Interacting fidelity defects in the replicative DNA polymerase of bacteriophage RB69

The DNA polymerases (gp43s) of the related bacteriophages T4 and RB69 are B family (polymerase alpha class) enzymes that determine the fidelity of pha ge DNA replication. A T4 whose gene 43 has been mutationally inactivated ca n be replicated by a cognate RB69 gp43 encoded by a recombinant plasmid in T l-infected Escherichia coli. We used this phage-plasmid complementation a ssay to obtain rapid and sensitive measurements of the mutational specifici ties of mutator derivatives of the RB69 enzyme, RB69 gp43s lacking proofrea ding 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 specifi c mutations in the T4 rII gene and on forward mutation in the T4 rI gene. T he results reveal that Tyr567 is a key determinant of the fidelity of base selection and that the Pol and fro functions are strongly coupled in this B family enzyme, In vitro assays show that the Pol(Y567A) EXO- enzyme genera tes 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.