SUMMARY Phage T4 has provided countless contributions to the paradigms of genetics and biochemistry. Its complete genome sequence of 168,903 bp encodes about 300 gene products. T4 biology and its genomic sequence provide the best-understood model for modern functional genomics and proteomics. Variations on gene expression, including overlapping genes, internal translation initiation, spliced genes, translational bypassing, and RNA processing, alert us to the caveats of purely computational methods. The T4 transcriptional pattern reflects its dependence on the host RNA polymerase and the use of phage-encoded proteins that sequentially modify RNA polymerase; transcriptional activator proteins, a phage sigma factor, anti-sigma, and sigma decoy proteins also act to specify early, middle, and late promoter recognition. Posttranscriptional controls by T4 provide excellent systems for the study of RNA-dependent processes, particularly at the structural level. The redundancy of DNA replication and recombination systems of T4 reveals how phage and other genomes are stably replicated and repaired in different environments, providing insight into genome evolution and adaptations to new hosts and growth environments. Moreover, genomic sequence analysis has provided new insights into tail fiber variation, lysis, gene duplications, and membrane localization of proteins, while high-resolution structural determination of the “cell-puncturing device,” combined with the three-dimensional image reconstruction of the baseplate, has revealed the mechanism of penetration during infection. Despite these advances, nearly 130 potential T4 genes remain uncharacterized. Current phage-sequencing initiatives are now revealing the similarities and differences among members of the T4 family, including those that infect bacteria other than Escherichia coli. T4 functional genomics will aid in the interpretation of these newly sequenced T4-related genomes and in broadening our understanding of the complex evolution and ecology of phages—the most abundant and among the most ancient biological entities on Earth.

General recombination is essential for growth of phage T4, because origin initiation of DNA replication is inactivated during development, and recombination-dependent initiation is necessary for continuing DNA replication. The requirement of recombination for T4 growth has apparently been a driving force to acquire and maintain multiple recombination mechanisms. This requirement makes this phage an excellent model to analyze several recombination mechanisms that appear redundant under optimal growth conditions but become essential under other conditions, or at different stages of the developmental program. The most important substrate for wild-type T4 recombination is single-stranded DNA generated by incomplete replication of natural or artificial chromosomal ends, or by nucleolytic degradation from induced breaks, or nicks. Recombination circumvents the further erosion of such ends. There are multiple proteins and multiple pathways to initiate formation of recombinants (by single-strand annealing or by strand invasion) and to convert recombinational intermediates into final recombinants ("cut and paste" or "cut and package"), or to initiate extensive DNA replication by "join-copy" or "join-cut-copy" mechanisms. Most T4 recombination is asymmetrical, favoring the initiation of replication. In wild-type T4 these pathways are integrated with physiological changes of other DNA transactions: mainly replication, transcription, and packaging. DNA replication and packaging enzymes participate in recombination, and recombination intermediates supply substrates for replication and packaging. The replicative recombination pathways are also important for transmission of intron DNA to intronless genomes ("homing"), and are implicated in horizontal transfer of foreign genes during evolution of the T-even phages. When horizontal transfer involves heteroduplex formation and repair, it is intrinsically mutagenic and contributes to generation of species barriers between phages.

