[structure of a region in the genome of bacteriophage n15, necessary for formation of hairpins at ends of the linear plasmid prophage].a fragment containing telrl site of bacteriophage n15 has been cloned in the vector plasmid puc19. the nucleotide sequence of a small region from ecorv-psti fragment has been defined by maxam-gilbert technique. the analysis of the obtained sequence has shown the telrl site to be a nonideal palindrome (the size of 56 nucleotide ops) in which two nucleotide pairs differ in the positions 12 and 14 on both sides of the palindrome centre. the dna region with alteration of purines and pyrimidines (gc) ...19921454079
independence of bacteriophage n15 lytic and linear plasmid replication from the heat shock proteins dnaj, dnak, and grpe.the chromosome of the temperate bacteriophage n15 replicates as a linear plasmid with covalently closed ends (or hairpins) when it forms a lysogen. i found that, in contrast to the cases for lambda and the low-copy-number plasmids f and p1, both phage and plasmid replication of n15 are independent of the heat shock proteins dnaj, dnak, and grpe.19911917885
[non-chromosomal localization of phage n15]. 19694922266
[cos-region of temperate coliphage n15].the cohesive termini including the cos region (altogether 414 bp) of the dna of the temperate coliphage n15 are sequenced. the termini are complementary 12-nucleotide single-stranded 5'-extended dnas. the sequence of the left terminus is 5'-gggcggcgtccg-3', that of the right 5'cggacgccgccc-3'. ten nucleotides of the n15 termini are identical to those of phage lambda. the n15 and lambda sequences are notably homologous only within the 50 bp region from the left and right ends. phage n15 has a reg ...19989611756
the circle is broken: telomere resolution in linear replicons.linear dna molecules with covalently closed hairpin ends (telomeres) exist in a wide variety of organisms. telomere resolution, a dna breakage and reunion reaction in which replicated telomeres are processed into hairpin ends, is now known to be a common theme in poxviruses, borrelia burgdorferi and escherichia coli phage n15. candidate proteins that may perform this reaction have recently been identified in poxviruses. moreover, the first purification and definitive identification of a telomere ...200111587933
protelomerase uses a topoisomerase ib/y-recombinase type mechanism to generate dna hairpin ends.protelomerases are enzymes responsible for the generation of closed hairpin ends in linear dna. it is proposed that they use a breaking-and-rejoin type mechanism to affect dna rearrangement on specific dna sequences. in doing so, one strand turns around and becomes the complementary strand. using the purified enzyme from the escherichia coli phage n15 and the klebsiella phage phiko2 and synthetic oligonucleotide substrates, we directly demonstrate the location where the cutting/re-ligation occur ...200415001353
[structural organization and control of expression of the sop-operon of linear plasmid prophage n15].stable inheritance of bacterial chromosomes and low-copy-number plasmids depends on the active partition of replicated molecules between daughter cells. the partition mechanism is well known for circular plasmids f and p1. the mechanism of partition of linear replicons was studied with the example of bacteriophage n15, which persists as a linear plasmid with covalently closed ends on lysogeny, rather than integrating into the escherichia coli chromosome. since stable inheritance of n15 is due to ...200415125235
[expression regulation of the protelomerase gene of the bacteriophage n15].the n15 bacteriophage, when in the lysogenic state, does not integrate into the chromosome; in fact, it exists as a linear plasmid with the covalently closed ends. upon infection, the phage dna circularizes via its cohesive ends, after which a specific enzyme, the n15 protelomerase, cuts the circular molecule thus generating a linear plasmid with the covalently closed telomeres. protelomerase generates, as the replication of plasmid prophage proceeds, the hairpin telomeres in replicated molecule ...200415164718
repa protein of the bacteriophage n15 exhibits activity of dna helicase. 200415523829
functional characterization of the repa replication gene of linear plasmid prophage n15.the prophage of coliphage n15 is not integrated into the chromosome, but exists as a linear plasmid molecule with covalently closed ends. the only phage gene required for replication of circular n15 miniplasmids is repa (gene 37). here we show that repa-driven replication of the n15-based circular and linear miniplasmids is independent of host dnab helicase protein, but requires the host dnag primase. replication of phage n15 dna during lytic growth following infection does not depend on either ...200616129583
the sxt conjugative element and linear prophage n15 encode toxin-antitoxin-stabilizing systems homologous to the tad-ata module of the paracoccus aminophilus plasmid pami2.a group of proteic toxin-antitoxin (ta) cassettes whose representatives are widely distributed among bacterial genomes has been identified. these cassettes occur in chromosomes, plasmids, bacteriophages, and noncomposite transposons, as well as in the sxt conjugative element of vibrio cholerae. the following four homologous loci were subjected to detailed comparative studies: (i) tad-ata from plasmid pami2 of paracoccus aminophilus (the prototype of this group), (ii) gp49-gp48 from the linear ba ...200717158670
n15: the linear phage-plasmid.the lambdoid phage n15 of escherichia coli is very unusual among temperate phages in that its prophage is not integrated into chromosome but is a linear plasmid molecule with covalently closed ends. upon infection the phage dna circularises via cohesive ends, then phage-encoded enzyme, protelomerase, cuts at an inverted repeat site and forms hairpin ends (telomeres) of the linear plasmid prophage. replication of the n15 prophage is initiated at an internally located ori site and proceeds bidirec ...201021185326
