Presentation on theme: "Thermus aquaticus DNA Polymerase Adam O’Leary October 21, 2014."— Presentation transcript:
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 growing DNA strand Results in two identical replicas of initial DNA
Importance lies in the polymerase chain reaction (PCR) Taq polymerase isolated from T. aquaticus Thermophilic bacterium (hot springs and hydrothermal vents) Displays high thermostability ( ˚C) Can withstand high temperature constraints of PCR Denaturation of helical DNA (94-96˚C) Annealing (68˚C) Elongation (72˚C)
Polymerase Chain Reaction Stable throughout the thermocycling events Not fully achievable by other DNA polymerases Revolutionized scientific industry Highly valuable tool in molecular biology, biochemistry, etc. Forensic science
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 : 3`-5` exonuclease (proofreading) domain - Red : 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,
Nuclease Domain Comprised of amino acids 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,
3`-5` exonuclease Domain Comprised of amino acids 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,
Polymerase Domain Comprised of amino acids 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, 487–91.
Critical amino Acids Critical amino acids Glu742 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.
Mutation Studies 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.
Mutation of E742 and A743
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