共查询到20条相似文献,搜索用时 31 毫秒
1.
Ravin NV 《FEMS microbiology letters》2003,223(1):1-6
The prophage of 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 circularizes via cohensive ends. A phage-encoded enzyme, protelomerase, then cuts at another site, telRL, and forms hairpin ends (telomeres). Purified protelomerase alone processes circular and linear plasmid DNA containing the target site telRL to produce linear double-stranded DNA with covalently closed ends in vitro. N15 protelomerase is necessary for replication of the linear prophage through its action as a telomere-resolving enzyme. Replication of circular N15-based miniplasmids requires the only gene repA that encodes multidomain protein homologous to replication proteins of bacterial plasmids replicated by theta-mechanism, particularly, phage P4 alpha-replication protein. Replication of the N15 prophage is initiated at an internal ori site located within repA. Bidirectional replication results in formation of the circular head-to-head, tail-to-tail dimer molecule. Then the N15 protelomerase cuts both duplicated telomeres generating two linear plasmid molecules with covalently closed ends. The N15 prophage replication thus appears to follow the mechanism distinct from that employed by poxviruses and could serve as a model for other prokaryotic replicons with hairpin ends, and particularly, for linear plasmids and chromosomes of Borrelia burgdorferi. 相似文献
2.
Bidirectional replication from an internal ori site of the linear N15 plasmid prophage 总被引:4,自引:0,他引:4
下载免费PDF全文
![点击此处可从《Nucleic acids research》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The prophage of coliphage N15 is not integrated into the chromosome but exists as a linear plasmid molecule with covalently closed hairpin ends (telomeres). Upon infection the injected phage DNA circularizes via its cohesive ends. Then, a phage-encoded enzyme, protelomerase, cuts the circle and forms the hairpin telomeres. N15 protelomerase acts as a telomere-resolving enzyme during prophage DNA replication. We characterized the N15 replicon and found that replication of circular N15 miniplasmids requires only the repA gene, which encodes a multidomain protein homologous to replication proteins of bacterial plasmids replicated by a theta-mechanism. Replication of a linear N15 miniplasmid also requires the protelomerase gene and telomere regions. N15 prophage replication is initiated at an internal ori site located within repA and proceeds bidirectionally. Electron microscopy data suggest that after duplication of the left telomere, protelomerase cuts this site generating Y-shaped molecules. Full replication of the molecule and subsequent resolution of the right telomere then results in two linear plasmid molecules. N15 prophage replication thus appears to follow a mechanism that is distinct from that employed by eukaryotic replicons with this type of telomere and suggests the possibility of evolutionarily independent appearances of prokaryotic and eukaryotic replicons with covalently closed telomeres. 相似文献
3.
Coliphage N15 is a temperate bacteriophage whose prophage is a linear plasmid molecule with covalently closed ends (telomeres). The N15 prophage provided the first example of such DNA in prokaryotes and, up to now, it is the only known example of a linear plasmid in Escherichia coli. The linear N15 mature phage DNA has single-stranded cohesive ends. The phage and plasmid prophage DNAs are circularly permuted. The nucleotide structure of the telomere-forming site tel RL in phage DNA corresponds to the structures of the terminal hairpin loops. It suggests a unique mechanism for conversion of the circular phage DNA to the linear plasmid form, which is performed by the prokaryotic telomerase (protelomerase). The results of a comparison of the protelomerase with integrases lead us to suggest that these proteins may have evolved from a common ancestor. The mechanism of plasmid N15 replication is unknown. We propose that the protelomerase participates in linear plasmid replication, acting as a resolvase of replicative intermediates that are tail-to-tail linear dimers. The sequence analysis of the N15 DNA showed that it represents an evolutionary 'link' between plasmids F, P1, P4 and lambdoid bacteriophages. 相似文献
4.
Dorokhov BD Strakhova TS Ravin NV 《Molekuliarnaia genetika, mikrobiologiia i virusologiia》2004,(2):28-32
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 molecules. We identified the promoter of the protelomerase gene and demonstrated that it could be repressed presumably due to its binding with 3 tosL sites overlapping the promoter. We also found the transformation efficiency of E. coli cells of linear DNA with hairpin telomeres to be approximately 100-fold lower versus the circular DNA of the same size. At the same time, presence of the N15 prophage or of the protelomerase-expressing vector enhances, in a strain being transformed, the efficiency of its transformation by linear DNA up to a level ensured in transformation by circular plasmids. We believe that protelomerase, while binding with the hairpin telomeres, protects the latter from degradation by cellular nucleases. 相似文献
5.
