Interacting fidelity defects in the replicative DNA polymerase of bacteriophage RB69.

Abstract:

The DNA polymerases (gp43s) of the related bacteriophages T4 and RB69 are B family (polymerase alpha class) enzymes that determine the fidelity of phage DNA replication. A T4 whose gene 43 has been mutationally inactivated can be replicated by a cognate RB69 gp43 encoded by a recombinant plasmid in T4-infected Escherichia coli. We used this phage-plasmid complementation assay to obtain rapid and sensitive measurements of the mutational specificities of mutator derivatives of the RB69 enzyme. RB69 gp43s lacking proofreading function (Exo(-) enzymes) and/or substituted with alanine, serine, or threonine at the conserved polymerase function residue Tyr(567) (Pol(Y567(A/S/T)) enzymes) were examined for their effects on the reversion of specific mutations in the T4 rII gene and on forward mutation in the T4 rI gene. The results reveal that Tyr(567) is a key determinant of the fidelity of base selection and that the Pol and Exo functions are strongly coupled in this B family enzyme. In vitro assays show that the Pol(Y567A) Exo(-) enzyme generates mispairs more frequently but extends them less efficiently than does a Pol(+) Exo(-) enzyme. Other replicative DNA polymerases may control fidelity by strategies similar to those used by RB69 gp43.

Polymerases:

Topics:

Nucleotide Incorporation, Exonuclease Activity, Kinetic Parameters, Fidelity

One line summary:

In RB69 polymerase Tyrosine 567 is an integral part of base selection, as RB69 Y567A is less accurate and slower at replicating than an RB69 mutant without exonuclease capabilities

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
RB69 Y567A Bebenek A2001 Frameshift Error Rate 4.5 Mutation frequency (relative to WT) Technique: RB69 rII 131 reversion
RB69 Y567A Bebenek A2001 Nucleotide Substitution Rate 820 Mutation frequency (relative to WT) Technique: RB69 rII UV256 reversion
RB69 Y567A Bebenek A2001 Nucleotide Substitution Rate 1400 Mutation frequency (relative to WT) Technique: RB69 rII UV375 reversion
RB69 Y567S Bebenek A2001 Frameshift Error Rate 14 Mutation frequency (relative to WT) Technique: RB69 rII 131 reversion
RB69 Y567S Bebenek A2001 Nucleotide Substitution Rate 1200 Mutation frequency (relative to WT) Technique: RB69 rII UV256 reversion
RB69 Y567S Bebenek A2001 Nucleotide Substitution Rate 1600 Mutation frequency (relative to WT) Technique: RB69 rII UV375 reversion
RB69 Y567T Bebenek A2001 Frameshift Error Rate 32 Mutation frequency (relative to WT) Technique: RB69 rII 131 reversion
RB69 Y567T Bebenek A2001 Nucleotide Substitution Rate 2600 Mutation frequency (relative to WT) Technique: RB69 rII UV256 reversion
RB69 Y567T Bebenek A2001 Nucleotide Substitution Rate 810 Mutation frequency (relative to WT) Technique: RB69 rII UV375 reversion
RB69 D222AD327A Bebenek A2001 Frameshift Error Rate 520 Mutation frequency (relative to WT) Technique: RB69 rII 131 reversion
RB69 D222AD327A Bebenek A2001 Nucleotide Substitution Rate 600 Mutation frequency (relative to WT) Technique: RB69 rII UV256 reversion
RB69 D222AD327A Bebenek A2001 Nucleotide Substitution Rate 2600 Mutation frequency (relative to WT) Technique: RB69 rII UV375 reversion

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