Single-stranded DNA Synthesis Research Articles

Effect of Rifampicin and Chloramphenicol on Progeny Single-stranded DNA Synthesis of BacteriophageϕX174

Neither bacteriophage ϕX174 single-stranded DNA synthesis nor phage growth was affected by rifampicin (200 μg/ml) once it started, whereas a low concentration of chloramphenicol (30 μg/ml) inhibited the phage growth when added in a late phase of infection. When rifampicin was added at a stage where double-stranded duplex (RF) DNA replication proceeded preferentially in the presence of chloramphenicol, or even after chloramphenicol was removed before the addition of rifampicin, both single-stranded DNA synthesis and phage growth were inhibited. These results suggest that RNA synthesis sensitive to rifampicin was necessary to initiate single-stranded DNA synthesis, but no longer needed once ϕX174 DNA synthesis started.

Functions of gene C and gene D products of bacteriophage phi X 174.

Phage-related materials existing in cells infected with various mutants of bacteriophage phi chi 174 were investigated. A novel species of replicative-form (RF) DNA was found in cells infected with a phage mutant of gene B, C, D, F, or G. This species, called RFI, sedimented at a position between RFI and RFII in a neutral sucrose gradient. It was converted to RFI upon denaturation in alkali, denaturation in formamide and subsequent renaturation, or RNase treatment at low ionic strength. In cells infected with a phage mutant of gene C, RFI was derived from pulse-labeled RFII after a short chase. TLLS INFECTED WITH A MUTANT OF GENE B, D, or F. A possible function of the C gene product of phi chi 174 could be to prevent the conversion of RFII to RFI, thereby maintaining the availability of RFII to act as the template for single-stranded viral DNA synthesis. A protein complex containing no DNA, which sedimented with an S value of 108 in a sucrose gradient and contained virion proteins F, G, and H, and nonvirion protein D, was found in cells infected with the gene C mutant. A possible function of protein D was considered as a scaffolding protein for assembly of phage structural proteins.

Gene A protein of bacteriophage S13 is required for singel-stranded DNA synthesis.

The product of gene A of the small icosahedral DNA phage S13 has been found to be needed for single-stranded DNA synthesis in vivo in addition to its previously known role in progeny replicative-form DNA synthesis.

Replication process of the parvovirus H-1. VII. Electron microscopy of replicative-form DNA synthesis.

The geometry of replicative form (RF) DNA synthesis of the H-1 parvovirus was studied with the electron microscope using formamide or aqueous variations of the Kleinschmidt spreading procedure. H-1 DNA was isolated from human or hamster cells infected with a temperature-sensitive mutant, ts1, which is deficient in progeny single-stranded DNA synthesis at the restrictive temperature (S.L. Rhode, 1976), thus minimizing possible confusion between RF and progeny DNA replicative intermediates (RIs). The purity of the isolated H-1 DNA, as determined by gel electrophoresis, ethidium bromide staining, autoadiography, and digestion with endo R-EcoRI, was high. H-1 RF DNA'S WERE LINEAR DOUBLE-STRANDED MOLECULES, 1.53 MUM IN LENGTH. H-1 RIs of RF DNA replication were double-stranded, Y-shaped molecules, with the same length as RF DNAs. The replication origin was localized no more than 0.15 genome lengths from one end of the RF DNA, with replication proceeding toward the other end at a uniform rate. Similar RF and RI molecules of dimer size were also observed. The length of H-1 single-stranded DNA extracted from purified virions was measured relative to that of phiX174 and it had a very similar contour length, so that the molecular weight of H-1 single-stranded DNA would be at least 1.48 X 10(6) to 1.59 X 10(6) (Berkowitz and Day, 1974).

Effect of rifampicin and chloramphenicol on progeny single-stranded DNA synthesis of bacteriophage .PHI.X174.

Neither bacteriophage øX174 single-stranded DNA synthesis nor phage growth was affected by rifampicin (200 μg/ml) once it started, whereas a low concentration of chloramphenicol (30 μg/ml) inhibited the phage growth when added in a late phase of infection. When rifampicin was added at a stage where double-stranded duplex (RF) DNA replication proceeded preferentially in the presence of chloramphenicol, or even after chloramphenicol was removed before the addition of rifampicin, both single-stranded DNA synthesis and phage growth were inhibited. These results suggest that RNA synthesis sensitive to rifampicin was necessary to initiate single-stranded DNA synthesis, but no longer needed once ø X 174 DNA synthesis started.

Bacterial rep- mutations that block development of small DNA bacteriophages late in infection.

