The 2.8 A resolution crystal structure of the bacteriophage RB69 gp43, a member of the eukaryotic pol alpha family of replicative DNA polymerases, shares some similarities with other polymerases but shows many differences. Although its palm domain has the same topology as other polymerases, except rat DNA polymerase beta, one of the three carboxylates required for nucleotidyl transfer is located on a different beta strand. The structures of the fingers and thumb domains are unrelated to all other known polymerase structures. The editing 3'-5' exonuclease domain of gp43 is homologous to that of E. coli DNA polymerase I but lies on the opposite side of the polymerase active site. An extended structure-based alignment of eukaryotic DNA polymerase sequences provides structural insights that should be applicable to most eukaryotic DNA polymerases.

The T4-related bacteriophages are a group of bacterial viruses that share morphological similarities and genetic homologies with the well-studied Escherichia coli phage T4, but that diverge from T4 and each other by a number of genetically determined characteristics including the bacterial hosts they infect, the sizes of their linear double-stranded (ds) DNA genomes and the predicted compositions of their proteomes. The genomes of about 40 of these phages have been sequenced and annotated over the last several years and are compared here in the context of the factors that have determined their diversity and the diversity of other microbial genomes in evolution. The genomes of the T4 relatives analyzed so far range in size between ~160,000 and ~250,000 base pairs (bp) and are mosaics of one another, consisting of clusters of homology between them that are interspersed with segments that vary considerably in genetic composition between the different phage lineages. Based on the known biological and biochemical properties of phage T4 and the proteins encoded by the T4 genome, the T4 relatives reviewed here are predicted to share a genetic core, or "Core Genome" that determines the structural design of their dsDNA chromosomes, their distinctive morphology and the process of their assembly into infectious agents (phage morphogenesis). The Core Genome appears to be the most ancient genetic component of this phage group and constitutes a mere 12-15% of the total protein encoding potential of the typical T4-related phage genome. The high degree of genetic heterogeneity that exists outside of this shared core suggests that horizontal DNA transfer involving many genetic sources has played a major role in diversification of the T4-related phages and their spread to a wide spectrum of bacterial species domains in evolution. We discuss some of the factors and pathways that might have shaped the evolution of these phages and point out several parallels between their diversity and the diversity generally observed within all groups of interrelated dsDNA microbial genomes in nature.

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 ...

We describe the use of a phylogenetic approach to analyze the modular organization of the single-chained (898 amino acids) and multifunctional DNA polymerase of phage T4. We have identified, cloned in expression vectors, and sequenced the DNA polymerase gene (gene 43) of phage RB69, a distant relative of T4. The deduced primary structure of the RB69 protein (RB69 gp43) differs from that of T4 gp43 in discrete clusters of short sequence that are interspersed with clusters of high similarity between the two proteins. Despite these differences, the two enzymes can substitute for each other in phage DNA replication, although T4 gp43 does exhibit preference to its own genome. A 55-amino acid internal gp43 segment of high sequence divergence between T4 and RB69 could be replaced in RB69 gp43 with the corresponding segment from T4 without loss of replication function. The reciprocal chimera and a deletion mutant of the T4 gp43 segment were both inactive for replication and specifically inhibitory ("dominant lethal") to the T4 wild-type allele. The results show that phylogenetic markers can be used to construct chimeric and truncated forms of gp43 that, although inactive for replication, can still exhibit biological specificity.In DNA replication, DNA polymerases bear the major responsibility for copying genomes with high accuracy. As a group, these enzymes display a variety of molecular types, but most are unified by exhibiting two catalytic functions that control fidelity: primer/template-dependent nucleotidyl transferase (polymerase) and DNA 3Ј exonuclease (proofreading function) (Kornberg and Baker, 1992). In bacteriophage T4, the two functions are part of the same polypeptide chain, product of phage gene 43 (gp43), whereas in some biological systems the polymerase and DNA 3Ј exonuclease activities are specified by separate protein subunits, e.g. Escherichia coli DNA polymerase III holoenzyme (Kelman and O'Donnell, 1995). Another E. coli enzyme, DNA polymerase I, resembles T4 gp43 in size and in possessing polymerase and DNA 3Ј exonuclease functions in the same polypeptide chain; however, unlike T4 gp43, polymerase I also has an N-terminal 5Ј to 3Ј exonuclease function. A third E. coli DNA polymerase, polymerase II, resembles T4 gp43 in biochemical properties and amino acid sequence motifs but is a little smaller in size than the phage enzyme (Cai et al., 1995). One group of DNA polymerases, the reverse transcriptases, lack editing function altogether (Skalka and Geoff, 1993). T4 gp43 also bears a sequence-specific RNA-binding autogenous translational repressor function (Andrake et al., 1988) that only partially overlaps the DNA binding function of the enzyme (Pavlov and Karam, 1994).Typically, replication DNA polymerases work in complex with other proteins, which provide accessory functions that help meet a number of requirements and overcome a variety of constraints inherent to the semiconservative duplication of long supercoiled and condensed double-helical DNA genomes. In the case of T4, the interfacing of repli...

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