Studies on the Recombination Genes of Bacteriophage T4: Suppression of uvsX and uvsY Mutations by uvsW Mutations

Genes uvsW, uvsX and uvsY are dispensable for T4 growth but are implicated in recombination and in the repair of damaged DNA. We found that large-plaque mutants arose efficiently from small-plaque uvsX and uvsY mutants at 42 degrees and were pseudorevertants containing a new mutation in uvsW. Using reconstructed double mutants, we confirmed that a mutation in uvsW partially increases the burst size and UV resistance of uvsX and uvsY mutants. At 41 degrees the uvsW mutation completely restores the arrest in DNA synthesis caused by mutations in genes uvsX, uvsY and 46, but at 30 degrees it only partially restores DNA synthesis in a gene 46 mutant and does not restore DNA synthesis in uvsX and uvsY mutants. Restored DNA synthesis at 41 degrees was paralleled by the overproduction of single-stranded DNA and gene 32 protein. Based on these findings, we propose that the uvsW gene regulates the production of single-stranded DNA and we discuss the phenotype of uvsW mutants and their suppression of some uvsX and uvsY phenotypes. Infection of restrictive cells with am uvsW mutants revealed a defect in the synthesis of a protein of molecular weight 53,000 daltons, suggesting that this protein is the uvsW gene product.     

Amplification of snap-back DNA synthesis reactions by the uvsX recombinase of bacteriophage T4.

The uvsX protein of bacteriophage T4 is a recA-type recombinase. This protein has previously been shown to help initiate DNA replication on a double-stranded DNA template by catalyzing synapsis between the template and a homologous DNA single strand that serves as primer. Here, we demonstrate that this replication-initiating activity of the uvsX protein greatly amplifies the snap-back (hairpin-primed) DNA synthesis that is catalyzed by the T4 DNA polymerase holoenzyme on linear, single-stranded DNA templates. Amplification requires the presence of uvsX protein, the DNA polymerase holoenzyme, T4 gene 32 protein, and a T4 DNA helicase, in a reaction that is modulated by the T4 uvsY protein (an accessory protein to the uvsX recombinase). The reaction products consist primarily of large networks of double-stranded and single-stranded DNA. With alkali or heat treatment, these networks resolve into dimer-length single-stranded DNA chains that renature instantaneously to reform a monomer-length double helix. A simple model can explain this uvsX protein-dependent amplification of snap-back DNA synthesis; the mechanism proposed makes several predictions that are confirmed by our experiments.     

Purification and properties of the bacteriophage T4 gene 61 RNA priming protein

The bacteriophage T4 gene 61 protein is required, together with the gene 41 protein and single-stranded DNA, for the synthesis of the pentaribonucleotides that are used as primers for the start of each new Okazaki DNA fragment during T4 DNA replication. Using this priming activity as an assay, we have purified the 61 protein to essential homogeneity in milligram amounts. The priming activity was identified with the product of T4 gene 61 by using two-dimensional polyacrylamide gel electrophoresis to compare all of the T4-induced proteins in wild-type and mutant infections; the purified protein co-migrates with the only detectable protein missing in a 61- mutant infection. The purified 61 protein is shown to bind to the T4 helix-destabilizing protein (gene 32 protein) and to both single-stranded and double-stranded DNA. We have failed to detect any ribonucleotide polymerizing activity in either the 61 protein or the 41 protein alone; both the 61 and 41 proteins must be present to observe any synthesis of oligoribonucleotides.     

