Mutational and pH studies of the 3' --> 5' exonuclease activity of bacteriophage T4 DNA polymerase.

Abstract:

The 3' --> 5' exonuclease activity of proofreading DNA polymerases requires two divalent metal ions, metal ions A and B. Mutational studies of the 3' --> 5' exonuclease active center of the bacteriophage T4 DNA polymerase indicate that residue Asp-324, which binds metal ion A, is the single most important residue for the hydrolysis reaction. In the absence of a nonenzymatic source of hydroxide ions, an alanine substitution for residue Asp-324 reduced exonuclease activity 10-100-fold more than alanine substitutions for the other metal-binding residues, Asp-112 and Asp-219. Thus, exonuclease activity is reduced 10(5)-fold for the D324A-DNA polymerase compared with the wild-type enzyme, while decreases of 10(3)- to 10(4)-fold are detected for the D219A- and D112A/E114A-DNA polymerases, respectively. Our results are consistent with the proposal that a water molecule, coordinated by metal ion A, forms a metal-hydroxide ion that is oriented to attack the phosphodiester bond at the site of cleavage. Residues Glu-114 and Lys-299 may assist the reaction by lowering the pK(a) of the metal ion-A coordinated water molecule, whereas residue Tyr-320 may help to reorient the DNA from the binding conformation to the catalytically active conformation.

Polymerases:

Topics:

Mutational Analysis, Kinetic Parameters, Fidelity, Exonuclease Activity

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
T4 Elisseeva E1999 kcat 200 /second Reaction: 3-5' Exonuclease; Substrate: DNA template; Technique: Degradation of labeled single-stranded DNA
T4 D324A Elisseeva E1999 kcat 0.01 /second Reaction: 3-5' Exonuclease; Substrate: DNA template; Technique: Degradation of labeled single-stranded DNA (9.5)
T4 Y320FD324A Elisseeva E1999 kcat 0.1 /second Reaction: 3-5' Exonuclease; Substrate: DNA template; Technique: Degradation of labeled single-stranded DNA

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