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

Derbyshire V, Grindley ND, Joyce CM

The EMBO journal (1991), Volume 10, Page 17

PubMed entry Full article text

Incomplete polymerases:

Abstract:

We have used site-directed mutagenesis to change amino acid side ...

Polymerases:

Eco Pol I,Klenow fragment,KF D355A,KF L361A,KF L361M,KF D424A,KF D424E,KF D424N

Topics:

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

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	The 3'-5' exonuclease of DNA polymerase I of Escherichia coli: contribution of each amino acid at the active site to the reaction.	3-5' Exonuclease (proofreading)	Yes
Klenow fragment	The 3'-5' exonuclease of DNA polymerase I of Escherichia coli: contribution of each amino acid at the active site to the reaction.	Cloned or native	Cloned in E. coli
Klenow fragment	The 3'-5' exonuclease of DNA polymerase I of Escherichia coli: contribution of each amino acid at the active site to the reaction.	Tagged	No
Klenow fragment	The 3'-5' exonuclease of DNA polymerase I of Escherichia coli: contribution of each amino acid at the active site to the reaction.	Full length or truncated	Full length
Klenow fragment	The 3'-5' exonuclease of DNA polymerase I of Escherichia coli: contribution of each amino acid at the active site to the reaction.	Specific Activity	9000 units/mg	Technique: Polymerase assay (poly[d(AT)]
Klenow fragment	The 3'-5' exonuclease of DNA polymerase I of Escherichia coli: contribution of each amino acid at the active site to the reaction.	K_M	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	The 3'-5' exonuclease of DNA polymerase I of Escherichia coli: contribution of each amino acid at the active site to the reaction.	k_cat	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	The 3'-5' exonuclease of DNA polymerase I of Escherichia coli: contribution of each amino acid at the active site to the reaction.	k_cat	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	The 3'-5' exonuclease of DNA polymerase I of Escherichia coli: contribution of each amino acid at the active site to the reaction.	Specific Activity	9000 units/mg	Technique: Polymerase assay (poly[d(AT)]
KF L361A	The 3'-5' exonuclease of DNA polymerase I of Escherichia coli: contribution of each amino acid at the active site to the reaction.	k_cat	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	The 3'-5' exonuclease of DNA polymerase I of Escherichia coli: contribution of each amino acid at the active site to the reaction.	k_cat	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	The 3'-5' exonuclease of DNA polymerase I of Escherichia coli: contribution of each amino acid at the active site to the reaction.	Specific Activity	1.1E+04 units/mg	Technique: Polymerase assay (poly[d(AT)]
KF L361M	The 3'-5' exonuclease of DNA polymerase I of Escherichia coli: contribution of each amino acid at the active site to the reaction.	k_cat	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	The 3'-5' exonuclease of DNA polymerase I of Escherichia coli: contribution of each amino acid at the active site to the reaction.	Specific Activity	9000 units/mg	Technique: Polymerase assay (poly[d(AT)]
KF D424A	The 3'-5' exonuclease of DNA polymerase I of Escherichia coli: contribution of each amino acid at the active site to the reaction.	k_cat	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	The 3'-5' exonuclease of DNA polymerase I of Escherichia coli: contribution of each amino acid at the active site to the reaction.	Specific Activity	1.1E+04 units/mg	Technique: Polymerase assay (poly[d(AT)]
KF D424E	The 3'-5' exonuclease of DNA polymerase I of Escherichia coli: contribution of each amino acid at the active site to the reaction.	k_cat	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	The 3'-5' exonuclease of DNA polymerase I of Escherichia coli: contribution of each amino acid at the active site to the reaction.	k_cat	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	The 3'-5' exonuclease of DNA polymerase I of Escherichia coli: contribution of each amino acid at the active site to the reaction.	Specific Activity	1.3E+04 units/mg	Technique: Polymerase assay (poly[d(AT)]
KF D355A	The 3'-5' exonuclease of DNA polymerase I of Escherichia coli: contribution of each amino acid at the active site to the reaction.	k_cat	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	The 3'-5' exonuclease of DNA polymerase I of Escherichia coli: contribution of each amino acid at the active site to the reaction.	Specific Activity	1.2E+04 units/mg	Technique: Polymerase assay (poly[d(AT)]
KF D424N	The 3'-5' exonuclease of DNA polymerase I of Escherichia coli: contribution of each amino acid at the active site to the reaction.	k_cat	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)