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 bidirectionally resulting in formation of duplicated telomeres. Then the N15 protelomerase cuts duplicated telomeres generating two linear plasmid molecules with hairpin telomeres. Stable inheritance of the plasmid prophage is ensured by partitioning operon similar to the F factor sop operon. Unlike F sop, the N15 centromere consists of four inverted repeats dispersed in the genome. The multiplicity and dispersion of centromeres are required for efficient partitioning of a linear plasmid. The centromeres are located in N15 genome regions involved in phage replication and control of lysogeny, and binding of partition proteins at these sites regulates these processes. Two N15-related lambdoid Siphoviridae phages, φKO2 in Klebsiella oxytoca and pY54 in Yersinia enterocolitica, also lysogenize their hosts as linear plasmids, as well as Myoviridae marine phages VP882 and VP58.5 in Vibrio parahaemolyticus and ΦHAP-1 in Halomonas aquamarina. The genomes of all these phages contain similar protelomerase genes, lysogeny modules and replication genes, as well as plasmid-partitioning genes, suggesting that these phages may belong to a group diverged from a common ancestor. 相似文献
6.
The termini of linear chromosomes are protected by specialized DNA structures known as telomeres that also facilitate the complete replication of DNA ends. The simplest type of telomere is a covalently closed DNA hairpin structure found in linear chromosomes of prokaryotes and viruses. Bidirectional replication of a chromosome with hairpin telomeres produces a catenated circular dimer that is subsequently resolved into unit-length chromosomes by a dedicated DNA cleavage-rejoining enzyme known as a hairpin telomere resolvase (protelomerase). Here we report a crystal structure of the protelomerase TelK from Klebsiella oxytoca phage varphiKO2, in complex with the palindromic target DNA. The structure shows the TelK dimer destabilizes base pairing interactions to promote the refolding of cleaved DNA ends into two hairpin ends. We propose that the hairpinning reaction is made effectively irreversible by a unique protein-induced distortion of the DNA substrate that prevents religation of the cleaved DNA substrate. 相似文献
7.
PY54, a linear plasmid prophage of Yersinia enterocolitica with covalently closed ends 总被引:1,自引:0,他引:1
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 pY54 has covalently closed ends forming telomere-like hairpins. The equivalent DNA sequence of the phage genome is a 42 bp perfect palindrome. Downstream from the palindrome, an open reading frame (ORF) was identified that revealed strong DNA homology to the telN gene of Escherichia coli phage N15 encoding a protelomerase. Similar to PY54, the N15 prophage is a linear plasmid with telomeres. The N15 protelomerase has cleaving/joining activity generating the telomeres by processing a 56 bp palindrome (telomere resolution site tel RL). To study the activity of the PY54 protein, the telN-like gene was cloned and expressed in E. coli. A 77 kDa protein was obtained and partially purified. The protein was found to process recombinant plasmids containing the 42 bp palindrome. Telomere resolution of plasmids under in vivo conditions was also investigated in Yersinia infected with PY54. Processing required a plasmid containing the palindrome as well as adjacent DNA sequences from the phage including an additional inverted repeat. Regions on the phage genome important for plasmid maintenance were defined by the construction of linear and circular miniplasmid derivatives of pY54, of which the smallest miniplasmid comprises a 4.5 kb DNA fragment of the plasmid prophage. 相似文献
8.
Jan Deneke Gunter Ziegelin Rudi Lurz Erich Lanka 《The Journal of biological chemistry》2002,277(12):10410-10419
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 approximately 50 bp protected by TelN. Surface plasmon resonance studies demonstrate that this extended footprint is caused by two TelN molecules bound to telRL. Stable TelN-target DNA complexes are achieved with telRL; however, the additional sequences of tos stabilize the TelN-target complexes. TelO alone is not sufficient for specific stable complex formation. However, processing can occur, i.e. generation of the linear covalently closed DNA. Within the context of telRL, sequences of telO are involved in specific TelN-telRL complex formation, in processing itself, and/or in recognition of the processing site. The sequence of the central (CG)(3) within telO that is part of a 14-bp stretch proposed to have Z-DNA conformation is essential for processing but not for formation of specific TelN-telRL complexes. The concerted action of both TelN molecules at the target site is the basis for telomere resolution. Capturing of reaction intermediates demonstrates that TelN binds covalently to the 3'-phosphoryl of the cleaved strands. 相似文献
9.