We show that bacteriophage T4 has two alternative mechanisms to initiate DNA replication: one dependent on Escherichia coli RNA polymerase (RNA nucleotidyltransferase, EC 2.7.7.6), and one dependent on general recombination. Continued DNA synthesis under recombination-defective conditions was sensitive to rifampin, an inhibitor ofRNA polymerase. On the other hand, DNA synthesis accelerated in spite of the presence of rifampin if recombination occurred.Replication ofbacteriophage T4 DNA is initiated on linear DNA molecules at one or several preferred origins (1-9). Soon after the first initiation, many more replication forks are initiated, leading to a rapid acceleration ofDNA synthesis (10, 11). During this acceleration period, a complex network of DNA is formed (12)(13)(14)(15)(16)(17). This process requires recombination functions (9, 18). Most mutations that inhibit recombination not only prevent for, mation of this network but also arrest DNA synthesis prematurely (19)(20)(21)(22). This indicates that recombination and the continuation ofDNA replication are interdependent (for review see ref. 23). The underlying reasons have remained unknown because of observations seemingly inconsistent with simple explanations: the DNA arrest phenotype ofcertain recombinationdefective mutants is overcome by additional mutations in genes 33 and 55 (20-22, 24), which code for RNA polymerase accessory proteins (25)(26)(27).To explain these and other apparently contradictory results, we have proposed that phage T4 uses different modes to initiate DNA replication: initiation from specific origin sequences, which we define as "primary" initiation, and subsequent "secondary" initiation from recombinational intermediates (7,8,28).For several reasons (7,8,29,30) we suspected that host RNA polymerase (RNA nucleotidyltransferase, EC 2.7.7.6) is required for primary initiation although it has been shown that late wild-type DNA replication does not depend on RNA polymerase (31, 32). After the onset of DNA replication, gene 33 and 55 products associate with RNA polymerase (25-27) to effect the switch to late gene expression (33-35) by altering promoter recognition (36). If unmodified host RNA polymerase were required for primary origin initiation, the association with gene 33 and 55 products would shut offreinitiation from primary origins and make the alternative recombinational initiation indispensable for growth of wild-type T4. This hypothesis accounts for the observations mentioned above: premature arrest of DNA synthesis in recombination-defective mutants (46-47-) and restoration of DNA synthesis by additional mutations in genes 33 and 55. Although the rate of DNA synthesis under these conditions is similar to that ofwild-type T4, no branched concatemers are formed (20)(21)(22). Instead, the DNA replicates as linear molecules of unit length, which we suspected to require continued initiation from primary origins by RNA polymerase. Therefore, this replication should remain sensitive to inhibitors of RNA polymerase at late...

. In wild-type T4, timing of these pathways is integrated with the developmental program and related to transcription and packaging of DNA. In primase mutants, which are defective in origin-dependent lagging-strand DNA synthesis, the late pathway can bypass the lack of primers for lagging-strand DNA synthesis. The exquisitely regulated synthesis of endo VII, and of two proteins from its gene, explains the delay of recombination-dependent DNA replication in primase (as well as topoisomerase) mutants, and the temperature-dependence of the delay. Other proteins (e.g., the singlestranded DNA binding protein and the products of genes 46 and 47) are important in all recombination pathways, but they interact differently with other proteins in different pathways. These homologous recombination pathways contribute to evolution because they facilitate acquisition of any foreign DNA with limited sequence homology during horizontal gene transfer, without requiring transposition or site-specific recombination functions. Partial heteroduplex repair can generate what appears to be multiple mutations from a single recombinational intermediate. The resulting sequence divergence generates barriers to formation of viable recombinants. The multiple sequence changes can also lead to erroneous estimates in phylogenetic analyses.The purpose of looking back is not, of course, merely to obtain satisfaction from reflecting on past triumphs; rather, it is to discover as many clues as possible to the likely developments of the future.Glenn T. Seaborg T he tight interrelationship between homologous recombination and DNA replication was first evident in T4 and the related T-even phages. Because DNA of T4 and its host E. coli differ in base composition and modifications and because the host DNA is rapidly degraded after phage infection, molecular aspects of T4 replication and recombination could be readily investigated by biochemical, biophysical, and genetic methods. Early characterization of mutations in most essential genes (1) and the almost complete dependence of replication and recombination on phage-encoded proteins (2) allowed analyses of recombination and replication proteins, as well as ''reality checks'' of results obtained with genetic and biochemical methods (3). The following idiosyncrasies of T4 chromosomes revealed the importance of DNA ends and recombination-dependent DNA replication. Ends of T4 chromosomes are cut during packaging from branched concatemers, which are generated by recombination-dependent replication. A ''headful mechanism'' packages a complete genome and Ϸ3% DNA repeated at each end as ''terminal redundancy,'' thereby generating the random circular permutation of chromosomal ends (4). Some smaller T4 particles, formed because of assembly errors, package incomplete genomes whose ends are also randomly circularly permuted (5). Multifactor crosses revealed stimulation of recombination by their DNA ends, regardless of map positions (5, 6). Moreover, different segregation patterns of alleles in patch vs. splice re...

No abstract