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Published byHarvey Reed Modified over 8 years ago
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Thermus aquaticus DNA polymerase 1 : structual basis for nucleotide incorporation (2KTQ, 3KTQ)
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Taq DNA Polymerase I: Mediates Replication Highly Thermostable Three Main Functions: - 5’ 3’ Polymerase Activity - 3’ 5’ exonuclease activity mediating proof reading - 5’ 3’ exonuclease activity, during DNA repair. Template Dependant Catalyzes the addition of the nucleotide to the 3’ primer chain. Function divided between two fragments - Larger Fragment (Klenow Fragment) - Small Fragment
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Klenow Fragment (Klentaq): larger fragment formed by subtilisin cleavage 5’ 3’ Polymerase Activity 3’ 5’ exonuclease activity Right hand. Active ternary complex (enz + DNA + dNTP) Switches between two conformations during nucleotide incorporation (open and closed) Polymerase domain Exonuclease domain
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Structure determination : 2KTQ(open form) Resolution : 2.3 A o R value: 22.4% R-free : 28.8% 3KTQ (Closed form) Resolution: 2.3 A R value: 21.8% R-free: 28.8% Obtained from selenomethionine derived protein. Phase problem solved my MAD.
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Open complex (2KTQ) Closed complex (3KTQ)
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Active site: D785 E786 D610 Y611 Tyr 671 O-Helix
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O-Helix : Closed complex Open Complex R659 K663 F667 Y671 R659 K663 F667 Y671
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Tyr 671: Tyr 671:. Closed ComplexOpen Complex
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Closed complexOpen complex Tyr 671 CTP G A G
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Active Site: Y611 D610 D685 E786 Y611 D610 D685 E786
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