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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...

The genomes of bacteriophages T4 and RB69 are phylogenetically related but diverge in nucleotide sequence at many loci and are incompatible with each other in vivo. We describe here the biological implications of divergence in a genomic segment that encodes four essential DNA replication proteins: gp45 (sliding clamp), gp44/62 complex (clamp loader), and gp46 (a recombination protein). We have cloned, sequenced, and expressed several overlapping segments of the RB69 gene 46-45.2-(rpbA)-45-44-62cluster and compared its features to those of the homologous gene cluster from T4. The deduced primary structures of all four RB69 replication proteins and gp45.2 from this cluster are very similar (80 to 95% similarity) to those of their respective T4 homologs. In contrast, the rpbA region (which encodes a nonessential protein in T4) is highly diverged (∼49% similarity) between the two phage genomes and does not encode protein in RB69. Expression studies and patterns of high divergence of intercistronic nucleotide sequences of this cluster suggest that T4 and RB69 evolved similar transcriptional and translational control strategies for the cistrons contained therein, but with different specificities. In plasmid-phage complementation assays, we show that posttranslationally, RB69 and T4 homologs of gp45 and the gp44/62 complex can be effectively exchanged between the two phage replicase assemblies; however, we also show results which suggest that mixed clamp loader complexes consisting of T4 gp62 and RB69 gp44 subunits are not active for phage DNA replication. Thus, specificity of the gp44-gp62 interaction in the clamp loader marks a point of departure between the T4 and RB69 replication systems.

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