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.
View Volume: https://doi.org/10.9734/bpi/rabr/v1
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