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DNA Polymerases: An Insight into Their Active Sites and Catalytic Mechanism | Chapter 02 | Recent Advances in Biological Research Vol. 1

Introduction: DNA polymerases are cardinal enzymes, which play a vital role in preserving as well as maintaining the blueprint of life in all living cells. Furthermore, in-depth analyses of DNA and RNA polymerases, which are the crucial catalysts of life, not only reveal fundamental information about their emergence but also on the evolution of life on the planet earth.

Aim: To analyze the active sites of various prokaryotic and eukaryotic DNA polymerases and propose a plausible mechanism of action for the polymerases with the Escherichia coli DNA polymerase I as a model system.

Study Design: Bioinformatics, Biochemical, Genetic, Site-Directed Mutagenesis (SDM) analyses and X-ray crystallographic data were analyzed.

Place and Duration of Study: Department of Molecular Microbiology, School of Biotechnology, Madurai Kamaraj University, Madurai – 625 021, India from 2007 to 2012.

Methodology: The advanced version of T-COFFEE was used to analyze both prokaryotic and eukaryotic DNA polymerase sequences. Along with this bioinformatics data, X-ray crystallographic and biochemical, SDM analysis data were also used to confirm the possible amino acids in the active sites of different types of polymerases from various sources.

Results: Multiple sequence analyses of various polymerases from different sources showed only a few highly conserved motifs among these enzymes except eukaryotic epsilon polymerases where a large number of highly conserved sequences were found. Possible catalytic/active site regions in all these polymerases showed a highly conserved catalytic amino acid K/R and the YG/A pair. A distance conservation is also observed between the active sites. Furthermore, two highly conserved Ds and DXD motifs are also observed and implicated in catalysis.

Conclusion: The highly conserved amino acid K/R acts as the proton abstractor in catalysis and the YG/A pair acts as a “steric gate” and along with a completely conserved R, select only dNTPS for polymerization reactions. The two highly conserved Ds act as the “charge shielder” of dNTPs and orient the alpha phosphate of incoming dNTPs to the 3’-OH end of the growing primer. Multiple sequence analyses have shown that a basic amino acid K/R and an YG pair are highly conserved in almost all DNA polymerases except in error-prone polymerases where the YG pair is not found at the expected distance from the catalytic K/R. SDM, biochemical and X-ray crystallographic analyses of DNA polymerase I from E. coli have also suggested their involvement in substrate binding and catalysis. Large numbers of highly/completely conserved monos, diads, triads are also found among different groups of DNA polymerases and they may play an important role in folding the proteins to the correct 3D structure. Based on these results, a mechanism of action is proposed for the polymerization reactions as well as for the proof-reading function of DNA polymerase I from E. coli as a model enzyme. A similar mechanism may be followed by other polymerases as the almost completely conserved K/R and YG pair are present in all of them.

Biography of author(s)

Dr. Peramachi Palanivelu
Department of Molecular Microbiology, School of Biotechnology, Madurai Kamaraj University, Madurai – 625 021, India.



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