Several related mutants of Escherichia coli C have been isolated that block the growth of the small icosahedral DNA phages phiX174 and S13 late in infection. Phage G6 is also blocked, at a stage not yet known. Growth of the filamentous phage M13, though not blocked, is affected in these strains. These host mutations co-transduce with ilv at high frequency, as do rep- mutations. However, the new mutants, designated groL-, differ from previously studied rep- mutants in that they permit synthesis of progeny replicative-form DNA. The groL- mutants are blocked in synthesis of stable single-stranded DNA of phiX174 and related phages. They are gro+ for P2. Evidence that groL- mutations and rep- mutations are in the same gene is presented. Spontaneous mutants (ogr) of phiX174, S13, and the G phages can grow on groL- strains. The ogr mutations are located in the phage's major capsid gene, F, as determined by complementation tests. There are numerous sites for mutation to ogr. Some mutations in genes A and F interfere with the ogr property when combined with an ogr mutation on the same genome. The ogr mutations are cis acting in a groL- cell; i.e., an ogr mutant gives very poor rescue of a non-ogr mutant. The wild-type form of each G phage appears to be naturally in the ogr mutant state for one or more groL- strains. It is suggested that a complex between F and rep proteins is involved in phage maturation. The A protein appears to interact with this complex.

Replication of bacteriophage M13: X. M13 replication in a mutant of Escherichia coli defective in the 5′ → 3′ exonuclease associated with DNA polymerase I

The 5′ → 3′ exonuclease associated with DNA polymerase I is required for the growth of bacteriophage M13. Both phage production and the synthesis of progeny single-stranded DNA are temperature-sensitive in Escherichia coli polAex1, a mutant temperature-sensitive in the 5′ → 3′ exonuclease associated with DNA polymerase I. During replicative form to single-stranded DNA synthesis, this strain is defective in making M13 supercoiled DNA and accumulates two classes of abnormal replicative forms; circular replicative form molecules ranging in size from 0.2 to 0.5 times unit-length and structures that sediment at the rate of unit-length replicative form DNA in neutral sucrose gradients but which are partially single-stranded and contain viral strand fragments ranging in size from 0.1 to 0.7 times unit-length.

Gene A product of phi X174 is required for site-specific endonucleolytic cleavage during single-stranded DNA synthesis in vivo.

A functional gene A product of phi X174 was found to be required at the stage of single-stranded DNA synthesis. A precursor complex that synthesizes single-stranded DNA (50S complex [Fujisawa and Hayashi, 1976]) was isolated from cells infected with wild-type or with temperature-sensitive gene A mutant phage. Proper cleavage of the single-stranded viral DNA did not occur in cells infected with the temperature-sensitive gene A mutant under restrictive conditions. This resulted in (i) accumulation of linear viral DNA molecules of 2 units in length in the 50S complex and (ii) cessation of elongation of viral-strand DNA after one complete cycle of single-stranded DNA synthesis.

The role of Escherichia coli dnaG function in coliphage M13 DNA synthesis.

Examination of the role of Escherichia coli dnaG function in different stages of M13 phage DNA synthesis by ultracentrifugal analysis of intracellular phage DNA in a thermosensitive dnaG mutant shows that: (a) the formation of parental double-strand replicative-form DNA (rfDNA) from the infecting virus is independent of dnaG function; (b) the synthesis of progeny rfDNA requires dnaG product; (c) after a pool of rfDNA is made up, dnaG function is not required for the progeny single-strand DNA (ssDNA) synthesis. The ssDNAs produced under nonpermissive condition are mostly circular and biologically functional.

Propidium diiodide-cesium chloride density gradient centrifugation for buoyant separations of duplex DNA molecules containing single-stranded regions.

At increasing dye concentrations in propidium diiodide-CsCl density gradients, the relative buoyant density shift was largest for open-circular duplex phiX174 DNA (RFII), next largest for single-stranded viral DNA, and least for closed-circular duplex DNA (RFI). These differential relative buoyant density shifts permitted discrete separations of these phiX DNA forms. Further, in propidium diiodide-CsCl density gradients, the distinctive density of single-stranded DNA permitted separations of rolling-circle intermediates with a single-stranded tail that occur during single-stranded phiX DNA synthesis. It is suggested that single-strandedness in a duplex DNA structure influences its buoyand density shift due to differential dye binding to the single-stranded region and is an additional physical basis for relative buoyant separations of DNA molecules in propidium diiodide-CsCl density gradients.

The role of Escherichia coli dna A gene and its integrative suppression in M13 coliphage DNA synthesis.

