Analysis of the interruptions in bacteriophage T5 DNA

Jacquemin‐Sablon, Alain; Richardson, Charles C.

doi:10.1016/0022-2836(70)90316-5

Cited by 87 publications

(38 citation statements)

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“…The strand which would exhibit the lower poly(G) binding capacity in a CsCl gradient migrates slightly more rapidly than the other strand to yield a doublet on the gel even though the molecular weights are the same (16). Jacquemin-Sablon and Richardson have reported that, after in vitro repair, the normally nicked strand of T5 DNA has a slightly lower affinity for poly(U,G) than does the intact one (11). In the experiments reported here, splitting of the 35.3 X 106 dalton band into a doublet occurred simultaneously with the sharp increase of the sealing index (Figs.…”

Section: Methodsmentioning

confidence: 99%

“…The intact single strand has a molecular weight of 35.3 X 106, and the two largest fragments of the nicked strand have molecular weights of 17.2 X 106 and 14.5 X 101 (10). The interruptions are only nicks in the phosphodiester backbone rather than gaps (with missing bases) because they can be repaired in vitro by the purified bacteriophage T4 polynucleotide ligase in the absence of a DNA polymerase (11). The locations of the nicks have been mapped by various techniques including electron microscopy (8) and gel electrophoresis (10), but their function in the viral growth cycle remains unknown.…”

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confidence: 99%

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In Vivo Repair of the Single-Strand Interruptions Contained in Bacteriophage T5 DNA

Herman¹,

Moyer²

1974

Proc. Natl. Acad. Sci. U.S.A.

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Bacteriophage T5 is known to contain several unique single-strand interruptions in only one strand of the duplex DNA. Analysis of labeled parental phage DNA from infected Escherichia coli shows that these nicks are repaired in vivo to yield intact doublestranded molecules. Sealing begins at about 6 min after infection and is independent of DNA replication. Repair may be an ordered process that starts at a unique end of the molecule.Bacteriophage T5 is a virulent DNA phage of Escherichia coli. T5 injects its DNA into a host cell by a two-step process (1). Attachment of the phage to a sensitive cell is followed by injection of only 8% of the molecule [the first step transfer portion; (fst) ] (2). This limited portion of the molecule must be transcribed and translated before the remainder of the molecule can be injected into the cell (3). The presence of certain plasmids can make E. coli a nonpermissive host for T5. If a cell contains the collb factor, only the earliest (class I) TSspecific proteins are synthesized. Class II and class III phage genes which. include the genes necessary for DNA replication and phage assembly, respectively, are not expressed and the infection is abortive (4).The viral DNA is a linear, nonpermuted, double-stranded molecule of approximately 71 X 106 daltons (5) and is about 9% terminally repetitious (6). This DNA contains a series of genetically determined (7) single-strand interruptions in only one chain of the duplex molecule (8) (Fig. 1). The intact single strand has a molecular weight of 35.3 X 106, and the two largest fragments of the nicked strand have molecular weights of 17.2 X 106 and 14.5 X 101 (10). The interruptions are only nicks in the phosphodiester backbone rather than gaps (with missing bases) because they can be repaired in vitro by the purified bacteriophage T4 polynucleotide ligase in the absence of a DNA polymerase (11). Parental T5 DNA has been examined after infection of noncolicinogenic cells, or colIb+ cells in which DNA replication fails to occur. The results presented here suggest that, shortly after the complete injection of the viral DNA, repair of the nicks occurs in both cell types. Repair, therefore, takes place prior to DNA replication and before the appearance of concatemeric forms. These experiments further suggest that repair may occur in a specific order. MATERIALS AND METHODSBactera and Phage. E. coli K12 W3110 (thy-) was a gift from J. Cairns. E. coli K12 W3110 (thy-, colIb+) and bacteriophage T5st(0) have been described previously (4).Lysis of Infected Cells. At each time point, a 10-ml aliquot of infected cells was removed and viral metabolism rapidly stopped by a modification of the method of Miller and Kozinski (12). Chloramphenicol was also present in the "stop mix" at 100 ,sg/ml of final concentration. The infected cells were pelleted in the cold and lysed by the procedure of Miller and Kozinski (12). The lysis buffer was modified to also contain nicotinamide mononucleotide at 1 mg/ml (13).Gel Electrophoresis.

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Section: Methodsmentioning

confidence: 99%

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confidence: 99%

In Vivo Repair of the Single-Strand Interruptions Contained in Bacteriophage T5 DNA

Herman¹,

Moyer²

1974

Proc. Natl. Acad. Sci. U.S.A.

