The 3'-5' exonuclease of DNA polymerase I of Escherichia coli: contribution of each amino acid at the active site to the reaction.

Abstract:

We have used site-directed mutagenesis to change amino acid side chains that have been shown crystallographically to be in close proximity to a DNA 3' terminus bound at the 3'-5' exonuclease active site of Klenow fragment. Exonuclease assays of the resulting mutant proteins indicate that the largest effects on exonuclease activity result from mutations in a group of carboxylate side chains (Asp355, Asp424 and Asp501) anchoring two divalent metal ions that are essential for exonuclease activity. Another carboxylate (Glu357) within this cluster seems to be less important as a metal ligand, but may play a separate role in catalysis of the exonuclease reaction. A second group of residues (Leu361, Phe473 and Tyr497), located around the terminal base and ribose positions, plays a secondary role, ensuring correct positioning of the substrate in the active site and perhaps also facilitating melting of a duplex DNA substrate by interacting with the frayed 3' terminus. The pH-dependence of the 3'-5' exonuclease reaction is consistent with a mechanism in which nucleophilic attack on the terminal phosphodiester bond is initiated by a hydroxide ion coordinated to one of the enzyme-bound metal ions.

Polymerases:

Topics:

Exonuclease Activity, Historical Protein Properties (MW, pI, ...), Kinetic Parameters, Mutational Analysis

One line summary:

Structure-function analysis using a panel of 3'-5'exo active site mutations.

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
Klenow fragment Derbyshire V1991 3-5' Exonuclease (proofreading) Yes
Klenow fragment Derbyshire V1991 Cloned or native Cloned in E. coli
Klenow fragment Derbyshire V1991 Tagged No
Klenow fragment Derbyshire V1991 Full length or truncated Full length
Klenow fragment Derbyshire V1991 Specific Activity 9000 units/mg Technique: Polymerase assay (poly[d(AT)]
Klenow fragment Derbyshire V1991 KM 0.56uM Reaction: 3-5' Exonuclease; Technique: Steady State (ssDNA: poly(dT)); Experimental conditions: pH (pH 7.5), Mg++ (6mM), Temp (37°C)
Klenow fragment Derbyshire V1991 kcat 0.09 /second Reaction: 3-5' Exonuclease; Technique: Steady State (ssDNA: poly(dT)); Experimental conditions: pH (pH 7.5), Mg++ (6mM), Temp (37°C)
Klenow fragment Derbyshire V1991 kcat 0.12 /second Reaction: 3-5' Exonuclease; Technique: Steady State (ssDNA: poly(dA)); Experimental conditions: pH (pH 7.5), Mg++ (8mM), Temp (37°C)
KF L361A Derbyshire V1991 Specific Activity 9000 units/mg Technique: Polymerase assay (poly[d(AT)]
KF L361A Derbyshire V1991 kcat 0.044 /second Reaction: 3-5' Exonuclease; Technique: Steady State (ssDNA, poly(dA)); Experimental conditions: pH (pH 7.5), Mg++ (8mM), Temp (37°C)
KF L361A Derbyshire V1991 kcat 4E-05 /second Reaction: 3-5' Exonuclease; Technique: Steady State (dsDNA, assuming WT has rate 0.001/s); Experimental conditions: pH (pH 7.5), Mg++ (6mM), Temp (37°C)
KF L361M Derbyshire V1991 Specific Activity 1.1E+04 units/mg Technique: Polymerase assay (poly[d(AT)]
KF L361M Derbyshire V1991 kcat 8.3E-05 /second Reaction: 3-5' Exonuclease; Technique: Steady State (dsDNA, assuming WT has rate 0.001/s); Experimental conditions: pH (pH 7.5), Mg++ (6mM), Temp (37°C)
KF D424A Derbyshire V1991 Specific Activity 9000 units/mg Technique: Polymerase assay (poly[d(AT)]
KF D424A Derbyshire V1991 kcat 1.3E-08 /second Reaction: 3-5' Exonuclease; Technique: Steady State (dsDNA, assuming WT has rate 0.001/s); Experimental conditions: pH (pH 7.5), Temp (37°C)
KF D424E Derbyshire V1991 Specific Activity 1.1E+04 units/mg Technique: Polymerase assay (poly[d(AT)]
KF D424E Derbyshire V1991 kcat 0.01 /second Reaction: 3-5' Exonuclease; Technique: Steady State (ssDNA, poly(dA)); Experimental conditions: pH (pH 7.5), Mg++ (8mM), Temp (37°C)
KF D424E Derbyshire V1991 kcat 4E-05 /second Reaction: 3-5' Exonuclease; Technique: Steady State (dsDNA, assuming WT has rate 0.001/s); Experimental conditions: pH (pH 7.5), Mg++ (6mM), Temp (37°C)
KF D355A Derbyshire V1991 Specific Activity 1.3E+04 units/mg Technique: Polymerase assay (poly[d(AT)]
KF D355A Derbyshire V1991 kcat 8.3E-08 /second Reaction: 3-5' Exonuclease; Technique: Steady State (dsDNA substrate, assuming WT has rate 0.001/s); Experimental conditions: pH (pH 7.5), Mg++ (6mM), Temp (37°C)
KF D424N Derbyshire V1991 Specific Activity 1.2E+04 units/mg Technique: Polymerase assay (poly[d(AT)]
KF D424N Derbyshire V1991 kcat 2.5E-08 /second Reaction: 3-5' Exonuclease; Technique: Steady State (dsDNA, assuming WT has rate 0.001/s); Experimental conditions: pH (pH 7.5), Mg++ (6mM), Temp (37°C)

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