The DNA polymerases (gp43s) of the related bacteriophages T4 and RB69 are B family (polymerase ␣ 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.Bacteriophage RB69 is a relative of phage T4, with which it shares many similarities in genetic organization (1, 2) and structures and functions of the phage-encoded DNA replication proteins (3,4). Replication fidelity in T4 and presumably also in RB69 is determined almost exclusively by the fidelities of the phage-encoded DNA polymerase and its associated proofreading 3Ј-5Ј exonuclease (5). This useful simplicity reflects the fact that T4 DNA replication appears to be devoid of DNA mismatch repair; phage T4 is not subject to the action of the several Escherichia coli mismatch repair systems (6) and seems unable to repair mutational heteroduplexes on its own. Screens for T4 mutator mutations have failed to uncover evidence for the involvement of mismatch repair in mutagenesis, and the mutational dose response to base analogues does not display the mismatch repair-dependent lag seen in E. coli (5).The DNA polymerases of phages T4 and RB69 (gp43, product of phage gene 43) are members of the polymerase ␣ class or B family of DNA polymerases, which includes the replicative polymerases ␣, ␦, and ⑀ of eukaryotic cells and the polymerases of several of their DNA viruses (7). Some archaeons also encode gp43-like B family enzymes (8 -10). As such, T4 gp43 and RB69 gp43 are attractive subjects for studies of mechanisms of replication by this class of enzymes, particularly because of the amenability of the phage system to combined genetic and biochemical analyses (11)(12)(13)(14). A recently determined crystal structure of RB69 gp43 reveals five discrete domains termed N, Exo, Palm, Fingers, and Thumb (15). This structure is in the "open" configuration and provides a preliminary framework for understanding the dynamics of DNA polymerase interactions with the DNA primer template, with incoming dNTPs, and ...

Usher syndrome is characterized by profound hearing loss and retinal degeneration. A splice-site mutation, 216G-->A, in exon 3 of USH1C is associated with Acadian Usher type IC. This mutation was reported to create an in-frame deletion of 39 base pairs (bp), resulting in an unstable transcript. By RT-PCR analysis of 216A and 216G constructs transfected into HeLa cells and also of patient cell lines, we have demonstrated a frame-shift deletion of 35 bp, not 39 bp. Thus, the instability of the USH1C mRNA is explained by the 216G-->A out-of-frame splice site mutation.

DNA polymerase (gp43) of phage T4 plays two biological roles, one as an essential DNA binding replication enzyme and the other as an mRNA-specific autogenous translational repressor. Binding of T4 gp43 to its mRNA target (translational operator RNA) interferes with gp43-DNA interactions, but it is unclear how the protein determinants for binding DNA are affected by the dynamics of gp43-mRNA interactions. We have used RB69 gp43, a natural variant of the T4 enzyme whose crystal structure has been determined to identify protein sites that respond to the interaction with specific RNA. We used protein phosphorylation markers, photocross-linking studies, protease sensitivity assays, and mutational analyses to examine the effects of operator RNA on the enzyme's five structural domains (N, exo, palm, fingers, and thumb). Our studies suggest that this RNA affects gp43-DNA interactions through global effects on protein structure that occlude DNA-binding sites but leave the enzyme accessible to interactions with the sliding clamp (RB69 gp45) and possibly other polymerase accessory proteins. We discuss the possible biological significance of putative RNA-binding motifs in the N and palm domains of RB69 gp43.

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