Genetic and crystallographic studies of the 3',5'-exonucleolytic site of DNA polymerase I.

Abstract:

Site-directed mutagenesis of the large fragment of DNA polymerase I (Klenow fragment) yielded two mutant proteins lacking 3',5'-exonuclease activity but having normal polymerase activity. Crystallographic analysis of the mutant proteins showed that neither had any alteration in protein structure other than the expected changes at the mutation sites. These results confirmed the presumed location of the exonuclease active site on the small domain of Klenow fragment and its physical separation from the polymerase active site. An anomalous scattering difference Fourier of a complex of the wild-type enzyme with divalent manganese ion and deoxythymidine monophosphate showed that the exonuclease active site has binding sites for two divalent metal ions. The properties of the mutant proteins suggest that one metal ion plays a role in substrate binding while the other is involved in catalysis of the exonuclease reaction.

Polymerases:

Topics:

Historical Protein Properties (MW, pI, ...), Kinetic Parameters, Mutational Analysis, Structure and Structure/Function

Note:

No PDB file

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
Klenow fragment Derbyshire V1988 Cloned or native Cloned in E. coli
Klenow fragment Derbyshire V1988 Residues Involved in Catalysis of 3-5' Exo D355, E357, D424, D501
Klenow fragment Derbyshire V1988 Tagged No
Klenow fragment Derbyshire V1988 Full length or truncated Full length
Klenow fragment Derbyshire V1988 Other Important Residues 2 Mn ions show metal A and B positions
Klenow fragment Derbyshire V1988 Specific Activity 1.07E+04 units/mg Technique: Polymerase assay (poly[d(AT)]
KF D424A Derbyshire V1988 Other Important Residues Metal B not bound
KF D424A Derbyshire V1988 Specific Activity 9300 units/mg Technique: Polymerase assay (poly[d(AT)]
KF D424A Derbyshire V1988 Specific Activity 1.3E-05 units/mg Technique: Exo activity (arbitrary units) (relative to WT = 1)
KF D355AE357A Derbyshire V1988 Other Important Residues No binding of metals A and B and dNMP
KF D355AE357A Derbyshire V1988 Specific Activity 1.11E+04 units/mg Technique: Polymerase assay (poly[d(AT)]
KF D355AE357A Derbyshire V1988 Specific Activity 1.4E-05 units/mg Technique: Exo activity (arbitrary units) (Relative to WT = 1)

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