Discontinuous replication of colicin E1 plasmid deoxyribonucleic acid.

The plasmid pML 21, which was found to contain approximately 49% of the Col E1 genome was used to determine the template origin of single-stranded deoxyribonucleic acid (DNA) fragments (4 to 32% of the Col E1 units length) associated with Col E1 dna replicative intermediates. The results of DNA hybridization competition experiments indicate that the single-stranded fragments derive from the full length of the Col E1 DNA template as expected for Okazaki fragments and the plasmid pML 21 contains the replication origin of Col E1 DNA.     

Ribonucleotide-deoxyribonucleotide linkages at the origin of DNA replication of colicin E1 plasmid

We have shown by nearest-neighbor analysis that ribonucleotide-DNA linkages exist on 6 S L-strand² fragments, the first DNA fragments made on Colicin E1 plasmid, at the origin of replication. The linkages rApdA and rApdC predominate. This is in agreement with the previous finding that located the 5′ end of the 6 S L-fragments on the plasmid DNA sequence (Tomizawa et al., 1977). Most of the molecules that contain ribonucleotides have a single rA residue and a large fraction of this residue is phosphorylated at the 5′ end. A total of 20 to 40% of the 6 S L-fragments contain at least one ribonucleotide.     

Fine cleavage map of a small colicin E1 plasmid carrying genes responsible for replication and colicin E1 immunity.

A small plasmid (pAO2, 1 megadalton) carrying genes responsible for replication and colicin E1 immunity has been constructed from colicin E1 plasmid (A. Oka, K. Sugimoto, and M. Takanami, Proc. Mol. Biol. Jpn., p. 113-115, 1976). pAO2 DNA was cleaved into unique fragments with seven restriction endonucleases (R.HaeII,R.HaeIII,R.HapII,R.HhaI,R.AluI,R.HgaI, and R.HinfI). R.HaeII cleaved pAO2 DNA at two sites, R.HaeIII at four sites, R.HapII at nine sites, R.HhaI at eight sites, R-AluI at nine sites, R.HgaI at two sites, and R.HinfI at four sites, respectively. The order of HaeIII fragments of pAO2 was deduced from the physical map of colicin E1 plasmid previously reported (A. Oka and M. Takanami, Nature (London) 264:193-196, 1976). HapII, HhaI, and AluI fragments of pAO2 were assigned by analyzing overlapping sets of fragments arising upon digestion of individual HaeIII fragments with one of R.HapII, R.HhaI, or R.AluI, and upon their reciprocal digestion. The cleavage sites for R.HaeII, R.HgaI, and R.HinfI were localized on HapII, HhaI, and AluI fragments by combined digestion. On the basis of these data and estimates of the size of each fragment, a fine cleavage map of pAO2 was constructed.     

Replication of colicin E1 plasmid DNA in vivo requires no plasmid-encoded proteins.

A derivative of bacteriophage lambda containing a colicin E1 plasmid replicon was constructed by recombinant DNA techniques. This phage, lambdacol100, has two functional modes of DNA replication; it can replicate via either plasmid or phage replication systems. lambdacol100 has been used to introduce the colicin E1 plasmid replicon into Escherichia coli previously treated with chloramphenicol to block protein synthesis. Under these conditions, lambdacol100 DNA is replicated normally as a colicin E1 plasmid. This suggests that colicin E1 plasmid replication in vivo does not require any plasmid-encoded proteins.     

Mapping of colicin E2 and colicin E3 plasmid deoxyribonucleic acid EcoR-1-sensitive sites.

Colicin plasmids E2 and E3 (Col E2 and Col E3) deoxyribonucleic acid (DNA) has been shown to contain, respectively, two and three EcoR1 restriction endonuclease-sensitive sites. This was determined by measuring the DNA fragments generated after EcoR1 endonuclease treatment by agarose gel electrophoresis and electron microscopy. The structure of heteroduplex Col E2-col E3 DNA molecules formed from EcoR1-generated fragments permitted a localization of the EcoR1-sensitive sites on the plasmid chromosomes.     

