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

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

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

scherichia 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

A DNA-unwinding enzyme induced in bacteriophage-T4-infected Escherichia coli cells

A single-stranded DNA-dependent ATP gamma-phosphohydrolase of Mr 56000 induced after infection of Escherichia coli cells with bacteriophage T4, probably the ATPase dependent on gene dda of the phage, was isolated. Studies on the enzyme show that in the presence of ATP and M2+ ions it is capable of dissociating partially double-stranded fd bacteriophage DNA into the single strands and that some 3000 enzyme copies are required to unwind the 6400-nucleotides-long DNA. Unwinding is inhibited by reducing the length of the single-stranded portion of DNA to two nucleotides. In addition it can be inhibited by sulfhydryl reagents which block the ATPase or by trapping free enzyme molecules in the assay system. The results suggest that unwinding is initiated near the single-stranded portion of the DNA and is driven by the ATPase. It further appears that the enzyme unwinds by adsorbing to the DNA. Affinity of the enzyme for double-standed DNA is not detectable by DNA binding assay

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

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

DNA Unwinding Enzyme II of Escherichia coli 2. Characterization of the DNA Unwinding Activity

The DNA‐stimulated 75000‐Mr ATPase described in the preceding paper is shown to be a further catalytic DNA unwinding principle (DNA unwinding enzyme II) made in Escherichia coli cells (the first being the 180000‐Mr ATPase of the cells: DNA unwinding enzyme I). Unwinding depends, strictly, on the supply of ATP. It occurs only under conditions permitting ATP dephosphorylation and it proceeds as long as enzyme molecules are permitted to enter the enzyme. DNA complex. The enzyme binds specifically to single‐stranded DNA yielding a complex of only limited stability. These results are interpreted in terms of a distributive mode of action of the enzyme. It is argued that chain separation starts near a single‐stranded DNA region and that, forced by continued adsorption of enzyme molecules to the DNA, it develops along the duplex. This mechanism is different from that deduced previously for DNA unwinding enzyme I. Complicated results were obtained using ATPase prepared from rep3 mutant cells

Ein fädiger DNS-Phage und ein sphärischer RNS-Phage III Biologisches Verhalten

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

Enzymic Unwinding of DNA 2. Chain Separation by an ATP‐Dependent DNA Unwinding Enzyme

The DNA‐stimulated ATPase characterized in the accompanying paper is shown to be a DNA unwinding enzyme. Substrates employed were DNA · RNA hybrid duplexes and DNA · DNA partial duplexes prepared by polymerization on fd phage single‐stranded DNA template. The enzyme was found to denature these duplexes in an ATP‐dependent reaction, without delectably degrading. EDTA, an inhibitor of the Mg2+ ‐requiring ATPase, was found to prevent denaturation suggesting that dephosphorylation of the ATP and not only its presence is required. These results together with those from enzyme‐DNA binding studies lead to ideas regarding the mode of enzymic action. It is proposed that the enzyme binds, in an initial step, to a single‐stranded part of the DNA substrate molecule and that from here, energetically supported by ATP dephosphorylation, it invades double‐stranded parts separating base‐paired strands by processive, zipper‐like action. It is further proposed that chain separation results from the combined action of several enzyme molecules and that a tendency of the enzyme to aggregate with itself reflects a tendency of the molecules to cooperate. Various functions are conceivable for the enzyme