Thermus aquaticus DNA polymerase 1 : structual basis for nucleotide incorporation (2KTQ, 3KTQ)

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

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

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.

Open complex (2KTQ) Closed complex (3KTQ)

Active site: D785 E786 D610 Y611 Tyr 671 O-Helix

O-Helix : Closed complex Open Complex R659 K663 F667 Y671 R659 K663 F667 Y671

Tyr 671: Tyr 671:. Closed ComplexOpen Complex

Closed complexOpen complex Tyr 671 CTP G A G

Active Site: Y611 D610 D685 E786 Y611 D610 D685 E786

Thank You