Functional consequences and exonuclease kinetic parameters of point mutations in bacteriophage T4 DNA polymerase.

Abstract:

Three groups of T4 DNA polymerase mutants were prepared and characterized. In the first group, Ala and Asn were substituted for four acidic residues in the exonuclease domain that were chosen on the basis of their sequence alignment with the Klenow fragment from Escherichia coli DNA polymerase I. Two divalent metal ions required for catalyzing the 3'-5' exonuclease reaction are ligated by carboxyl groups from these conserved Asp and Glu residues. The Ala and Asn replacements have a profound effect on the exonuclease activity of T4 DNA polymerase and also have a significant, but less pronounced influence on its polymerase activity which is located in a domain distal to the exonuclease region. The kcat values for the exonuclease reaction were reduced by 3-4 orders of magnitude by these replacements, but the values of Km(app) did not differ greatly from the wild-type enzyme. The second group consists of replacements of other residues, that are conserved in the exonuclease domain of eukaryotic DNA polymerases, but do not contribute to divalent metal ion coordination. Many of these alterations resulted in decreased exonuclease and/or polymerase activity. Mutants in the third group have substitutions of conserved residues in the polymerase domain which diminished polymerase and altered exonuclease activities. Our results, combined with structural data on crystals of protein N388, a truncated form of T4 DNA polymerase (Wang et al., 1996), show that: (i) the reduction in the relative specific exonuclease activities of mutants in the first group was significantly less than that of mutants in the Klenow fragment, despite the nearly identical geometric arrangement of the metal liganding groups in two proteins; (ii) altered residues, that affect exonuclease and/or polymerase activities in mutants of the second group, cluster within a small area of the exonuclease domain, suggesting that this area may be directly or indirectly involved in polymerase activity; (iii) mutations in the third group, which affect polymerase and exonuclease activities, may participate in DNA and dNTP binding. Our results point to the functional interdependence of the polymerase and exonuclease domains in T4 DNA polymerase, a property not observed with the Klenow fragment.

Polymerases:

Topics:

Mutational Analysis, Modulators/Inhibitors, Kinetic Parameters, Structure and Structure/Function, Nucleotide Incorporation, Exonuclease Activity, Enzyme Substrate Interactions

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
T4 Abdus Sattar AK1996 KM 6.9uM Reaction: 3-5' Exonuclease; Substrate: DNA template
T4 Abdus Sattar AK1996 KM 0.4uM Reaction: 3-5' Exonuclease; Substrate: DNA template
T4 Abdus Sattar AK1996 kcat 65.1 /second Reaction: 3-5' Exonuclease; Substrate: DNA template
T4 Abdus Sattar AK1996 kcat 0.16 /second Reaction: 3-5' Exonuclease; Substrate: DNA template
T4 D112A Abdus Sattar AK1996 KM 7.7uM Reaction: 3-5' Exonuclease; Substrate: DNA template
T4 D112A Abdus Sattar AK1996 KM 1.6uM Reaction: 3-5' Exonuclease; Substrate: DNA template
T4 D112A Abdus Sattar AK1996 kcat 0.0078 /second Reaction: 3-5' Exonuclease; Substrate: DNA template
T4 D112A Abdus Sattar AK1996 kcat 0.0037 /second Reaction: 3-5' Exonuclease; Substrate: DNA template
T4 D112N Abdus Sattar AK1996 KM 4uM Reaction: 3-5' Exonuclease; Substrate: DNA template
T4 D112N Abdus Sattar AK1996 kcat 0.001 /second Reaction: 3-5' Exonuclease; Substrate: DNA template
T4 E114A Abdus Sattar AK1996 KM 5uM Reaction: 3-5' Exonuclease; Substrate: DNA template
T4 E114A Abdus Sattar AK1996 KM 2uM Reaction: 3-5' Exonuclease; Substrate: DNA template
T4 E114A Abdus Sattar AK1996 kcat 2 /second Reaction: 3-5' Exonuclease; Substrate: DNA template
T4 E114A Abdus Sattar AK1996 kcat 0.08 /second Reaction: 3-5' Exonuclease; Substrate: DNA template
T4 D219AD324A Abdus Sattar AK1996 KM 6.7uM Reaction: 3-5' Exonuclease; Substrate: DNA template
T4 D219AD324A Abdus Sattar AK1996 kcat 0.0034 /second Reaction: 3-5' Exonuclease; Substrate: DNA template
T4 D219A Abdus Sattar AK1996 KM 12.8uM Reaction: 3-5' Exonuclease; Substrate: DNA template
T4 D219A Abdus Sattar AK1996 KM 0.3uM Reaction: 3-5' Exonuclease; Substrate: DNA template
T4 D219A Abdus Sattar AK1996 kcat 0.096 /second Reaction: 3-5' Exonuclease; Substrate: DNA template
T4 D219A Abdus Sattar AK1996 kcat 6E-05 /second Reaction: 3-5' Exonuclease; Substrate: DNA template
T4 D324A Abdus Sattar AK1996 KM 6.3uM Reaction: 3-5' Exonuclease; Substrate: DNA template
T4 D324A Abdus Sattar AK1996 KM 0.4uM Reaction: 3-5' Exonuclease; Substrate: DNA template
T4 D324A Abdus Sattar AK1996 kcat 0.0062 /second Reaction: 3-5' Exonuclease; Substrate: DNA template
T4 D324A Abdus Sattar AK1996 kcat 7E-05 /second Reaction: 3-5' Exonuclease; Substrate: DNA template

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