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“…Of'Nick-Free T5' DNA For the sealing of the nicks in the T5' DNA which was first described by Richardson et al [38] and Jacquemin-Sablon and Richardson [4], we used highly purified T4-induced ligase. Under the conditions described in Materials and Methods this enzyme worked most efficiently if the reaction was carried out at 15 "C. The ligated DNA was analyzed either by analytical band centrifugation (Fig.…”

Section: Preparation and Characterizationmentioning

confidence: 99%

“…One strand of the linear duplex is intact [3,4] whereas the other contains 4 to 5 interruptions (henceforth called "nicks") whose locations along the DNA molecule have been determined [3,5-71. Another unique feature of phage T5 is its mode of infection: in a first step, only 8 % of the genome is injected into the host cell, and protein synthesis directed by this fraction of the DNA, the first-step-transfer DNA, is a prerequisite for the transfer of the residual 92% of the chromosome [8,9].…”

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Structure and Function of the Genome of Coliphage T5. Transcription in vitro of the "Nicked" and "Nick-Free" T5+ DNA

Knopf

1975

Eur J Biochem

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Purified T5' DNA in nicked form and after repair of the specific single-strand interruptions with DNA ligase was used in transcription studies using Escherichia coli RNA polymerase (holoenzyme) in the presence and absence of E. coli termination protein rho. The transcriptional products were analyzed with respect to their size distribution and the sequences transcribed from the different templates. The results indicate that the single-strand breaks in the DNA of bacteriophage T5, though in genetically defined positions, do not have any specific effect on transcription in vitro. Furthermore, the E. coli rho protein, although it depresses net RNA synthesis and reduces the average molecular weight of the transcripts, seems to act in a non-specific way in this system.The chromosome of bacteriophage T5 is unique in that it possesses single-strand interruptions in genetically defined positions [1,2]. One strand of the linear duplex is intact [3,4] whereas the other contains 4 to 5 interruptions (henceforth called "nicks") whose locations along the DNA molecule have been determined [3,5-71. Another unique feature of phage T5 is its mode of infection: in a first step, only 8 % of the genome is injected into the host cell, and protein synthesis directed by this fraction of the DNA, the first-step-transfer DNA, is a prerequisite for the transfer of the residual 92% of the chromosome [8,9]. Furthermore, after infection bacteriophage T5 expresses its genes in a well-defined sequence resulting in at least three classes of proteins [lo] and corresponding groups of RNA [ 11,121. The specific location of the nicks in the T5 DNA molecule has led to a number of hypotheses about their possible function. We were interested in whether or not those nicks play a role in the transcriptional process of the T5' genome. We have therefore investigated the template properties of T5+ DNA in a transcriptional system in vitro using DNA extracted from phage T5' in nicked form and after the nicks have been sealed by DNA ligase. In these experiments Escherichia coli RNA polymerase (holoenzyme) and Enzymes. DNA ligase (EC 6.5

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“…In some instance the purification was carried further according to Jacquemin-Sablon and Richardson [17] in order to remove traces of nuclease activity. Calf intestine alkaline phosphatase (Boehringer Mannheim GmbH (Mannheim, Germany) was free from RNase and DNase activity as tested with the techniques reported below.…”

Section: Enzymesmentioning

confidence: 99%

RNA Ligase Activity in Phage-Infected Bacteria and Animal Cells

1974

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An RNA ligase which is dependent on ATP and Mg2+ for maximal activity with 5'-32P-labeled poly(1) * poly(C) as substrate has been purified from Escherichia coli infected with phage T4. Ligase activity was demonstrated by the transfer of alkaline phosphatase sensitive 5'-32P termini into an alkaline phosphatase resistant form. The formation of phosphodiester bond was verified by hydrolysis of the reaction product and determining the transfer of 32P from the 5' position to the 2'(3') position by electrophoresis. An increase in sedimentation rate of poly(1) -poly(C) was also observed after reaction with RNA ligase. The enzyme also showed an ATP-PPi exchange activity. The activity on the synthetic polyribonucleotides poly(A), poly(U), poly(A) * poly(U) and poly(1) * poly(C) was examined and an increased efficiency of the substrate was obtained when . An RNA ligase activity must probably also be involved in the formation of the covalent circles of double-stranded RNA observed in the electron microscope among the replicative forms of EMC virus RNA [8]. A rolling circle model for the replication of viral RNA has been suggested for foot and mouth disease virus RNA [9] and this replication mechanism would probably also require an RNA ligase. In contrast to the animal cell no evidence for RNA recombination has been obtained in E. coli in spite of several attempts to recombine RNA phages [lo]. However, i t has been reported that the purified T4 phage-induced DNA ligase can use a ribo-deoxyribose heteropolymer as substrate [ll, 121.The present study reports attempts to isolate an RNA ligase from mammalian cells, E . coli and T4 phage-infected E . coli. The latter system was included Abbreviations. poly(U), poly(uridy1ic acid) ; poly(A), poly(adeny1ic acid) ; poly(1) -poly(C), poly(riboinosinic acid)

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Analysis of the interruptions in bacteriophage T5 DNA

Cited by 87 publications

References 19 publications

In Vivo Repair of the Single-Strand Interruptions Contained in Bacteriophage T5 DNA

In Vivo Repair of the Single-Strand Interruptions Contained in Bacteriophage T5 DNA

Structure and Function of the Genome of Coliphage T5. Transcription in vitro of the "Nicked" and "Nick-Free" T5+ DNA

RNA Ligase Activity in Phage-Infected Bacteria and Animal Cells

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