10.
Huang WM DaGloria J Fox H Ruan Q Tillou J Shi K Aihara H Aron J Casjens S 《The Journal of biological chemistry》2012,287(30):25551-25563
Agrobacterium tumefaciens C58, the pathogenic bacteria that causes crown gall disease in plants, harbors one circular and one linear chromosome and two circular plasmids. The telomeres of its unusual linear chromosome are covalently closed hairpins. The circular and linear chromosomes co-segregate and are stably maintained in the organism. We have determined the sequence of the two ends of the linear chromosome thus completing the previously published genome sequence of A. tumefaciens C58. We found that the telomeres carry nearly identical 25-bp sequences at the hairpin ends that are related by dyad symmetry. We further showed that its Atu2523 gene encodes a protelomerase (resolvase) and that the purified enzyme can generate the linear chromosomal closed hairpin ends in a sequence-specific manner. Agrobacterium protelomerase, whose presence is apparently limited to biovar 1 strains, acts via a cleavage-and-religation mechanism by making a pair of transient staggered nicks invariably at 6-bp spacing as the reaction intermediate. The enzyme can be significantly shortened at both the N and C termini and still maintain its enzymatic activity. Although the full-length enzyme can uniquely bind to its product telomeres, the N-terminal truncations cannot. The target site can also be shortened from the native 50-bp inverted repeat to 26 bp; thus, the Agrobacterium hairpin-generating system represents the most compact activity of all hairpin linear chromosome- and plasmid-generating systems to date. The biochemical analyses of the protelomerase reactions further revealed that the tip of the hairpin telomere may be unusually polymorphically capable of accommodating any nucleotide. 相似文献
11.
12.
The vaccinia virus DNA telomere, which contains a covalently closed hairpin structure, has been cloned in a yeast plasmid vector. Restriction mapping indicates that the cloned vaccinia telomere is maintained in yeast not in its native hairpin configuration but as an inverted repeat structure, within a circular plasmid, with the sequences of the viral hairpin now at the axis of symmetry of an imperfect palindrome. As such, the cloned telomere resembles the telomeric replicative intermediate observed during vaccinia virus DNA replication. Small deletions and duplications in the viral inverted repeats of different clones suggest a model in which the observed circular plasmids were generated in yeast by the replication of hybrid linear DNA molecules consisting of the linearized yeast vector flanked by two hairpin-containing vaccinia termini. 相似文献
13.
Beatriz Zabala Jens A. Hammerl Romilio T. Espejo Stefan Hertwig 《Journal of virology》2009,83(18):9313-9320
Vibrio parahaemolyticus O3:K6 pandemic strains recovered in Chile frequently possess a 42-kb plasmid which is the prophage of a myovirus. We studied the prototype phage VP58.5 and show that it does not integrate into the host cell chromosome but replicates as a linear plasmid (Vp58.5) with covalently closed ends (telomeres). The Vp58.5 replicon coexists with other plasmid prophages (N15, PY54, and ΦKO2) in the same cell and thus belongs to a new incompatibility group of telomere phages. We determined the complete nucleotide sequence (42,612 nucleotides) of the VP58.5 phage DNA and compared it with that of the plasmid prophage. The two molecules share the same nucleotide sequence but are 35% circularly permuted to each other. In contrast to the hairpin ends of the plasmid, VP58.5 phage DNA contains 5′-protruding ends. The VP58.5 sequence is 92% identical to the sequence of phage VHML, which was reported to integrate into the host chromosome. However, the gene order and termini of the phage DNAs are different. The VHML genome exhibits the same gene order as does the Vp58.5 plasmid. VHML phage DNA has been reported to contain terminal inverted repeats. This repetitive sequence is similar to the telomere resolution site (telRL) of VP58.5 which, after processing by the phage protelomerase, forms the hairpin ends of the Vp58.5 prophage. It is discussed why these closely related phages may be so different in terms of their genome ends and their lifestyle.Most temperate bacteriophages integrate into the host chromosome during lysogeny. However, there are some phages (telomere phages) whose prophages are linear plasmids with covalently closed ends. Members of this group of phages are the siphoviruses N15, PY54, and ΦKO2 isolated from Escherichia coli, Yersinia enterocolitica, and Klebsiella oxytoca, respectively, and the recently