An F+ derivative of Escherichia coli E508 thermosensitive in dna A function (involved in DNA synthesis initiation), its revertant and an Hfr derivative of E508 (ts) in which the temperature-sensitive phenotype is suppressed by integrative suppression have been compared for their ability to support M13 phage DNA synthesis at the nonpermissive temperature. Upon infection at the nonpermissive temperature, both the revertant and the Hfr strain support normal phage replication while the temperature-sensitive mutant does not. However, when infection is carried out at a permissive temperature and the temperature is shifted up after infection, phage synthesis occurs in the temperature-sensitive mutant also, but in lesser quantity than in the revertant strain. Analysis of intracellular labeled phage DNA indicates: (a) parental replicative form DNA synthesis is not dependent on dna A function; (b) progeny replicative form DNA synthesis is strongly inhibited in the temperature-sensitive dna A mutant at the nonpermissive temperature; (c) progeny single-strand DNA synthesis does not absolutely require dna A function; (d) progeny single-strand DNA is present in the circular form. The implication of the host DNA replication in M13 DNA synthesis is discussed.

Bacteriophage phiX174 single-stranded viral DNA synthesis in temperature-sensitive dnaB and dna C mutants of Escherichia coli.

We asked if phiX174 single-stranded DNA synthesis could reinitiate at the nonpermissive temperature in dnaB and dnaC temperature-sensitive host mutants. The rates of single-stranded DNA synthesis were measured after the removal of chlorampheicol that had been added at various times after infection to specifically stop this stage of phiX174 DNA synthesis. Reinitiation was not defective in either mutant host. Our data suggested that the reinitiation of the single-stranded stage of phiX174 DNA synthesis in these experiments was analogous to the normal initiation of this stage of phiX174 DNA synthesis in infections without chloramphenicol. Assuming this to be the case, we conclude that the host cell dnaB and dnaC proteins are not essential for the normal initiation of the single-stranded synthesis stage of phiX174 DNA synthesis. In related experiments we observed that in the dnaC mutant host at the permissive temperature, phiX174 replicative form DNA synthesis continued at its initial rate even during the single-stranded DNA synthesis stage. This indicates that these two stages of phiX174 DNA synthesis are not necessarily mutually exclusive.

Process of infection with bacteriophage phi chi 174. XL. Viral DNA replication of phi chi 174 mutants blocked in progeny single-stranded DNA synthesis.

Mutation in several different cistrons of bacteriophage phi chi 174 blocks net progeny single-stranded DNA synthesis at the late period of infection (15). For the study of the functions of these cistrons in single-stranded DNA synthesis, asymmetric replication of replicative form DNA was examined at the late period of infection with amber mutants of these cistrons. While the normal, rapid process of asymmetric single-stranded viral DNA synthesis is blocked at the late period of these mutant infections, an asymmetric synthesis of the viral strand of replicative-form DNA is observed in this period, though at a reduced level, together with degradation of prelabeled viral strand. Some intermediate replicative-form molecules were also detected. Asymmetric synthesis of the viral strand of replicative-form DNA at the late period of phi chi infection is completely inhibited in the presence of a low concentration (35mug/ml) of chloramphenicol (which also blocks net single-stranded viral DNA synthesis). These results are discussed in terms of the possible role of the specific viral proteins for normal single-stranded DNA synthesis.

The process of infection with bacteriophage φX174: XXXIX. The structure of a DNA form with restricted binding of intercalating dyes observed during synthesis of φX single-stranded DNA

A DNA form with restricted binding of intercalating dyes (propidium iodide or ethidium bromide) has been found in bacteriophage φX-infected cells during the period of single-stranded DNA synthesis. In the electron microscope, this DNA form is seen to be a double-stranded DNA ring with two single-stranded DNA tails protruding from the same portion of the ring; it is composed of a linear φX DNA strand, longer than one φX genome, and a single-stranded ring complementary to φX DNA. Base-pairing of these two tails in partially complementary regions restricts unwinding of the double-stranded DNA ring and consequently intercalation and binding of the dyes. It is postulated that these molecules originate from a previously reported precursor of φX DNA, namely a double-stranded ring with a single-stranded tail, by branch migration.

Purification and properties of bacteriophage phi X 174 gene D product.

Bacteriophage phi X 174 gene D product, a protein required for single-stranded DNA synthesis by the phage, has been purified to near homogeneity. The protein is very abundant; approximately 10(5) monomers are present per infected cell when lysis is delayed. The protein has a monomer molecular weight of 15,200 and is normally a tetramer; however, it can form very large aggregates at high concentrations. Amino acid analysis shows an excess of arginine over lysine and a relatively high number of nonpolar residues. The protein carries a net negative charge at neutral pH. The first eight amino acids of the protein sequence have been determined; they are Ser-Gln-Val-Thr-Glu-Gln-Arg-Val. The carboxy-terminal residue is methionine. The protein has not yet been shown to bind directly to any single-stranded DNA; it does not adsorb to denatured calf thymus DNA-cellulose.

