Replication of bacteriophage G13 DNA in dna mutants of Escherichia coli.

Host functions required for replication of microvirid phage G13 DNA were investigated in vivo, using thermosensitive dna mutants of Escherichia coli. In dna+ bacteria, conversion of viral single-stranded DNA into double-stranded replicative form (stage I synthesis) was resistant to 150 microgram/ml of chloramphenicol or 200 microgram/ml of rifampicin. Although multiplication of G13 phage was severely inhibited at 42--43 degrees C even in dna+ host, considerable amount of parental replicative form was synthesized at 43 degrees C in dna+, dnaA or dnaE bacteria. In dnaB and dnaG mutants, however, synthesis of parental replicative form was severely inhibited at the restrictive temperature. Interestingly enough, stage I replication of G13 DNA was, unlike that of phiX174, dependent on host dnaC(D) function. Moreover, the stage I synthesis of G13 DNA in dnaZ was thermosensitive in nutrient broth but not in Tris/casamino acids/glucose medium. In contrast with the stage I replication, synthesis of G13 progeny replicative form was remarkably thermosensitive even in dna+ or dnA cells.     

Replication of bacteriophage M13 IX. Requirement of the Escherichia coli dnaG function for M13 duplex DNA replication.

Temperature-shift experiments with an Escherichia coli dnaG strain indicate a requirement for the dnaG function for M13 phage production only at an early stage of infection. Mutant cells infected at nonpermissive temperature form the parental RF (SS leads to RF) but do not replicate further. A shift to nonpermissive temperature after infection inhibits RF leads to RF replication but not RF leads to SS synthesis. The synthesis of both strands of the duplex RF was inhibited equally after a temperature shift during RF leads to RF replication. We infer that the dnaG protein is required for M13 production only during RF replication and that it is required for the synthesis of both strands of the RF.     

Replication of M-13 DNA in plasmolysed Escherichia coli cells. Structure of a replicative intermediate with restricted binding of intercalating dyes.

DNA molecules with restricted binding of intercalating dyes are observed as replicative intermediates during the replication of bacteriophage M-13 duplex DNA in a cellular system in vitro prepared by plasmolysis of M-13-am5-infected Escherichia coli cells. Restriction of dye binding is abolished by heating the DNA to 80 degrees C, but can be recovered by slow cooling of the heat-treated DNA. Radioactive pulse-label incorporated by these molecules is found exclusively in elongated viral strands of more than one genome length. In the electron microscope this DNA fraction is seen to contain a significant number of duplex DNA rings with two single-stranded tails protruding from the same region of the ring. It is proposed that these structures arise by branch migration during the isolation of replicating molecules containing only one single-stranded tail. The topological constraint in these molecules is most likely caused by base-pairing between partially complementary regions of the two single-stranded tails.     

Two-triplet CGA repeats impede DNA replication in bacteriophage M13 in Escherichia coli

Expansion and contraction instabilities associated with CAG, CGG, GAA and CGA (GAC) repeats propagation cause more than a dozen human genetic diseases and cancers. In this work, the propagation behavior of a bacteriophage M13 carrying a calf prochymosin cDNA fragment with a (CGA)2 repeat in a small hairpin forming region is reported. Such a M13 derivative when propagated in Escherichia coli, produces small plaques by decreasing phage yield and also mitigates the inhibition on host cell growth, compared to those control bacteriophages either containing a "CTGCTA" sequence or wildtype, suggesting that CGA2 repeat impedes DNA replication in vivo. Moreover, an increased internal free energy is found associated with (CGA)2 sequence compared to those "CTGCTA" and wildtype, which ruled out a possibility of CGA2 repeat effects on propagation is through influencing the hairpin structure formation.     

Replication of colicinogenic factor E1 DNA in plasmolysed Escherichia coli cells. Coupling of DNA replication and RNA synthesis.

Plasmolysed chloramphenicol-treated Escherichia coli cells carrying the colicinogenic factor E1 utilize deoxynucleoside triphosphates for the semi-conservative synthesis of Col E1 DNA. Col E1 DNA replication in plasmolysed cells can be dissociated into two temporally separated processes: (a) a rifampicin-sensitive RNA synthesis, which is stimulated by adenosine 3':5'-monophosphate (cyclic AMP) and requires all four ribonucleoside triphosphates and (b) an ATP-dependent DNA synthesis, which is inhibited by arabinosylnucleoside triphosphates and sulfhydryl-blocking reagents. Thes two processes exhibit different sensitivities to inhibition by polyamines and actinomycin D.     

Hypervariable DNA fingerprinting in Escherichia coli: minisatellite probe from bacteriophage M13.

Extensive restriction-fragment-length polymorphism was revealed in Escherichia coli strains by using a region of the bacteriophage M13 genome as a DNA hybridization probe. This variation was observed across natural strains, in clinical samples, and to a lesser extent in laboratory strains. The sequence in M13 which revealed this fingerprint pattern was a region of the gene III coat protein, which contains two clusters of a 15-base-pair repeat. Oligonucleotides made to a consensus of these repeats also revealed the fingerprint profile. While this consensus sequence has significant homology to the lambda chi site sequence, an oligonucleotide made of the chi sequence did not reveal polymorphic fingerprint patterns in E. coli. The strain variation revealed by the M13 and M13-derived oligonucleotide probes will be useful for bacterial characterization and should find use in studies of bacterial evolution and population dynamics. The findings raise questions about what these repeated sequences are and why they are so variable.     

Replication of bacteriophage M13. XV. Location of the specific nick in M13 replicative form II accumulated in Escherichia coli polAex1.

