Structure-function studies of the bacteriophage T4 DNA polymerase. Isolation of a novel suppressor mutant.

Abstract:

We describe here our first attempt in using suppressor mutations to study structure-function relationships of the bacteriophage T4 DNA polymerase. One intragenic suppressor mutation, J5(43) degrees, was isolated that suppresses the temperature sensitivity but not the mutator activity of tsM19, a DNA polymerase mutant. Thus, the substituted amino acid induced by the tsM19 lesion decreases DNA polymerase fidelity, even if the temperature sensitivity has been corrected by a second amino acid substitution in the DNA polymerase polypeptide. The isolation, mapping and characterization of the J5(43) degrees mutation as well as the purification and characterization of the tsM19-J5(43) degrees mutant DNA polymerase are presented. The suppressor isolation procedure has general applicability for the selection of suppressor mutations of other T4 DNA polymerase mutator mutants.

Polymerases:

Topics:

Mutational Analysis, Structure and Structure/Function, Fidelity, Nucleotide Incorporation, Methods

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
T4 Q731am Reha-Krantz LJ1985 Nucleotide Substitution Rate 0.6 Mutation frequency (relative to WT) Technique: T4 rII P7oc reversion assay (AT->GC, Host: supE (C600))
T4 Q731am Reha-Krantz LJ1985 Nucleotide Substitution Rate 0.2 Mutation frequency (relative to WT) Technique: T4 rII P7oc reversion assay (AT->GC, Host: supD (CR63))
T4 Q731am Reha-Krantz LJ1985 Nucleotide Substitution Rate 1.2 Mutation frequency (relative to WT) Technique: T4 rII P7oc reversion assay (AT->TA(CG), Host: supE (C600))
T4 Q731am Reha-Krantz LJ1985 Nucleotide Substitution Rate 2.2 Mutation frequency (relative to WT) Technique: T4 rII P7am reversion assay (GC->TA(CG), Host: supE (C600))
T4 Q731am Reha-Krantz LJ1985 Nucleotide Substitution Rate 0.1 Mutation frequency (relative to WT) Technique: T4 rII P7oc reversion assay (AT->TA(CG), Host: supD (CR63))
T4 Q731am Reha-Krantz LJ1985 Nucleotide Substitution Rate 0.4 Mutation frequency (relative to WT) Technique: T4 rII P7am reversion assay (GC->TA(CG), Host: supD (CR63))
T4 L340P Reha-Krantz LJ1985 Nucleotide Substitution Rate 238 Mutation frequency (relative to WT) Technique: T4 rII P7oc reversion assay (AT->TA(CG), Host: supD (CR63))
T4 L340P Reha-Krantz LJ1985 Nucleotide Substitution Rate 538 Mutation frequency (relative to WT) Technique: T4 rII P7am reversion assay (GC->TA(CG), Host: supD (CR63))
T4 L340P Reha-Krantz LJ1985 Nucleotide Substitution Rate 1189 Mutation frequency (relative to WT) Technique: ac forward mutational assay (Host: supE (MBM7007.2))
T4 L340P Reha-Krantz LJ1985 Nucleotide Substitution Rate 1744 Mutation frequency (relative to WT) Technique: ac forward mutational assay (Host: supD (MBM7007.1))
T4 L340P Reha-Krantz LJ1985 Nucleotide Substitution Rate 1980 Mutation frequency (relative to WT) Technique: T4 rII P7oc reversion assay (AT->GC, Host: supE (C600))
T4 L340P Reha-Krantz LJ1985 Nucleotide Substitution Rate 343 Mutation frequency (relative to WT) Technique: T4 rII P7oc reversion assay (AT->TA(CG), Host: supE (C600))
T4 L340P Reha-Krantz LJ1985 Nucleotide Substitution Rate 742 Mutation frequency (relative to WT) Technique: T4 rII P7am reversion assay (GC->TA(CG), Host: supE (C600))
T4 L340P Reha-Krantz LJ1985 Nucleotide Substitution Rate 941 Mutation frequency (relative to WT) Technique: T4 rII 199oc reversion assay (AT->GC, Host: supD (CR63))
T4 V325AL340P Reha-Krantz LJ1985 Nucleotide Substitution Rate 844 Mutation frequency (relative to WT) Technique: T4 rII P7oc reversion assay (AT->TA(CG), Host: supD (CR63))
T4 V325AL340P Reha-Krantz LJ1985 Nucleotide Substitution Rate 674 Mutation frequency (relative to WT) Technique: T4 rII P7am reversion assay (GC->TA(CG), Host: supD (CR63))