T4 DNA replication and viral gene expression

The normal dependence of “late” T4 gene expression on concurrent viral DNA replication is circumvented in cells infected with a triple mutant in which viral DNA polymerase, DNA ligase, and the exonuclease functions of genes 46 or 47 are defective. Acrylamide gel electrophoresis of labeled proteins from infected cells has made possible an extension of the analysis of replication-uncoupled T4 protein synthesis. We find a number of late T4 proteins synthesized: the products of genes 34, 37, 18, 23 and 24. Processing of the gene 23 product, normally headassembly dependent, occurs, but with considerably diminished efficiency compared to wild-type infection. Late T4 protein synthesis in replication-uncoupled infection retains a requirement for T4 gene 33 and gene 55 function. Finally, a number of “early” T4 gene products, normally shut off late in wildtype infection, continue to be synthesized late in replication-uncoupled infection, concurrently with the late proteins.     

Fine mapping of secondary structures of fd phage DNA in the region of the replication origin.

A synthetic heptaribonucleotide, GACCCCC, which is complementary to a unique site on fd bacteriophage DNA, primes DNA synthesis of fd by T4 bacteriophage DNA polymerase. The rate of the GACCCCC-primed DNA synthesis was not uniform as reflected by the appearance of discrete DNA fragments as replication intermediates on an alkaline agarose gel. After 10 minutes of synthesis a significant fraction of the DNA product ran as a single band with a length of about 1960 nucleotides. We have isolated this DNA fragment, hybridized back to unlabeled fd DNA template, and mapped the Taq I restriction fragments by urea polyacrylamide gel electrophoresis. This fine mapping procedure has located two major pause sites at fd nucleotide positions 5575 and 5674. These sites reside in the stem of two very stable hairpin helices near the origin of DNA replication of fd. Models for the functional roles of these two hairpin helices are presented.     

The UvsY protein of bacteriophage T4 modulates recombination-dependent DNA synthesis in vitro

An in vitro system containing the T4 gene 43, 45, 44/62, 32, dda, and uvsX proteins catalyzes DNA synthesis that is dependent on the synapsis step of homologous genetic recombination. The rate of DNA synthesis in this system is highly dependent on the concentration of the uvsX recombinase (a recA-like protein). Here we report the effect of the T4 uvsY protein, a recombination accessory protein, on this reaction. Low concentrations of uvsY protein greatly stimulate DNA synthesis at low concentrations of uvsX protein, but these same concentrations inhibit DNA synthesis at high concentrations of uvsX protein. As a result, the addition of small amounts of uvsY protein lowers the minimum concentration of uvsX protein needed for the reaction 8-fold, and it lowers the uvsX protein concentration for maximum activity 4-fold. The uvsY protein can affect either the initiation or elongation phase of DNA synthesis, depending on the concentration of uvsX protein present. The implications of these results for the function of the uvsY protein in T4 DNA replication in vivo are discussed.     

Selective stimulation by mitogens of incorporation of 35S-methionine into a family of proteins released into the medium by 3T3 cells

Serum and three mitogens for mouse embryo 3T3 cells—fibroblast growth factor from brain, fibroblast growth factor from pituitary, and epidermal growth factor—specifically stimulate the synthesis and release into the medium by these cells of a group of proteins that travel together on SDS gel electrophoresis and that are detected by ³⁵S-methionine labeling. These proteins, designated mitogen-releasable proteins (MRPs), have a median, monomer molecular weight on SDS polyacrylamide gel electrophoresis of 34,000 daltons (30,000–38,000 daltons). Our evidence indicates that these proteins comprise a family of glycoproteins, probably with a common polypeptide backbone. The observations supporting this conclusion are that MRPs give a diffuse pattern of bands upon SDS gel electrophoresis; travel as a single, diffuse band when resolved by electrophoresis in the absence of SDS; adsorb to a pea-lectin-sepharose column and can be eluted with α-methyl mannose; and can be labeled metabolically with ³H-mannose. In addition, in the presence of tunicamycin, MRPs are not made—instead, a smaller molecular weight (22,000 dalton), and apparently homogeneous, protein appears. We believe this 22,000 dalton protein to be the unglycosylated form of MRP. Further support for this idea comes from our observation that treatment of MRPs with endoglycosidase H produces a protein with a molecular weight slightly greater than 22,000 daltons. The effect of mitogens on DNA synthesis and MRP release are correlated in the following ways. First, serum factors are required for both responses. Second, in 3T3 cells transformed by SV40, Moloney and Kirsten viruses that do not synthesize DNA in response to FGF, MRPs are not released in response to FGF. Third, in untransformed 3T3 cells, the dose-response curves for fibroblast growth factor on MRP release and thymidine incorporation are closely correlated. Fourth, insulin, a poor mitogen for 3T3 cells, does not enhance MRP release. Fifth, stimulation of MRP release by epidermal growth factor or fibroblast growth factor is inhibited by hydroxyurea and butyrate, both inhibitors of DNA synthesis in these cells. Sixth, if the mitogen is removed at any time during the 20 hr preincubation period, the effect on MRP release observed between 20 and 24 hr is severely diminished.     

