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1.
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   

2.
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   

3.
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 circularises via cohesive ends. A phage-encoded enzyme, protelomerase, then cuts at another site, telRL, and forms hairpin ends (telomeres). We demonstrate that this enzyme acts in vivo on specific substrates, and show that it is necessary for replication of the linear prophage. We show that protelomerase is an end-resolving enzyme responsible for processing of replicative intermediates. Removal of protelomerase activity resulted in accumulation of replicative intermediates that were found to be circular head-to-head dimers. N15 protelomerase and its target site constitute a functional unit acting on other replicons independently of other phage genes; a mini-F or mini-P1 plasmid carrying this unit replicates as a linear plasmid with covalently closed ends. Our results suggest the following model of N15 prophage DNA replication. Replication is initiated at an internal ori site located close to the left end of plasmid DNA and proceeds bidirectionally. After replication of the left telomere, protelomerase cuts this sequence and forms two hairpin loops telL. After duplication of the right telomere (telR) the same enzyme resolves this sequence producing two linear plasmids. Alternatively, full replication of the linear prophage to form a circular head-to-head dimer may precede protelomerase-mediated formation of hairpin ends.  相似文献   

4.
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   

5.
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   

6.
Hairpin telomeres of bacterial linear chromosomes are generated by a DNA cutting–rejoining enzyme protelomerase. Protelomerase resolves a concatenated dimer of chromosomes as the last step of chromosome replication, converting a palindromic DNA sequence at the junctions between chromosomes into covalently closed hairpins. The mechanism by which protelomerase transforms a duplex DNA substrate into the hairpin telomeres remains largely unknown. We report here a series of crystal structures of the protelomerase TelA bound to DNA that represent distinct stages along the reaction pathway. The structures suggest that TelA converts a linear duplex substrate into hairpin turns via a transient strand-refolding intermediate that involves DNA-base flipping and wobble base-pairs. The extremely compact di-nucleotide hairpin structure of the product is fully stabilized by TelA prior to strand ligation, which drives the reaction to completion. The enzyme-catalyzed, multistep strand refolding is a novel mechanism in DNA rearrangement reactions.  相似文献   

7.
Ravin NV 《Plasmid》2011,65(2):102-109
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   

8.
The plasmid prophage N15: a linear DNA with covalently closed ends   总被引:1,自引:0,他引:1  
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   

9.
10.
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   

11.
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   

12.
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   

13.
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   

14.
Evolution of the linear DNA replicons of the Borrelia spirochetes.   总被引:9,自引:0,他引:9  
Members of the spirochete genus Borrelia carry numerous linear DNA replicons with covalently closed hairpin telomeres. The genome of one member of this genus, B. burgdorferi B31, has now been completely characterized and contains a linear chromosome, twelve linear plasmids and nine circular extra-chromosomal elements. The phylogenetic position of the Borrelia spirochetes strongly suggests that a progenitor with circular replicons acquired the ability to replicate linear DNA molecules.  相似文献   

15.
A combined genetic and physical map of the Agrobacterium tumefaciens A348 (derivative of C58) genome was constructed to address the discrepancy between initial single-chromosome genetic maps and more recent physical mapping data supporting the presence of two nonhomologous chromosomes. The combined map confirms the two-chromosome genomic structure and the correspondence of the initial genetic maps to the circular chromosome. The linear chromosome is almost devoid of auxotrophic markers, which probably explains why it was missed by genetic mapping studies.  相似文献   

16.
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   

17.
18.
The ResT telomere resolvase is responsible for maintaining the hairpin telomeres that cap the linear chromosome and minichromosomes of Borrelia burgdorferi. This enzyme acts at the tandem telomere junctions present within circular dimers resulting from DNA replication. ResT mediates the transesterification steps of resolution using a constellation of active site residues similar to that found in tyrosine recombinases and type IB topoisomerases. By combining this reaction mechanism with a hairpin binding module in its N-terminal domain, ResT reduces a fused telomere dimer into two hairpin monomers. ResT displays a split DNA binding specificity, with the N- and C-terminal domains targeting distinct regions of the telomere. This bi-specificity in binding is likely to be important in protein delivery, substrate selection and regulation of enzyme activity.  相似文献   

19.
Borrelia burgdorferi, a causative agent of Lyme disease, has a highly unusual segmented genome composed of both circular molecules and linear DNA replicons terminated by covalently closed hairpin ends or telomeres. Replication intermediates of the linear molecules are processed into hairpin telomeres via the activity of ResT, a telomere resolvase. We report here the results of limited proteolysis and mass spectroscopy to identify two main structural domains in ResT, separated by a chymotrypsin cleavage site between residues 163 and 164 of the 449 amino acid protein. The two domains have been overexpressed and purified. DNA electrophoretic mobility shift assays revealed that the C-terminal domain (ResT(164-449)) displays sequence-specific DNA binding to the box 3,4,5 region of the telomere, while the N-terminal domain (ResT(1-163)) exhibits sequence-independent DNA binding activity. Further analysis by DNase I footprinting supports a model for telomere resolution in which the hairpin binding module of the N-terminal domain is delivered to the box 1,2 region of the telomere through its tethering to ResT(164-449). Conversely, ResT(1-164) may play an important regulatory role by modulating both sequence-specific DNA binding activity and catalysis by the C-terminal domain.  相似文献   

20.
It was shown for some oncogenic Agrobacterium tumefaciens strains that agrocin 84 sensitivity is determined by the presence of a large closed circular DNA plasmid, called the Ti-plasmid. Whereas wild-type strain C58 is agrocin 84 sensitive, all Ti-plasmid cured derivatives were found to be fully resistant. Moreover all independently isolated agrocin 84 resistant colonies were stably non-oncogenic and plasmid negative. In a growth experiment carried out at 37 degrees C it was shown that the kinetics of appearance of non-oncogenic cells on the one hand and of agrocin 84 resistant cells on the other were identical. The fact that not all oncogenic, plasmid harbouring, Agrobacterium tumefaciens strains are sensitive to agrocin 84, points to the possibility that the genes determining agrocin 84 sensitivity are not essential for tumor-inducing ability.  相似文献   

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