M13 replicative form II (RFII) DNA was prepared from Escherichia coli RS5052 (polAex1) cells in the late stage of infection, and the DNA sequence at the discontinuity was examined. The data presented here suggest that the single discontinuity in the late stage of infection RFII maps at the same position as the gene II protein nicking site on fd RFI which was determined in vitro (Meyer et al., Nature (London) 278:365-367, 1979) and has a 5' terminal nucleotide sequence identical to that at the nick produced by gene II protein in vitro. The discontinuity in the in vivo RFII appears to be a single break in the phosphodiester backbone, leaving a 3' OH terminus. RFII molecules containing a gap, i.e., missing nucleotides at the site of discontinuity, were not detected.     

Replication of M13 duplex DNA in soluble extracts of Escherichia coli. Effect of helix-destabilising proteins.

Soluble extracts of M13-am5-infected Escherichia coli cells can carry out multiple rounds of M13 duplex DNA replication when supplemented with helix-destabilising protein of E. coli. Similarly addition of the helix-destabilising M13 gene 5 protein in low concentrations (up to 30 micrograms/ml) stimulates the replication of double-stranded M13 DNA. In contrast, higher concentrations of gene 5 protein (but not of E. coli helix-destabilising protein) cause a preferential inhibition of complementary strand synthesis resulting in a switch from double-strand replication to single-strand synthesis. Depending on the addition of the appropriate amounts of these two helix-destabilising proteins either stage of M13 DNA replication can now be studied with cell-free preparations.     

UNG-dependent correction of molecular heteroduplexes of M13 phage DNA in Escherichia coli cells

Correction of heteroduplex DNA obtained by hybridization of uracil-containing single-stranded M13mp18 phage DNA and "mutant" synthetic oligonucleotide with deletion of cytosine in SalGI site was studied in ung+ and ung- E. coli strains. Uracil-containing DNA was prepared after growth of phage in an E. coli strain dut- ung-. The DNA was hybridized with "mutant" oligonucleotide then complementary DNA chain was synthesized by T4 DNA polymerase. Ung+ and ung- E. coli cells were transformed by DNA. In all experiments mutation frequency in ung+ was higher than in ung- cells (approximately 6-fold) and reached 11-50%. Absolute number of mutants was higher in ung+ cells. The results indicate that high level of mutagenesis depends on uracil repair system polarizing the correction of heteroduplex DNA.     

Replication of bacteriophage phiK duplex replicative-form DNA in dnaB and dnaC mutants of Escherichia coli.

We have directly tested the effects of host cell DNA synthesis mutations on bacteriophage phiK replicative-form (RF) DNA replication in vivo. We observed that phiK RF DNA replication continued at normal rates in both dnaB and dnaC mutant hosts under conditions in which the activities of the dnaB and dnaC gene products were shown to be markedly reduced. This suggests that these two host proteins are not essential for normal phiK RF DNA replication. In control experiments we observed markedly reduced rates of phiK RF DNA replication in temperature-sensitive dnaG and dnaE host mutants, indicating that the products of these genes are essential. Thus, the mechanism of DNA chain initiation in vivo on the duplex RF DNA templates of isometric phages such as phiK apparently is different from that on the similar templates of isometric phages such as phiX174. The implications of this difference are discussed in the text.     

Replication of bacteriophage M13. XII. In vivo cross-linking of a phage-specific DNA binding protein to the single-stranded DNA of bacteriophage M13 by ultraviolet irradiation.

Ultraviolet irradiation of bacteriophage M13-infected Escherichia coli induces the formation of a covalent crosslink between progeny single-stranded DNA and the M13 DNA binding protein, the product of gene 5. The crosslinked complex is readily isolated from detergent-treated lysates by sucrose-gradient velocity sedimentation and CsCl equilibrium sedimentation in the presence of detergent. The crosslinked complex produced with optimal levels of irradiation sediments 1.06 times faster than uncomplexed M13 single-stranded DNA, has a buoyant density of approximately 1.62 to 1.64 g/cm³ and a protein to DNA mass ratio of 2 mg protein per mg DNA. Cleavage of the crosslinked complex with cyanogen bromide or trypsin yields products similar to those produced by cleavage of purified M13 gene 5 protein. The crosslink is located close to the carboxyl terminus of the protein.     

Replication of bacteriophage M13. XIV. Differential inhibition of the replication of M13 and M13 miniphage in a mutant of Escherichia coli defective in the 5'' leads to 3'' exonuclease associated with DNA polymerase I.

Previous studies have shown that M13 single-strand synthesis is inhibited at nonpermissive temperature in Escherichia coli polAexl, a temperature-sensitive mutant defective in the 5' leads to 3' exonuclease activity of polymerase I (T.-C. Chen and D. S. Ray, J. Mol. Biol. 106:589-604, 1976). Under these conditions the formation of covalently closed replicative form (RF) molecules is greatly reduced, and miniature forms of RF accumulate. We show here that the accumulation of mini-RFs is the consequence of a differential inhibition of the replication of unit-length phage and preexisting miniphage rather than a de novo production of miniphage. Mini-RFs do not accumulate even after as many as nine cycles of growth in the mutant host infected only with unit-length phage. Mixed infections of the mutant host with plaque-purified unit-length phage and a single cloned miniphage show that discontinuities in the mini-RFs are joined with higher efficiency than are those contained in unit-length RFs. After a shift to nonpermissive temperature during single-strand synthesis in cells infected with plaque-purified phage alone, M13 RFs are found largely as RFII molecules (RF form having one or more single-strand discontinuities) containing only a single discontinuity in the viral strand. The inability of the accumulated unit-length RFII molecules to actively replicate may reflect the presence of either a bound protein or RNA primer on the 5' terminus of the viral strand and provides further support for the existence of distinct initiation and termination events in the synthesis of the viral strand.