Assembly of bacteriophage T4 tail fibers: Identification and characterization of the nonstructural protein gp57

Summary Formation of both the tail fiber and the baseplate of bacteriophage T4 depends on the product of T4 gene 57. A single amber mutation in that gene causes loss of two T4-specific proteins. Their molecular weights are 18,000 and about 6,000, respectively, based on their electrophoretic mobilities in SDS-polyacrylamide gels. E. coli carrying a cloned T4 DNA fragment of about 700 basepairs, which directs the synthesis of the smaller protein only, specifically supports the growth of gene 57 amber mutants. We conclude that the small protein is a functional product of gene 57.Abbreviations Am ampicillin - Cm chloramphenicol - Tet tetracycline - SDS sodium dodecyl sulfate - bp basepairs - wt wildtype - Su suppressor - Km kanamycin - ds double stranded - ss single stranded - SDS-PAGE SDS-polyacrylamide gel electrophoresis     

Identification of the gene controlling the synthesis of the major bacteriophage T5 membrane protein.

After infection of Escherichia coli B by bacteriophage T5, a major new protein species, as indicated by polyacrylamide gel electrophoresis, appears in the cells' membranes. Phage mutants with amber mutations in the first-step-transfer portion of their DNA have been tested for their ability to induce membrane protein synthesis after they infect E. coli B. We have found that phage with mutations in the Al gene of T5 do not induce the synthesis of the T5-specific major membrane protein, whereas phage that are mutant in the A2 gene do induce its synthesis. We conclude that gene Al must function normally for T5-specific membrane protein biosynthesis to occur and that only the first 8% (first-step-transfer piece) of the DNA need be present in the cell for synthesis to occur.     

Studies on structural proteins of the rat liver ribosomes. I. Molecular wights of the proteins of large and small subunits.

Structural proteins of active 60-S and 40-S subunits of rat liver ribosomes were analysed by two-dimensional polyacrylamide gel electrophoresis. 35 and 29 spots were shown on two-dimensional gel electrophoresis of proteins from large and small subunits, respectively. It was noted that the migration distances of stained proteins with Amido black 10B remained unchanged in the following sodium dodecyl sulfate-acrylamide gel electrophoresis, although some minor degradation and/or aggregation products were observed in the case of several ribosomal proteins, especially of those with high molecular weights. This finding made it possible to measure the molecular weight of each ribosomal protein in the spot on two-dimensional gel electrophoresis by following sodium dodecyl sulfate-acrylamide gel electrophoresis. The molecular weights of the protein components of two liver ribosomal subunits were determined by this 'three-dimensional' polyacrylamide gel electrophoresis. The molecular weights of proteins of 40-S subunits ranged from 10 000 to 38 000 and the number average molecular weight was 23 000. The molecular weights of proteins of 60-S subunits ranged from 10 000 to 60 000 and the number average molecular weight was 23 900.     