described myoviruses ΦHAP-1 of Halomonas aquamarina and VP882 of Vibrio parahaemolyticus (6, 20, 23, 26, 37). Despite their different origins (enterobacteria versus marine bacterium) and morphologies, all known telomere phages share similar genome organizations and some protein similarities. The linear DNA of each phage is a circular permutation of the respective linear plasmid prophage. For the generation of the terminal hairpins of the linear plasmid, the protelomerase (Tel) is essential (8). This enzyme has cleaving/joining activity; its target is a large palindromic DNA sequence called the telomere resolution site (telRL) located upstream of tel on the phage genome. After cleaving telRL by staggered cuts, the resulting self-complementary single-stranded DNA overhangs fold back and are rejoined by the protelomerase (9). Besides tel, all telomere phages possess the gene repA, encoding a multifunctional replication protein. repA of N15 and PY54 was shown to harbor the prophage replication origin and to function as a circular minimal replicon (35, 42). Compatibility studies demonstrated that the N15 and ΦKO2 plasmids belong to the same incompatibility group, whereas the PY54 plasmid is able to coexist with these two prophages in doubly lysogenic E. coli and Y. enterocolitica hosts (19).There are some reports on the presence of tel and repA in prophages (VP882, VHML, and Vp58.5) of marine Vibrio strains (28, 41). V. parahaemolyticus phage VP882 is a close relative of the Halomonas phage ΦHAP-1 (26). VHML was isolated from a toxin-producing Vibrio harveyi strain, pathogenic for some crustaceans and fish (30). Similarly to ΦHAP-1 and VP882, VHML has a myovirus-like morphology. The phage contains genes for products similar to Tel and RepA, suggesting that its prophage is a linear plasmid with terminal hairpins. However, it was surmised that VHML integrates into the Vibrio chromosome (28, 29). Phage VP58.5 was isolated from a V. parahaemolyticus strain belonging to the serovar O3:K6 pandemic clonal complex (41). During the last several years, this clone has been associated with many seafood-borne diarrhea outbreaks in Southeast Asia and South America, particularly Chile (5, 12, 13, 15). Up to 33% of the Chilean isolates harbored a 42-kb plasmid which was shown to be the prophage of a myovirus inducible by mitomycin C. VP58.5 is the prototype of these phages.In this work we demonstrate that VP58.5 is closely related to the V. harveyi phage VHML but that its prophage is a linear plasmid with covalently closed ends. The Vp58.5 prophage belongs to a new incompatibility group of telomere phages. 相似文献
14.
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 resolvase has been reported for phage N15. This protein is the prototype for a new class of DNA enzyme that performs a unique reaction. Advances in the study of telomere resolution in poxviruses, B. burgdorferi and E. coli phage N15 are discussed. 相似文献
15.
Replication and resolution of cloned poxvirus telomeres in vivo generates linear minichromosomes with intact viral hairpin termini. 总被引:17,自引:11,他引:6
下载免费PDF全文
![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The covalently closed terminal hairpins of the linear duplex-DNA genomes of the orthopoxvirus vaccinia and the leporipoxvirus Shope fibroma virus (SFV) have been cloned as imperfect palindromes within circular plasmids in yeast cells and recombination-deficient Escherichia coli. The viral telomeres inserted within these recombinant plasmids are equivalent to the inverted-repeat structures detected as telomeric replicative intermediates during poxvirus replication in vivo. Although the telomeres of vaccinia and SFV show little sequence homology, the termini from both viral genomes exist as AT-rich terminal hairpins with extrahelical bases and alternate "flip-flop" configurations. Using an in vivo replication assay in which circular plasmid DNA was transfected into poxvirus-infected cells, we demonstrated the efficient replication and resolution of the cloned imperfect palindromes to bona fide hairpin termini. The resulting linear minichromosomes, which were readily purified from transfected cells, were shown by restriction enzyme mapping and by electron microscopy to have intact covalently closed hairpin termini at both ends. In addition, staggered unidirectional deletion derivatives of both the cloned vaccinia and SFV telomeric palindromes localized an approximately 200-base-pair DNA region in which the sequence organization was highly conserved and which was necessary for the resolution event. These data suggest a conserved mechanism of the resolution of poxvirus telomeres. 相似文献
16.
