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Description of the structure and function of the T7 DNA Polymerase

Who discovered the first DNA Pol? When? Arthur Kornberg, 1956
Directionality of DNA replication? 5'-->3'
2 main functions of DNA Polymerase? -Fidelity of DNA replication -Ligation of dNTPs to primer strand
What factors affect the fidelity of DNA replication? -Selection of the correct dNTP based on the template strand -Excision of incorrect bases by the exonuclease activity of the polymerase
What is processivity? The ability of an enzyme to complete several catalytic cycles without dissociating from its substrate
What is the overall architecture of the T7 DNA Polymerase? The shape of a right hand with fingers, palm, and thumb domains
What domain contributes to the processivity of the enzyme? Thioredoxin domain
What metal ions are most likely involved in DNA Polymerization? How many of them are there? Mg2+, 2
Where is the DNA-binding groove located? Between the fingers, thumb, and palm domains
Which domain is found at the N-terminus of the protein? Exonuclease
Which domain is found at the C-terminus of the protein/ Polymerase
Where is the thioredoxin domain located? On top of the thumb
What strictly conserved residues are necessary for enzyme function? Where are they located? -3 acidic residues (Asp475, Asp654, Glu655) -In the active site in the DNA-binding groove
What does the B-factor of the active site tell us about the region around the active site of the protein? there is little to no movement in this area to ensure affinity for DNA
What is the Klenow fragment? The historical name for the part of the enzyme that contains catalytic activity.
What domains are present in the Klenow fragment? The polymerase and exonuclease domains
What enzyme was used to isolate the klenow fragment? Subtilisin
What purpose does the thioredoxin domain play in DNA replication? Provides processivity to the activity of DNA Polymerase
Where does polymerization occur? At the confluence of the fingers and the palm domains
What interactions stabilize the enzyme-DNA complex? Electrostatic interactions with the PO4 backbone
Why does the enzyme interact with the phosphate backbone? To avoid sequence-dependent interaction with DNA that would hinder processivity
What shape is the helical axis of the primer-template heteroduplex DNA? What causes this? The heteroduplex DNA adopts an S-shape due to extensive interaction with the thumb and fingers domains
Is t he heteroduplex DNA in A- or B-form? No. It is slightly contorted so that the minor groove is widened relative to B-DNA.
What role does His607 play in the activity of the enzyme? Base-stacks with the template base to hold it in optimal configuration for base-pairing with the incoming dNTP
What effect does template distortion have? Positions the template base to Watson-Crick base-pair with the incoming dNTP.
How do the authors account for the higher-than-expected fidelity of the enzyme? The active site is shielded from water, increasing the dielectric constant of the environment
What factors contribute to exclude water from the active site? -The O-helix of the fingers domain, Tyr526, and the 3' base of the primer
What stabilizes the incoming dNTP? The helical dipole of the L-helix is oriented such that the negative charge of the triphosphate group of the incoming NTP is stabilized
Why is the solved complex considered non-covalent? The most recently incorporated base is a ddNTP which does not allow incorporation of the next base.
What is the mechanism by which the incoming dNTP is ligated to the primer strand? DNA polymerases catalyze the phosphoryl transfer through nucleophilic attack of the primer 3' hydroxyl on the alpha-phosphate of a dNTP
How do DNA polymerases discriminate so heavily against the incorporation of RNTPs? Realistically, we don't know. However, the authors suggest the presence of a hydrophobic pocket formed by Tyr526 and the aliphatic part of the side chain of Glu480. Glu480 is held in place by Tyr530. This pocket excludes the RNTP 2' hydroxyl
Which step of the catalytic cycle does the solved structure represent? The authors propose that the complex is in the activated transition state.
Created by: mbell133



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