The mechanism of homologous DNA strand exchange catalyzed by the bacteriophage T4 uvsX and gene 32 proteins

A strand exchange reaction between a single-stranded DNA circle and a homologous linear double-stranded DNA molecule is catalyzed by a mixture of two T4 bacteriophage proteins, the uvsX protein (a DNA-dependent ATPase that resembles the recA protein) and the gene 32 protein (a helix-destabilizing protein). The products are different from those formed in the corresponding recA protein-catalyzed reaction; rather than producing a linear single strand plus a nicked circular double-stranded (form II) DNA molecule as the final products, interlinked DNA networks are rapidly generated. Electron microscopy reveals that these networks form from multiple pairing reactions that involve the recombination intermediates. Since the uvsX protein is present in substoichiometric quantities, it presumably recycles to catalyze these successive pairing events. Recycling of the uvsX protein has been more directly examined in an assay that monitors the rate of uvsX protein-catalyzed branch migration. The branch migration reaction is rapidly inhibited by dilution of the uvsX protein or by the addition of a heterologous competitor DNA, showing that the uvsX protein-DNA filaments that catalyze strand exchange are dynamic structures. The evidence suggests that individual uvsX protein monomers are continuously entering and leaving the cooperatively formed filament in a cycle that is strongly affected by their ATP hydrolysis.     

Purification and characterization of the T4 bacteriophage uvsX protein

Gene uvsX of bacteriophage T4 encodes a 40,000-dalton protein that plays a key role in the major pathway for genetic recombination in T4-infected cells. Mutations at the uvsX locus lead to increased sensitivity to various DNA-damaging agents, reduced phage bursts, decreased genetic recombination, and early arrest of DNA synthesis. Like the Escherichia coli recA protein, the purified uvsX protein is a DNA-dependent ATPase that catalyzes pairing between homologous single- and double-stranded DNA molecules in vitro (Yonesaki, T., Ryo, Y., Minagawa, T., and Takahashi, H., (1985) Eur. J. Biochem. 148, 127-134). At physiological salt concentrations, the uvsX protein binds tightly and cooperatively to single-stranded DNA, covering about five nucleotides per protein monomer; at lower salt concentrations, a similar type of binding to double-stranded DNA is detected (Griffith, J., and Formosa, T., (1985) J. Biol. Chem. 260, 4484-4491). We show here that the ATPase activity of this protein is unusual in producing both ADP plus Pi and AMP plus PPi as products. Generating the fully active form of the ATPase is a cooperative process, apparently requiring that a protein monomer be bound to single-stranded DNA while surrounded by other ATP-bound monomers. The catalysis of homologous pairing by the uvsX protein is shown to be greatly stimulated by the presence of the T4 gene 32 protein, a helix-destablizing protein previously studied in this laboratory, and it requires continued ATP hydrolysis. We present a method that allows the purification of the uvsX protein to essential homogeneity. We also describe the complete purification of two proteins that bind to the uvsX protein: the T4 uvsY protein (16,000 daltons) and an E. coli host protein of 32,000 daltons whose gene is unknown. The host protein is likely to play a role in DNA metabolism, because it also binds to the T4 gene 32 protein and to DNA; the sequence of its amino-terminal 29 amino acids has been determined.     

Changes in the acid-soluble nucleotide composition of red cells of cattle at various ages.

DNA polymerase was extracted from HeLa cell mitochondria with high salt concentrations (1M) and Nonidet-P 40 (0.2%). Subsequently the enzyme was purified stepwise by DEAE-cellulose-, phosphocellulose-, hydroxyapatite-Ultrogel-, DNA-cellulose chromatography and preparative polyacrylamide gel electrophoresis. The purified enzyme exhibited a molecular weight between 100 000 – 110 000 and was devoid of endonuclease activity. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of this enzyme preparation revealed two protein bands suggesting that the mitochondrial DNA polymerase might consist of two subunits with the molecular weights of 45 000 and 60 000.     