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 occurs. We identified a pair of transient staggered cleavages six base-pairs apart centered around the axis of dyad symmetry of the target site. Two molecules of the protelomerase form a pair of protein-linked DNA intermediates at each 3' end of the cleaved openings leaving a 5'-OH. Then, in a process not yet clearly defined, the partners of the two initial openings are exchanged, and the transient breaks are resealed to generate hairpin ends. The formation of 3'-covalent DNA-protein intermediates is a hallmark of the topoisomerase IB type reaction, and we have thus shown experimentally that protelomerase is a member of the tyrosine-recombinase superfamily. In addition, by introducing single nicks in the substrates as perturbation, we found that the integrity of the nucleotide chain 4 bp away from the cutting site as well as this nucleotide's complementary location on the stem if the strands were to fold into a cruciform structure are required for activity, suggesting that these locations may be important substrate-protein contacts. We determined that N15 and phiKO2 protelomerases are monomers in solution and two molecules are needed to interact with the substrate to form two closed hairpin products. The target sites of protelomerases invariably consist of inverted repeats. Comparative studies using the related target sites of different protelomerases suggest that these proteins may require both sequence-specific and structure (possibly cruciform)-specific recognition for activity. 相似文献
17.
The genus Borrelia includes the causative agents of Lyme disease and relapsing fever. An unusual feature of these bacteria is a genome that includes linear DNA molecules with covalently closed hairpin ends referred to as telomeres. We have investigated the mechanism by which the hairpin telomeres are processed during replication. A synthetic 140 bp sequence having the predicted structure of a replicated telomere was shown to function as a viable substrate for telomere resolution in vivo, and was sufficient to convert a circular replicon to a linear form. Our results suggest that the final step in the replication of linear Borrelia replicons is a site-specific DNA breakage and reunion event to regenerate covalently closed hairpin ends. The telomere substrate described here will be valuable both for in vivo manipulation of linear DNA in Borrelia and for in vitro studies to identify and characterize the telomere resolvase. 相似文献
18.
19.
Casjens SR Gilcrease EB Huang WM Bunny KL Pedulla ML Ford ME Houtz JM Hatfull GF Hendrix RW 《Journal of bacteriology》2004,186(6):1818-1832
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 a mosaic relationship that is typical of different members of double-stranded DNA tailed-phage groups. Although the head, tail shaft, and lysis genes are not recognizably homologous between these phages, other genes such as the plasmid partitioning, replicase, prophage repressor, and protelomerase genes (and their putative targets) are so similar that we predict that they must have nearly identical DNA binding specificities. The phiKO2 virion is unusual in that its phage lambda-like tails have an exceptionally long (3,433 amino acids) central tip tail fiber protein. The phiKO2 genome also carries putative homologues of bacterial dinI and umuD genes, both of which are involved in the host SOS response. We show that these divergently transcribed genes are regulated by LexA protein binding to a single target site that overlaps both promoters. 相似文献
20.
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 in different regions of the N15 genome essential for replication and the control of lysogeny.
We found that binding of SopB to the centromere could silence centromere-proximal promoters, presumably due to subsequent
polymerization of SopB along the DNA. Close to the IR4 centromere site we identified a promoter, P59, which was able to drive
the expression of phage late genes encoding structural proteins of virion. We found that, following binding to IR4, the N15
Sop proteins could induce repression of this promoter. The repression depended on SopB and was enhanced in the presence of
SopA. Sop-dependent silencing of centromere-proximal promoters may control gene expression in phage N15, particularly preventing
undesired expression of late genes in the N15 prophage. Thus, the phage N15 sop system not only ensures plasmid partitioning but is also involved in the genetic network controlling prophage replication
and the maintenance of lysogeny. 相似文献