Thermus aquaticus DNA Polymerase Adam O’Leary October 21, 2014

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

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 - 80˚C

Background Can withstand high temperature constraints of PCR Denaturation of initial helical DNA (94-96˚C) Annealing (68˚C) Elongation (72˚C) Stable throughout the thermocycling events Not fully achievable by other DNA polymerases Revolutionized scientific industry Highly valuable tool in molecular biology, biochemistry, etc.

Structure Composed of 832 a.a Not membrane associated PDB: 1TAU 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   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.

Structure: Nuclease Domain Comprised of amino acids 1-290 Responsible for primer excision Contain important amino acids for binding Mg2+ Asp142, Asp144: ligate first Mg2+ Asp116, Glu117, Asp120: ligate second Mg2+ 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.

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 Mg2+ 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.  

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 Mg2+ 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, 487–91.

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 (NH3+) or (CO-) Or stabilize negatively charged phosphate backbone (NH3+) 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.

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.

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

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