Bacteriophage T 7 gene 2 . 5 protein : An essential protein for DNA replication ( DNA binding protein / recombination / T 7 DNA

The product of gene 2.5 of bacteriophage T7, a single-stranded DNA binding protein, physically interacts with the phage-encoded gene S protein (DNA polymerase) and gene 4 proteins (helicase and primase) and stimulates their activities. Genetic analysis ofT7 phage defective in gene 2.5 shows that the gene 2.5 protein is essential for T7 DNA replication and growth. T7 phages that contain null mutants ofgene2.5 were constructed by homologous recombination. These gene 2.5 null mutants contain either a deletion ofgene2.5 (T7A2.5) or an insertion into gene 2.5 and cannot grow in Escherichia coli (efficiency of plating, <10-8). After infection of E. coli with T7A2.5, host DNA synthesis is shut off, and phage DNA synthesis is reduced to <1% ofphage DNA synthesis in wild-type T7-infected E. coli cells as measured by incorporation of [3H]thymidine. In contrast, RNA synthesis is essentially normal in T7A2.5-infected cells. The defects in growth and DNA replication are overcome by wild-type gene 2.5 protein expressed from a plasmid harboring the T7 gene 2.5. Single-stranded DNA binding proteins (SSBs), such as Escherichia coli SSB and T4 gene 32 protein, are essential components of DNA metabolism in prokaryotic cells (1-3). The gene 2.5 protein of bacteriophage T7, originally isolated based on its strong affinity for single-stranded DNA and its ability to stimulate DNA synthesis by T7 DNA polymerase (4, 5), is thought to be analogous to these well characterized SSBs. Like E. coli SSB and T4 gene 32 protein, gene 2.5 protein has been implicated in T7 DNA replication, recombination, and repair (6-11). We purified gene 2.5 protein from cells overexpressing the gene and characterized its physical properties and interactions with DNA (6). Gene 2.5 protein exists as a dimer of two identical subunits of Mr 25,562. It binds specifically to single-stranded DNA with a stoichiometry of -7 nt bound per monomer of gene 2.5 protein and extends the length of the DNA molecules as measured by electron microscopy. The binding constant of gene 2.5 protein for single-stranded DNA is -2.5 x 106 M-1, as determined by fluorescence quenching and nitrocellulose filter binding assays (6). Fluorescence studies suggest that tyrosine residue(s) on gene 2.5 protein interacts with single-stranded DNA, whereas tryptophan residues do not (6). In T7 DNA replication, the gene 2.5 protein has the potential to play one of several essential roles. At the T7 replication fork, three proteins, the products of T7 genes 4 and 5 and the host trxA gene, account for the fundamental reactions (12, 13). Gene 5 protein is a DNA polymerase, catalyzing the polymerization of nucleotides with low processivity (14, 15). E. coli thioredoxin, the product of the trxA gene, binds to gene S protein in a 1:1 stoichiometry and confers processivity on the polymerization reaction by increasing the affinity of the enzyme for a primer/template (14, The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 15). Both direct and indirect evidence support an interaction between these essential replication proteins and the T7 gene 2.5 protein. T7 gene 2.5 protein stimulates DNA synthesis catalyzed by the T7 DNA polymerase/thioredoxin complex on singlestranded DNA templates (4, 5, 11, 14) and increases the processivity ofthe reaction (11). It has been shown by affinity chromatography and fluorescence emission anisotropy that T7 DNA polymerase and gene 2.5 protein physically interact with a dissociation constant of 1.1 ,uM (11). Similarly, interaction of the T7 helicase/primase with gene 2.5 protein has been inferred from the ability ofgene 2.5 protein to stimulate synthesis of primers (10, 16). The T7 helicase/primase-gene 2.5 protein interaction has also been confirmed by affinity chromatography (11). We have recently found (unpublished data) that the C-terminal acidic domain of gene 2.5 protein is required for T7 growth in vivo and it participates in gene 2.5 dimerization and protein-protein interactions. The role of gene 2.5 protein in recombination is not as well understood. Sadowski et al. (17) demonstrated that extracts of T7 phage-infected cells contain an activity that promotes renaturation of complementary single strands and suggested that this activity resided in the T7 SSB. Recent studies (S. Tabor and C.C.R., unpublished results) have, in fact, demonstrated that the gene 2.5 protein facilitates renaturation of homologous single-stranded DNA even more efficiently than does E. coli recA protein, E. coli SSB, or T4 gene 32 protein. A determination of the definitive role of the T7 gene 2.5 protein in DNA replication and recombination in vivo is dependent on genetic analysis of gene 2.5 mutants. Previously, T7 phage containing mutations in gene 2.5 have been isolated based on their inability to grow on E. coli strains that have a defective SSB (8). These T7 mutant phages contain an amber mutation in gene 2.5 that leads to synthesis of a shortened polypeptide -90% of the length of the wild-type protein (9). These gene 2.5 mutant phages can grow on wild-type E. coli strains but not on strains expressing a temperature-sensitive SSB at the nonpermissive temperature. Furthermore, these T7 phages are defective in recombination (8). Recently, however, a mutational analysis has identified T7 gene 2.5 mutants that cannot grow even in E. coli strains producing wild-type SSB (F. W. Studier, personal communication). We show that T7 phages with a deletion ofgene 2.5 (T7A2.5) do not grow in wild-type E. coli and have no detectable T7 DNA replication; the T7A2.5 phages grow normally in E. coli strains expressing wild-type gene 2.5 from a plasmid. MATERIALS AND METHODS Bacterial Strains. E. coli HMS157 (FrecB21 recC22 sbcA5 endA gal thi sup) (laboratory collection), E. coli Abbreviations: SSB, single-stranded DNA binding protein; moi, multiplicity of infection. *Present address: Department of Microbiology, National Fisheries University of Pusan, Pusan, 608-737, Korea.