Identification of the pifC gene and its role in negative control of F factor pif gene expression

The pif region of the F factor includes two genes, pifA and pifB, that lead to abortive T7 infection. We have identified a new gene in this region, pifC, by constructing an in vitro fusion of pif DNA at 41.6 kilobases on the F factor physical map to the lacZ gene. A PifC-LacZ fusion protein of 149,000 daltons has been identified by immunoprecipitation and polyacrylamide gel electrophoresis. This allows us to assign the N terminus of pifC to 42.5 kilobases on the F map. Using fusions of pifC, pifA, and pifB to lacZ, we have studied the regulation of pif gene expression and have shown that the product of pifC negatively controls its own expression and that of pifA and pifB.     

Purification and characterization of the gene 1.2 protein of bacteriophage T7

Gene 1.2 of bacteriophage T7, located near the primary origin of DNA replication at position 15.37 on the T7 chromosome, encodes a 10,059-dalton protein that is essential for growth on Escherichia coli optA1 strains (Saito, H., and Richardson, C. C. (1981) J. Virol. 37, 343-351). In the absence of the T7 1.2 and E. coli optA gene products, the degradation of E. coli DNA proceeds normally, and T7 DNA synthesis is initiated at the primary origin. However, T7 DNA synthesis ceases prematurely and the newly synthesized DNA is degraded; no viable phage particles are released. The gene 1.2 protein has been purified to apparent homogeneity from cells in which the cloned 1.2 gene is overexpressed. Purification of the [35S] methionine-labeled protein was followed by monitoring the radioactivity of the protein and by gel electrophoresis. The purified protein has been identified as the product of gene 1.2 on the basis of molecular weight and partial amino acid sequence. We have found that extracts of E. coli optA1 cells infected with T7 gene 1.2 mutants are defective in packaging exogenous T7 DNA when such extracts are prepared late in infection. Purified gene 1.2 protein restores packaging activity to these defective extracts, thus providing a biological assay for gene 1.2 protein. No specific enzymatic activity has been found associated with the purified gene 1.2 protein.     

Identification and purification of a calcium-binding protein from Bacillus subtilis

A Ca(2+)-binding protein was identified in Bacillus subtilis in the log phase of growth. The molecular mass of this protein is about 38 kDa as estimated by polyacrylamide gel electrophoresis in the presence of SDS and by gel filtration. The protein was found to be resistant 10 min at 65 degrees C and was purified about 400 times, starting from heated crude extract, by conventional procedures. This novel protein is able to bind Ca2+ in the presence of an excess of MgCl2 and KCl both in solution and after SDS gel electrophoresis and electrotransfer. Since an impairment of the Ca2+ intake, in Bacillus subtilis, results in an impairment of chemotactic behavior (Matsushita, T. et al (1988) FEBS lett. 236, 437-440), 38 kDa protein may be involved in the regulation of chemotaxis.     

Insulin-like growth factor I/somatomedin-C: a rapid isolation procedure with FPLC

Insulin-like growth factor I (IGF I)/somatomedin-C (SM-C) was purified from lyophilized human serum by acid-ethanol extraction. The extract was precipitated with acetone-ethanol. The precipitate was purified by Sephadex G-50 chromatography. The protein peak within a molecular weight range of 5000-10 000 was further purified with FPLC-reversed phase chromatography using a Pep RPC HR 5/5 column (Pharmacia) with a solvent system of acetonitrile (CH3CN) and 0.1% trifluoroacetic acid (TFA) in water. The purification of IGF I was monitored by radioimmunoassay for SM-C. Purity was established by analytical isoelectric focusing and by SDS polyacrylamide gel electrophoresis. Analytical isoelectric focusing showed one single protein band with an apparent pI of 8.3 +/- 0.1. SDS polyacrylamide gel electrophoresis showed also one single protein band with an apparent molecular weight of 7000. Biological activity was demonstrated by measuring the (3H)thymidine incorporation into DNA of cultured arterial smooth muscle cells.