Defective DNA replication and repair associated with decreases in deoxyribonucleotide pools in a mouse cell mutant with thermolabile ubiquitin-activating enzyme E1.

Upon shift-up in temperature, mouse tsFS20 mutant cells with thermolabile ubiquitin-activating enzyme E1 immediately stopped DNA replication and showed cell cycle arrest in S-phase. In contrast, when the cells were permeabilized with lysolecithin after culture at the nonpermissive temperature, they exhibited a normal level of replicative DNA synthesis in vitro. In agreement with this, intracellular pools of deoxyribonucleoside triphosphates were significantly reduced in the cells cultured at the nonpermissive temperature. Even under the permissive conditions, tsFS20 cells were more sensitive to hydroxyurea and alkylating agents, and induced less mutation than the wild-type cells. These results suggest that the ubiquitin system affects DNA replication and repair.     

Strand specificity of mutagenic bypass replication of DNA containing psoralen monoadducts in a human cell extract.

Psoralens are mutagenic compounds of vegetable origin that are used as photosensitizing agents in the treatment of various skin diseases, blood cell cancer, and autoimmune disorders. To study the mechanism of mutagenicity of psoralens in humans, we examined the efficiency and fidelity of simian virus 40 origin-dependent replication in a human cell extract of M13mp2 DNA randomly treated with the psoralen derivative 4'-hydroxymethyl-4,5',8-trimethyl psoralen plus UVA irradiation. Replication of DNA treated with variable amounts of 4'-hydroxymethyl-4,5',8-trimethyl psoralen and a fixed UVA fluence was inhibited in a concentration-dependent manner. However, covalently closed monomer-length circular replication products were observed. Product analysis by renaturing agarose gel electrophoresis after cross-linking with 250- to 280-nm UV light indicated that approximately 1 of 9 psoralen monoadducts was bypassed during in vitro replication. Introduction of product DNA into Escherichia coli to score replication errors in the lacZalpha reporter gene demonstrated that replication of the damaged DNA was more mutagenic than was replication of undamaged DNA. Sequence analysis of lacZ mutants revealed that damage-dependent replication errors were predominantly T.A-->C.G transitions, transversions at C.G base pairs, and deletions of single A.T base pairs, the last occurring most frequently in homopolymeric runs. A comparison of error specificities with two substrates having the replication origin asymmetrically placed on opposite sides of the mutational target suggests that the lagging-strand replication apparatus is less accurate than the leading-strand replication apparatus for psoralen monoadduct-dependent deletion errors. A model is proposed based on the preferential loopout of the monoadducted base from the strand that templates retrograde discontinuous synthesis.     

Studies of colicin induction with an imm- col+ mutant of the plasmid colicin E1.

Summary A mutant of a derivative of the colicin E1 plasmid has been isolated that does not confer immunity to colicin E1 on its host (imm^-) although it is still capable of producing colicin (col⁺). Cells carrying the col⁺, imm^- plasmid are capable of forming colonies and grow best in liquid culture in the presence of trypsin. The induction of colicin synthesis by ultraviolet light has been analysed using this mutant plasmid. The results suggest that a) the expression of the col⁺ gene may be delayed for many generations after the inducing stimulus, b) although induced cells are usually killed they can reproduce and c) the capacity to produce colicin can be propagated and segregated into the progeny of an induced cell.     

P1 plasmid replication requires methylated DNA.

Plasmids driven by the plasmid replication origin of bacteriophage P1 cannot be established in Escherichia coli strains that are defective for the DNA adenine methylase (dam). Using a composite plasmid that has two origins, we show that the P1 origin cannot function even in a plasmid that is already established in a dam strain. An in vitro replication system for the P1 origin was developed that uses as a substrate M13 replicative-form DNA containing the minimal P1 origin. The reaction mixture contains a crude extract of E. coli and purified P1 RepA protein. In addition to being RepA dependent, synthesis was shown to be dependent on methylation of the dam methylase-sensitive sites of the substrate DNA. As the P1 origin contains five such sites in a small region known to be critical for origin function, it can be concluded that methylation of these sites is a requirement for initiation. This suggests that the postreplicational methylation of the origin may control reinitiation and contribute to the accuracy of the highly stringent copy-number control of the origin in vivo.     

