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1.
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   

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 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   

5.
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   

6.
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   

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 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   

9.
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   

10.
11.
Sequence-specific DNA-binding proteins must quickly and reliably localize specific target sites on DNA. This search process has been well characterized for monomeric proteins, but it remains poorly understood for systems that require assembly into dimers or oligomers at the target site. We present a single-molecule study of the target-search mechanism of protelomerase TelK, a recombinase-like protein that is only active as a dimer. We show that TelK undergoes 1D diffusion on non-target DNA as a monomer, and it immobilizes upon dimerization even in the absence of a DNA target site. We further show that dimeric TelK condenses non-target DNA, forming a tightly bound nucleoprotein complex. Together with theoretical calculations and molecular dynamics simulations, we present a novel target-search model for TelK, which may be generalizable to other dimer and oligomer-active proteins.  相似文献   

12.
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   

13.
Telomeres are essential structures that stabilize the ends of eukaryotic chromosomes and allow complete replication of linear DNA molecules. We examined the structure and replication of telomeres by observing the fate of the linear extrachromosomal rDNA of Tetrahymena after injection into unfertilized Xenopus eggs. The rDNA replicated efficiently as a linear extrachromosomal molecule, increasing in mass 30-50-fold by 15-20 h after injection. In addition, the molecules increased in length by addition of up to several kilobases of DNA to their termini. Sequence analysis demonstrated that the added DNA bore no resemblance to known telomeres. The junction between the rDNA and added DNA was apparently random, indicating that the addition reaction did not involve a site-specific recombination or integration event. Surprisingly, Southern blot analysis showed that the added DNA did not derive from Xenopus DNA, but rather from co-purifying and therefore co-injected Tetrahymena DNA. The nonspecific ligation of random DNA fragments to the rDNA termini suggests that microinjected Tetrahymena rDNA ends are not recognized as telomeres in Xenopus eggs.  相似文献   

14.
The Borrelia burgdorferi chromosome is linear, with telomeres characterized by terminal inverted repeats and covalently closed single-stranded hairpin loops. The replication mechanism of these unusual molecules is unknown. Previous analyses of bacterial chromosomes for which the complete sequence has been determined, including that of B. burgdorferi, revealed an abrupt switch in polarity of CG skew at known or putative origins of replication. We used nascent DNA strand analysis to physically map the B. burgdorferi origin to within a 2 kb region at the centre of the linear chromosome, and to show that replication proceeds bidirectionally from this origin. The results are consistent with replication models in which termination occurs at the telomeres after bidirectional, symmetrical elongation from the central origin. Sequences typical of origins of other bacterial chromosomes were not found at the origin of this spirochete. The most likely location of the replication origin of the linear chromosome is the 240 bp sequence between dnaA and dnaN where the switch in CG skew occurs.  相似文献   

15.
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   

16.
Cloning yeast telomeres on linear plasmid vectors   总被引:53,自引:0,他引:53  
J W Szostak  E H Blackburn 《Cell》1982,29(1):245-255
We have constructed a linear yeast plasmid by joining fragments from the termini of Tetrahymena ribosomal DNA to a yeast vector. Structural features of the terminus region of the Tetrahymena rDNA plasmid maintained in the yeast linear plasmid include a set of specifically placed single-strand interruptions within the cluster of hexanucleotide (C4A2) repeat units. An artificially constructed hairpin terminus was unable to stabilize a linear plasmid in yeast. The fact that yeast can recognize and use DNA ends from the distantly related organism Tetrahymena suggests that the structural features required for telomere replication and resolution have been highly conserved in evolution. The linear plasmid was used as a vector to clone chromosomal telomeres from yeast. One Tetrahymena end was removed by restriction digestion, and yeast fragments that could function as an end on a linear plasmid were selected. Restriction mapping and hybridization analysis demonstrated that these fragments were yeast telomeres, and suggested that all yeast chromosomes might have a common telomere sequence. Yeast telomeres appear to be similar in structure to the rDNA of Tetrahymena, in which specific nicks or gaps are present within a simple repeated sequence near the terminus of the DNA.  相似文献   

17.
Telomeres are specialized structures at the ends of linear chromosomes that were originally defined functionally based on observations first by Muller (1938) and subsequently by McClintock (1941) that naturally occurring chromosome ends do not behave as double-stranded DNA breaks, in spite of the fact that they are the physical end of a linear, duplex DNA molecule. Double-stranded DNA breaks are highly unstable entities, being susceptible to nucleolytic attack and giving rise to chromosome rearrangements through end-to-end fusions and recombination events. In contrast, telomeres confer stability upon chromosome termini, as evidenced by the fact that chromosomes are extraordinarily stable through multiple cell divisions and even across evolutionary time. This protective function of telomeres is due to the formation of a nucleoprotein complex that sequesters the end of the DNA molecule, rendering it inaccessible to nucleases and recombinases as well as preventing the telomere from activating the DNA damage checkpoint pathways. The capacity of a functional end-protective complex to form is dependent upon maintenance of sufficient telomeric DNA. We have learned a great deal about telomere structure and how this specialized nucleoprotein complex confers stability on chromosome ends since the original observations that defined telomeres were made. This review summarizes our current understanding of mammalian telomere replication, structure and function.  相似文献   

18.
Telomeres,telomerase, and stability of the plant genome   总被引:11,自引:0,他引:11  
Telomeres, the complex nucleoprotein structures at the ends of linear eukaryotic chromosomes, along with telomerase, the enzyme that synthesizes telomeric DNA, are required to maintain a stable genome. Together, the enzyme and substrate perform this essential service by protecting chromosomes from exonucleolytic degradation and end-to-end fusions and by compensating for the inability of conventional DNA replication machinery to completely duplicate the ends of linear chromosomes. Telomeres are also important for chromosome organization within the nucleus, especially during mitosis and meiosis. The contributions of telomeres and telomerases to plant genome stability have been confirmed by analysis of Arabidopsis mutants that lack telomerase activity. These mutants have unstable genomes, but manage to survive up to ten generations with increasingly shortened telomeres and cytogenetic abnormalities. Comparisons between telomerase-deficient Arabidopsis and telomerase-deficient mice reveal distinct differences in the consequences of massive genome damage, probably reflecting the greater developmental and genomic plasticity of plants.  相似文献   

19.
Suzanne S. Chan 《FEBS letters》2010,584(17):3773-6077
The linear nature of eukaryotic chromosomes leaves natural DNA ends susceptible to triggering DNA damage responses. Telomeres are specialized nucleoprotein structures that comprise the “end zone” of chromosomes. Besides having specialized sequences and structures, there are six resident proteins at telomeres that play prominent roles in protecting chromosome ends. In this review, we discuss this team of proteins, termed shelterin, and how it is involved in regulating DNA damage signaling, repair and replication at telomeres.  相似文献   

20.
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