Thermus aquaticus DNA Polymerase Adam OLeary October 21, 2014
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Background Taq DNA polymerase Enzymatic protein isolated from
Thermus aquaticus Function: replicate DNA during DNA replication in
T. aquaticus Moves along template DNA Adds free nucleotides to 3`
end of of growing DNA strand Results in two identical replicas of
initial DNA 2
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Background Importance lies in the polymerase chain reaction
(PCR) Taq polymerase isolated from T. aquaticus Thermophilic
bacterium (hot springs and hydrothermal vents) Displays high
thermostability 50 - 80C 3
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Background Can withstand high temperature constraints of PCR
Denaturation of initial helical DNA (94- 96C) Annealing (68C)
Elongation (72C) Stable throughout the thermocycling events Not
fully achievable by other DNA polymerases Revolutionized scientific
industry Highly valuable tool in molecular biology, biochemistry,
etc. 4
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Structure Composed of 832 a.a Not membrane associated No a.a.
with large (+) hydropathy index Alpha and Beta Sheets present Three
functional domains 1-290: 5`-3` nuclease domain - Yellow 291-423:
3`-5` exonuclease (proofreading) domain - Red 424-832: polymerase
domain - Green PDB: 1TAU Kim, Y., Eom, S.H., Lee, D.S., Suh, S.W.,
and Steitz, T.A. (1995) Crystal structure of Thermus aquaticus DNA
polymerase. Nature. 376, 612-616. 5
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Structure: Nuclease Domain Comprised of amino acids 1-290
Responsible for primer excision Contain important amino acids for
binding Mg 2+ Asp142, Asp144: ligate first Mg 2+ Asp116, Glu117,
Asp120: ligate second Mg 2+ Stabilize negatively charged phosphate
backbone of DNA Urs, U.; Ramachandran, M.; Krishna Murthy, H.,
(1999) Structure of Taq polymerase shows a new orientation for the
structure-specific nuclease domain. Acta Cryst. D55, 1971-1977.
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Structure: 3`-5` exonuclease Domain Comprised of amino acids
291-423 Typically, responsible for functioning as editing or
proofreading nuclease Removes incorrect nucleotide base pairs added
to growing DNA strand Non-functional in T. aquaticus Does not
possess catalitically critical carboxylate residues that bind Mg 2+
Result: low fidelity/accuracy and increased error rate during PCR 1
and 9000 nucleotides for single base substitutions Frameshift error
frequency of 1 and 41,000 Villbrant, B. ; Sobek, H. ; Frey, B.;
Schomburg, D., (2000) Domain exchange: chimeras of thermus
aquaticus DNA polymerase, escherichia DNA polymerase I and
thermotoga neapolitana DNA polymerase. Protein Engineering. 9,
645-54. 7
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Structure: Polymerase Domain Comprised of amino acids 424-832
Contains catalytic site where template DNA is bound Incorporates
nucleotides onto the newly formed strand Added nucleotides base
pair (hydrogen bond) to template DNA Uses two Mg 2+ cofactors
Prepares end of new DNA strand to bind with incoming phosphate of
dNTPs Stabilizes negative charge formed as two phosphates are
cleaved off dNTPs Saiki, RK; et al. (1988). Primer-directed
enzymatic amplification of DNA with a thermostable DNA polymerase.
Science 239, 48791. 8
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Structure: Polymerase Domain Critical amino acids included
Glu742 and Ala743 Responsible for stabilizing DNA Control extension
of newly forming DNA strand Hydrogen bond to nucleotide bases
through (NH 3 + ) or (CO - ) Or stabilize negatively charged
phosphate backbone (NH 3 + ) Yamagami, T., Ishino, S., Kawarabayas,
Y, and Ishino, Y. (2014) Mutant Taq DNA polymerases with improved
elongation ability as a useful reagent for genetic engineering.
Front Microbiol. 461,1-10. 9
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Mutation of E742 and A743 Numerous mutated forms of Taq DNA
polymerase purified Mutations focused on Glu742 and Ala743 Amino
acids replaced DNA affinity and primer elongation evaluated in
relation to PCR performance All mutations resulted in faster
ability of primer extension and overall higher DNA affinity
Yamagami, T., Ishino, S., Kawarabayas, Y, and Ishino, Y. (2014)
Mutant Taq DNA polymerases with improved elongation ability as a
useful reagent for genetic engineering. Front Microbiol. 461,1-10.
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Conclusion Isolated from thermophilic bacterium T. aquaticus
Widely used in the PCR DNA amplification technique Revolutionized
science Highly importance to molecular biology, biochemistry Also
forensic science Combined DNA Index System (CODIS) Identification
of criminals through comparison to DNA database Controversial High
commercial potential Great Taq Rip-Off Yellow Stone National Park
Benefits sharing agreement in effect 11
