DNA Polymerases: An Insight into Their Active Sites and Catalytic Mechanism

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.