A scalable pipeline for highly effective genetic modification of a malaria parasite.In malaria parasites, the systematic experimental validation of drug and vaccine targets by reverse genetics is constrained by the inefficiency of homologous recombination and by the difficulty of manipulating adenine and thymine (A+T)-rich DNA of most Plasmodium species in Escherichia coli. We overcame these roadblocks by creating a high-integrity library of Plasmodium berghei genomic DNA (>77% A+T content) in a bacteriophage N15-based vector that can be modified efficiently using the lambda Re ...201122020067
determinants of gas-phase disassembly behavior in homodimeric protein complexes with related yet divergent structures.the overall structure of a protein-protein complex reflects an intricate arrangement of noncovalent interactions. whereas intramolecular interactions confer secondary and tertiary structure to individual subunits, intermolecular interactions lead to quaternary structure-the ordered aggregation of separate polypeptide chains into multisubunit assemblies. the specific ensemble of noncovalent contacts dictates the stability of subunit folds, enforces protein-protein binding specificity, and determi ...201121486017
[sop proteins can cause transcriptional silencing of genes located close to the centromere sites of linear plasmid n15].stable inheritance of bacterial chromosomes and low copy number plasmids is ensured by accurate partitioning of replicated molecules between the daughter cells at division. partitioning of the prophage of the temperate bacteriophage n15, which exists as a linear plasmid molecule with covalently closed ends, depends on the sop locus, comprising genes sopa and sopb, as well as four centromere sites located in different regions of the n15 genome essential for replication and the control of lysogeny ...201020586190
n15 cro and lambda cro: orthologous dna-binding domains with completely different but equally effective homodimer interfaces.bacteriophage cro proteins bind to target dna as dimers but do not all dimerize with equal strength, and differ in fold in the region of the dimer interface. we report the structure of the cro protein from enterobacteria phage n15 at 1.05 a resolution. the subunit fold contains five alpha-helices and is closely similar to the structure of p22 cro (1.3 a backbone room mean square difference over 52 residues), but quite different from that of lambda cro, a structurally diverged member of this fami ...200818369196
tightly regulated, high-level expression from controlled copy number vectors based on the replicon of temperate phage n15.a new escherichia coli host/vector system has been developed to allow a dual regulation of both the plasmid copy number and gene expression. the new pn15e vectors are low copy number plasmids based on the replicon of temperate phage n15, comprising the repa replicase gene and cb repressor gene, controlling the plasmid copy number. regulation of pn15e copy number is achieved through arabinose-inducible expression of phage n15 antirepressor protein, anta, whose gene was integrated into the chromos ...200717433573
py54, a linear plasmid prophage of yersinia enterocolitica with covalently closed ends.py54 is a temperate phage isolated from yersinia enterocolitica. lysogenic yersinia strains harbour the py54 prophage as a plasmid (py54). the plasmid has the same size (46 kb) as the py54 genome isolated from phage particles. by electron microscopy, restriction analysis and dna sequencing, it was demonstrated that the phage and the plasmid dnas are linear, circularly permuted molecules. unusually for phages of gram-negative bacteria, the phage genome has 3'-protruding ends. the linear plasmid p ...200312753191
linear closed mini dna generated by the prokaryotic cleaving-joining enzyme teln is functional in mammalian cells.for application of dna in gene medicine plasmid or viral dna is usually used as a vector for the gene of interest. to generate dna with a minimum of foreign dna sequences, we used the prokaryotic telomerase, protelomerase teln, of bacteriophage n15. this is a novel enzyme with cleaving-joining activity, which is required for the formation of linear prophage dna with closed ends in lysogenic bacteria. acting on a telomere resolution site telrl, the protelomerase converts circular plasmid dna into ...200212395149
phage n15 telomere resolution. target requirements for recognition and processing by the protelomerase.the escherichia coli prophage n15 exists as a linear dna molecule with covalently closed ends. purified n15 protelomerase teln is the only protein required to convert circular dna substrates to the linear form with hairpin termini. within the center of the telomerase occupancy site tos, the target for teln is the 56-bp telrl consisting of the central 22-bp palindrome telo and two 14-bp flanking inverted sequence repetitions. dnase i footprinting of teln-telrl complexes shows a segment of approxi ...200211788606
crude protein extraction protocol for phage n15 protelomerase in vitro enzymatic assays.the phage n15 protelomerase enzyme (teln) is essential for the replication of its genome by resolution of its telrl domain, located within a telomerase occupancy site (tos), into hairpin telomeres. isolation of teln for in vitro processing of tos, however, is a highly complex process, requiring multiple purification steps. in this study a simplified protocol for crude total protein extraction is described that retains the tos-cleaving activity of teln for at least 4 weeks, greatly simplifying in ...201121396906
