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.