Isolation and characterization of a mutant ColE1 plasmid that allows constitutive colicin E1 synthesis.

It has been possible to isolate a ColE1 mutant which synthesizes colicin E1 constitutively. This result shows that there must be a gene(s) responsible for the regulation of colicin E1 synthesis.     

In vitro replication of a DNA fragment containing the vicinity of the origin of E. coli DNA replication.

Summary The restriction nuclease cleavage pattern of E. coli DNA synthesized in vitro in the cellophane membrane system (Schaller et al., 1972) is similar to the one obtained after labelling E. coli in vivo. This is shown for exponentially growing cells and for cells synchronized by amino acid starvation followed by thymine starvation. In synchronized cells a piece of some 180 kilobase pairs is labelled containing oriC and neighbouring regions at 82 min on the genetic map of E. coli. A pulse label in vitro is incorporated into the same piece of DNA, but the center of this region, i.e. the EcoR1 fragment of 8.6 kbp length which contains the oriC region (Marsh and Worcel, 1977; v. Meyenburg et al., 1977; Yasuda and Hirota, 1977) is missing.     

Mechanism of export of colicin E1 and colicin E3.

The mechanism of export of colicins E1 and E3 was examined. Neither colicin E1, colicin E3, Nor colicin E3 immunity protein appears to be synthesized as a precursor protein with an amino-terminal extension. Instead, the colicins, as well as the colicin E3 immunity protein, appear to leave the cells where they are made, long after their synthesis, by a nonspecific mechanism which results in increased permeability of the producing cells. Induction of ColE3-containing cells with mitomycin C leads to actual lysis of those cells, as some time after synthesis of the colicin E3 and its immunity protein has been completed. Induction of ColE1-containing cells results in increased permeability of the cells, but not in actual lysis, and most of the colicin E1 produced never leaves the producing cells. Intracellular proteins such as elongation factor G can be found outside of colicinogenic cells after mitomycin C induction, along with the colicin. Until substantial increases in permeability occur, most of the colicin remains cell associated, in the soluble cytosol, rather than in a membrane-associated form.     

Studies of colicin E1 plasmid functions by analysis of deletions and TnA insertions of the plasmid.

The further identification of regions of the colicin E1 plasmid that affect plasmid functions has been achieved by studying deletions and TnA insertions of the plasmid. Colicin production, colicin immunity, relaxation of plasmid deoxyribonucleic acid, and plasmid incompatibility functions have been examined. A strong correlation has been observed between the ability of colicin E1 plasmid deoxyribonucleic acid to be relaxed and the ability of that plasmid to be transferred by conjugation.     

Accumulation of single strand interruptions within the yeast 2 microns DNA plasmid during replication in a DNA ligase mutant

Summary We have investigated the fate of the yeast 2 m DNA plasmid in strains with a temperature sensitive mutation of DNA ligase. At the restrictive temperature the plasmid DNA collects as an open circular form with single strand interruptions. Both alpha factor pheromone, which arrests cells before the start of S phase, and hydroxyurea, which blocks progression through S phase, prevent the appearance of the open circular form. Thus, interrupted plasmid DNA does not accumulate in the absence of DNA replication. On average the interrupted molecules contain four to five interruptions per newly replicated strand. Most of the interruptions are nicks (breaks in a single phosphate ester bond) rather than gaps (absence of one or more nucleotides in a strand) as judged by the in vitro conversion of the interrupted molecules into a covalently closed form by DNA ligase. Mapping of the position of the interruptions reveals no predominate sites.