T4 V325AL340P Reha-Krantz LJ1985 Nucleotide Substitution Rate 2305 Mutation frequency (relative to WT) Technique: ac forward mutational assay (Host: supE (MBM7007.2))
T4 V325AL340P Reha-Krantz LJ1985 Nucleotide Substitution Rate 4160 Mutation frequency (relative to WT) Technique: ac forward mutational assay (Host: supD (MBM7007.1))
T4 V325AL340P Reha-Krantz LJ1985 Nucleotide Substitution Rate 480 Mutation frequency (relative to WT) Technique: T4 rII P7oc reversion assay ( AT->GC, Host: supE (C600))
T4 V325AL340P Reha-Krantz LJ1985 Nucleotide Substitution Rate 2500 Mutation frequency (relative to WT) Technique: T4 rII P7oc reversion assay (AT->TA(CG), Host: supE (C600))
T4 V325AL340P Reha-Krantz LJ1985 Nucleotide Substitution Rate 500 Mutation frequency (relative to WT) Technique: T4 rII P7am reversion assay (GC->TA(CG), Host: supE (C600))
T4 V325AL340P Reha-Krantz LJ1985 Nucleotide Substitution Rate 642 Mutation frequency (relative to WT) Technique: T4 rII P7oc reversion assay ( AT->GC, Host: supD (CR63))
T4 L340PQ731am Reha-Krantz LJ1985 Nucleotide Substitution Rate 6 Mutation frequency (relative to WT) Technique: T4 rII P7oc reversion assay (AT->TA(CG), Host: supD (CR63))
T4 L340PQ731am Reha-Krantz LJ1985 Nucleotide Substitution Rate 4 Mutation frequency (relative to WT) Technique: T4 rII P7am reversion assay (GC->TA(CG), Host: supD (CR63))
T4 L340PQ731am Reha-Krantz LJ1985 Nucleotide Substitution Rate 2930 Mutation frequency (relative to WT) Technique: ac forward mutational assay (Host: supE (MBM7007.2))
T4 L340PQ731am Reha-Krantz LJ1985 Nucleotide Substitution Rate 96 Mutation frequency (relative to WT) Technique: ac forward mutational assay (Host: supD (MBM7007.1))
T4 L340PQ731am Reha-Krantz LJ1985 Nucleotide Substitution Rate 1720 Mutation frequency (relative to WT) Technique: T4 rII P7oc reversion assay (AT->GC, Host: supE (C600))
T4 L340PQ731am Reha-Krantz LJ1985 Nucleotide Substitution Rate 1417 Mutation frequency (relative to WT) Technique: T4 rII P7oc reversion assay (AT->TA(CG), Host: supE (C600))
T4 L340PQ731am Reha-Krantz LJ1985 Nucleotide Substitution Rate 1600 Mutation frequency (relative to WT) Technique: T4 rII P7am reversion assay (GC->TA(CG), Host: supE (C600))
T4 L340PQ731am Reha-Krantz LJ1985 Nucleotide Substitution Rate 20 Mutation frequency (relative to WT) Technique: T4 rII P7oc reversion assay (AT->GC, Host: supD (CR63))
T4 V325AL340P/Q731am Reha-Krantz LJ1985 Nucleotide Substitution Rate 35 Mutation frequency (relative to WT) Technique: T4 rII P7oc reversion assay (AT->TA(CG), Host: supD (CR63))
T4 V325AL340P/Q731am Reha-Krantz LJ1985 Nucleotide Substitution Rate 17 Mutation frequency (relative to WT) Technique: T4 rII P7am reversion assay (GC->TA(CG), Host: supD (CR63))
T4 V325AL340P/Q731am Reha-Krantz LJ1985 Nucleotide Substitution Rate 4453 Mutation frequency (relative to WT) Technique: ac forward mutational assay (Host: supE (MBM7007.2))
T4 V325AL340P/Q731am Reha-Krantz LJ1985 Nucleotide Substitution Rate 223 Mutation frequency (relative to WT) Technique: ac forward mutational assay (Host: supD (MBM7007.1))
T4 V325AL340P/Q731am Reha-Krantz LJ1985 Nucleotide Substitution Rate 590 Mutation frequency (relative to WT) Technique: T4 rII P7oc reversion assay (AT->GC, Host: supE (C600))
T4 V325AL340P/Q731am Reha-Krantz LJ1985 Nucleotide Substitution Rate 1967 Mutation frequency (relative to WT) Technique: T4 rII P7oc reversion assay (AT->TA(CG), Host: supE (C600))
T4 V325AL340P/Q731am Reha-Krantz LJ1985 Nucleotide Substitution Rate 1867 Mutation frequency (relative to WT) Technique: T4 rII P7am reversion assay (GC->TA(CG), Host: supE (C600))
T4 V325AL340P/Q731am Reha-Krantz LJ1985 Nucleotide Substitution Rate 49 Mutation frequency (relative to WT) Technique: T4 rII P7oc reversion assay ( AT->GC, Host: supD (CR63))

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