recombineering linear dna that replicate stably in e. coli.the advent of recombineering technology in escherichia coli has revolutionized the way recombinant dna molecules are constructed. we present a novel application of recombineering to linearize dna by capping their ends with individual telomeres derived from bacteriophage n15, which exists as a linear prophage in e. coli. the n15 telomerase occupancy site was recombined into circular dna and resolved into individual telomeres by the phage n15 protelomerase enzyme. we demonstrate this technique by ...200817988739
interplay between the temperate phages py54 and n15, linear plasmid prophages with covalently closed ends.the objective of this study was to determine whether the temperate yersinia enterocolitica phage py54 may interact with the related escherichia coli phage n15 during both the lysogenic and the lytic cycle in the same cell. the py54 and n15 prophages are linear plasmids which have been shown to be compatible and stably replicating in e. coli and yersinia. in e. coli, the py54 prophage does not restrict n15 propagation. in contrast, n15 reduces by use of its cor gene the susceptibility of yersinia ...200717827299
[initiator protein dnaa of escherichia coli is a negative replication regulator of the linear phage-plasmid n15].temperate bacteriophage n 15 in the lysogenic state is incapable of integrating in the chromosome of escherichia coli and represents a linear plasmid with covalently closed ends. the phage repa gene, the product of which possesses activities of primase and helicase, ensures replication of n15 dna. the ori site of initiation of n15 replication contains binding sites for repa and a potential site of binding the bacterial initiator protein dnaa. it was shown in our work that replication of miniplas ...200717333938
escherichia coli with a linear genome.chromosomes in eukaryotes are linear, whereas those of most, but not all, prokaryotes are circular. to explore the effects of possessing a linear genome on prokaryotic cells, we linearized the escherichia coli genome using the lysogenic lambda-like phage n15. linear genome e. coli were viable and their genome structure was stable. there were no appreciable differences between cells with linear or circular genomes in growth rates, cell and nucleoid morphologies, genome-wide gene expression (with ...200717218953
sequence analysis of the genome of the temperate yersinia enterocolitica phage py54.the temperate yersinia phage py54 belongs to the unusual group of phages that replicate as linear plasmids with covalently closed ends. besides escherichia coli phage n15, py54 is the only member of this group to be identified. we have determined the complete sequence (46,339 bp) of the py54 genome. bioinformatic analyses revealed 67 open reading frames (orfs) with good coding potential located on both dna strands. the comparison of the deduced py54 gene products with known proteins encoded by o ...200312899832
the protelomerase of temperate escherichia coli phage n15 has cleaving-joining activity.escherichia coli phage n15 encodes the slightly acidic, 630-residue protein of 72.2 kda called protelomerase (teln). teln is a component of the n15 replication system proposed to be involved in the generation of the linear prophage dna. this linear dna molecule has covalently closed ends. the reaction converting circular plasmids into linear molecules was catalyzed in vitro. we demonstrate that the product of teln functions as the protelomerase in the absence of other n15-encoded factors. purifi ...200010884403
conversion of linear dna with hairpin telomeres into a circular molecule in the course of phage n15 lytic replication.the prophage of temperate coliphage n15 is not integrated into the bacterial chromosome but exists as a linear plasmid molecule with covalently closed ends. upon infection of an escherichia coli cell, the phage dna circularises via cohesive ends. a phage-encoded enzyme, protelomerase, then cuts at another site, telrl, and forms hairpin ends (telomeres) of linear plasmid prophage. bidirectional replication of a linear plasmid produces a molecule with duplicated telomeres that is subsequently reso ...200919523475
the pko2 linear plasmid prophage of klebsiella oxytoca.temperate bacteriophages with plasmid prophages are uncommon in nature, and of these only phages n15 and py54 are known to have a linear plasmid prophage with closed hairpin telomeres. we report here the complete nucleotide sequence of the 51,601-bp klebsiella oxytoca linear plasmid pko2, and we demonstrate experimentally that it is also a prophage. we call this bacteriophage phiko2. an analysis of the 64 predicted phiko2 genes indicate that it is a fairly close relative of phage n15; they share ...200414996813
genomic sequence and analysis of the atypical temperate bacteriophage n15.n15 is a temperate bacteriophage that forms stable lysogens in escherichia coli. while its virion is morphologically very similar to phage lambda and its close relatives, it is unusual in that the prophage form replicates autonomously as a linear dna molecule with closed hairpin telomeres. here, we describe the genomic architecture of n15, and its global pattern of gene expression, which reveal that n15 contains several plasmid-derived genes that are expressed in n15 lysogens. the tel site, at w ...200010860722
[characteristics of the bacteriophage n15 lysogenic conversion gene and identification of its product].the plasmids containing ecorv fragments of n15 phage dna and inhibiting the adsorption of t1, phi 80 and n15 phages were selected and characterized. the n15 lysogenic conversion gene (cor) was mapped in the sali-psti fragment of 700 bp in length which is localized near sali site with the coordinates 40.1 kb on the n15 plasmid prophage dna physical map. the cor gene was recloned on a multicopy vector in the both possible orientations and its expression was shown to occur most probably under contr ...19938486255
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