The mechanism of replication of øX174 DNA: XIII. Discontinuous synthesis of the complementary strand in an escherichia coli host with a temperature-sensitive polynucleotide ligase

Escherichia coli KS252, isolated by Konrad et al. (1973), carries a thermosensitive ligase by virtue of the lig ts7 mutation and is capable of supporting limited growth of bacteriophage oX174 at the restrictive temperature. Analysis of intermediates in oX DNA replication synthesized at 41°C yielded the following results. o (a) During the synthesis of the parental replicative form (RF) most of the [3H]thymidine incorporated into DNA after a short pulse appeared in 5 S to 9 S fragments as analysed by velocity sedimentation in alkaline sucrose. In vivo these fragments appeared to be precursors of full-length DNA; in vitro, as part of a replicative form molecule, they could be joined together by polynucleotide ligase. (b) The [3H]thymidine incorporated during a 30-s labelling period during RF replication was found in polydeoxynucleotide strands ranging from 5 S to 9 S to longer than unit-length. Competition annealing experiments revealed that the unit-length and longer oX174 DNA comprised mostly viral strand sequences while the 5 S to 9 S fragments were composed exclusively of complementary strand sequences. Although some of the viral strand DNA labelled by a 5-s pulse was longer than unit-length, most of it was unit-length. In replicating intermediates, viral strand sequences were found in part to be single-stranded, suggesting that synthesis of the viral strand preceded synthesis of the complementary strand. (c) During single-stranded DNA synthesis, no 5 S to 9 S DNA was found and much of the pulse-labelled DNA was longer than unit-length. We conclude that synthesis of the complementary strand, but not of the viral strand, of oX174 is obligatorily discontinuous and that the discontinuities found in the nascent DNA in the lig ts7 mutant are repaired by the E. coli NAD-dependent polynucleotide ligase.

Development of bacteriophage Ha 2, a ФX174 derivative, in Escherichia coli strains carrying a thermosensitive mutation in the dnaG gene

Ha2 is a derivative of HaHb, a phiX174 mutant able to grow on several Escherichia coli K 12 strains which are insensitive to phiX174 wild type. Ha2 was isolated after nitrous acid mutagenesis and selected as being able to give large plaques in equal number at 42 degrees C as well as 30 degrees C when plated on Escherichia coli C and CR34. Hosts carrying a thermosensitive mutation at the dnaG locus are unable to support the replication and maturation of Ha2 at the restrictive temperature. The first steps of Ha2 development: attachment, eclipse and penetration, are not affected by the dnaG mutation. The viral DNA penetrates the cell as single-strands, which are probably attached to a viral protein component. The following steps of viral replication depend on the presence of an active dnaG gene product: no formation of parental RF; no synthesis and replication of progeny RF were observed in the mutant strains at 42 degrees C. The synthesis of viral single-stranded DNA as well as phage formation are lowered after a shift to the restrictive conditions, suggesting that the dnaG gene product is also involved at these stages.

Segregation into the replication of bacteriophage M 13 DNA in minicells of Escherichia coli.

Minicells derived from E. coli x796(F+) are refractory to infection by phage M 13. However, after infection of the minicell-producing strain with M 13, phage DNA is found to segregate efficiently into newly formed minicells. The M 13 specific DNA present in minicells isolated several hours after infection consists of single stranded viral DNA and double stranded replicative forms in nearly equal amounts. M 13 DNA containing minicells are capable of carrying out at least one complete round of single stranded DNA synthesis as shown by the flow of label from replicative forms to free single strands.

Faulty DNA repair following ultraviolet irradiation in Fanconi's anemia.

Fibroblasts from a patient with Fanconi's anemia were deficient in their ability to excise UV-induced pyrimidine dimers from their DNA but were capable of single-strand break production and unscheduled DNA synthesis.

Studies on the structure of replicative intermediates in bacteriophage M13 single stranded DNA synthesis.

Pulse-labeled replicative intermediates in M 13 single stranded DNA synthesis can be separated by dye-buoyant density centrifugation into two major fractions: Supercoiled molecules (RI I) containing viral strands of more than one genome length, and "relaxed" molecules (RI II) with labeled DNA chains shorter than unit length. It is postulated that RI II molecules might be formed in vivo by site-specific nicking of RF I molecules.