Hildegard Dürwald

Endonuclease I-deficient and ribonuclease I-deficient Escherichia coli mutants

Abstract A method has been developed to select for Escherichia coli mutants which lack certain nucleases. Using this method, two classes of mutants have been isolated: mutants deficient in endonuclease I, and mutants deficient in RNase I. Extracts from endonuclease I mutants had 0.1 to 0.3% of the RNA-sensitive wild-type endonuclease and, using single-stranded DNA, no RNA-resistant endonuclease. The location of the endonuclease I mutation on the bacterial genetic map was shown to be near st on the side opposite to mtl . Extracts from RNase I mutants had 0.1% of the magnesium-independent wild-type RNase or lower activity. Mutants deficient in endonuclease I or RNase I were essentially identical to wild-type with respect to their general biological properties.

Enzymatic unwinding of DNA. III. Mode of action of Escherichia coli DNA unwinding enzyme.

The ATP-dependent 180,000 molecular weight DNA unwinding enzyme of Escherichia coli has been subjected to further study. The results support the following conclusions. The enzyme, the largest soluble peptide in E. coli , is a fibrous protein. The typical large aggregates which the enzyme forms are ordered structures. The enzyme accepts double-stranded DNA as a substrate only when the duplex DNA is covalently linked to single-stranded DNA. It initiates on such DNA by binding to the single-stranded region. Denaturation, an effect induced by ATP, then follows in this pre-formed enzyme-DNA complex. Denaturation requires dephosphorylation of the ATP cofactor; the mere presence of a recognized nucleoside triphosphate does not induce the enzyme to unwind. Some 80 enzyme molecules are required to unwind the double-stranded section of an fd bacteriophage DNA molecule, the typical substrate. The number of enzyme molecules required is not reduced when a non-enzymatic, single-stranded DNA binding protein is added to facilitate chain separation. There is sufficient enzyme in an E. coli cell to satisfy the needs of unwinding several duplexes. These results are interpreted in terms of the previously proposed scheme of processive enzymatic action: an ATP dephosphorylating enzyme proceeds “actively” along a bound DNA chain, thereby displacing a base-paired complementary chain. The unwinding agent is probably a multimeric form of the enzyme protein. The implications of this concept are discussed with respect to the mechanism that causes myosin to proceed along an actin filament.

DNA synthesis in nucleotide-permeable Escherichia coli cells: II. Synthesis of replicative form DNA of phage φX174

Escherichia coli cells were treated with mitomycin C to suppress host DNA synthesis, infected for five minutes with bacteriophage φX174, made permeable to nucleotides by treatment with ether and allowed to synthesize DNA from exogenous deoxynucleoside triphosphates. The product was 70 to 80% replicative form (RF) DNA of the phage according to sedimentation and annealing properties. RF synthesis occurred at the normal level in DNA polymerase-deficient cells and did not occur in these cells when they were infected with a gene VI amber mutant of the virus, which in the living cells does not replicate beyond the first RF. The structure of intermediates of RF synthesis in ether-treated cells is thought to be basically consistent with the rolling circle model of RF replication proposed by Knippers, Whalley & Sinsheimer (1969). In addition, the results suggest that a temporary interruption exists in the complementary strand just ahead of the growth point. Complementary strand material, and possibly also viral strand material, exists early after synthesis as short pieces having only a small fraction of the viral length. These pieces are subsequently joined together but can be induced to accumulate. This accumulation is suppressed by 0.2 mm-dATP but not detectably by any of the other deoxynucleoside triphosphates in high concentration, or by ATP or DNA ligase cofactor. Label in RF can be chased up to 80% into supertwisted molecules.

DNA synthesis in nucleotide-permeable Escherichia coli cells: VII. Conversion of φX174 DNA to its replicative form

Abstract On incubation with deoxynucleoside triphosphates and rATP, ether-treated (nucleotide-permeable) cells convert the single-stranded DNA of adsorbed bacteriophage φX174 particles to the double-stranded replicative forms. The main final product is the doubly-closed replicative form, RFI; a minor product is the relaxed form II. Interruptions in the nascent complementary strand of the viral DNA result in pieces corresponding to 5 to 10% of the unit length of the viral DNA. Pieces of similar size were previously seen in studies of the replication synthesis of Escherichia, coli DNA in ether-treated cells. Since the conversion of the single-stranded φX174 DNA to replicative form is known to be mediated entirely by host factors, it is argued that the viral single strands are replicated by macromolecular factors involed in the replication of E. coli DNA and that this is the reason why new φX174 DNA appears in short pieces. Possible consequences of this interpretation for an understanding of duplex replication are discussed. The joining of the short pieces of complementary φX174 DNA is inhibited at low deoxynucleoside triphosphate concentration (1 μM) but not by nicotinamide mononucleotide, which inhibits the NAD-dependent DNA ligase and blocks the conversion of RFII to RFI in ether-treated cells. The results are discussed with respect to previous studies on cell-DNA synthesis (Geider, 1972). It is argued that there are two polynucleotide joining mechanisms, of which only one requires NAD-dependent ligase action.

Ein fädiger DNS-Phage (fd) und ein sphärischer RNS-Phage (fr), wirtsspezifisch für männliche Stämme von E. coli

fd and fr adsorb to male strains of E. coli and infect female cells, after the fd-DNA (or the fr-RNA 15) have been deproteinized by phenol and after the cells have been converted to the form of spheroplasts. fd is very heat resistant, highly antigenic and poorly adsorbing. The latency period of intracellular multiplication is 10 min (in Tryptone broth at 37 °C). The most unusual property seems to be that fd is the only phage on record which is liberated by the host cell without destruction of the host cell. The evidence for this is threefold: 1. in infected cultures phage is liberated at the rate of about 300 phage particles per bacterium per cell generation, and the growth rate of these cultures is indistinguishable from that of controls. 2. In such cultures no significant amounts of bacterial enzyme are liberated. 3. In single burst experiments it appears that more than 60% of the individual cells have liberated around 450 phage particles after about 20 min. and later produce bacterial growth as shown by turbidity. Non-lytic infection gives rise to an unstable carrier state. The phage is lost from the cells if superinfection is prevented by the addition of fd-antiserum. Lytic mutants of fd have been recorded. fr in its properties is similar to f2 and the other RNA phages and seems to be liberated by bacteriolysis, fr, due to the chemical nature of its nucleobases, is highly sensitive to hydroxylamine in slightly alkaline solution. Infection with fr in rare cases is non-lytic and leads to an unstable carrier state.

Replication of the Single-Stranded DNA of Bacteriophage ϕX174 in Nucleotide-Permeable Cells

The replication of φX174 is initiated by conversion of single-stranded ring DNA (SS) into a double-stranded replicative form (RF) DNA molecule. The fact that this conversion of SS to RF is insensitive to high concentration of chloramphenicol suggests involvement of pre-existing host enzyme(s) in this step. The product of a viral gene is required only for the next stage in which the RF replicates to yield further RF (Sinsheimer, 1968).