stability (connecting Asp279 with dCTP) and apparent reaction activation energy. Minor relationship to the Dg#enz could be also attributed to the catalytic Mg - OD2 (Asp256) coordination bond stability showing correlation coefficient 0.74.
The presented study attributes significant role in the correct (Watson-Crick) nucleotide incorporation reaction to the interaction with Asn279 and with lover extend Asp256. However, in order to be able to fully evaluate the role of these residues in pol b fidelity the calculations of the mutation effects on the insertion of incorrect incoming deoxynucleotides should be performed.
1. D. K. Srivastava, I. Husain, C. L. Arteaga, and S. H. Wilson DNA polymerase b expression differences in selected human tumors and cell lines Carcinogenesis, 20, (1999), 1049 – 1054
This work was supported by the NIH grant 1U19CA105010