Progressive rearrangement of telomeric sequences added to both the ITR ends of the yeast linear pGKL plasmid

Relocation into the nucleus of the yeast cytoplasmic linear plasmids was studied using a monitor plasmid pCLU1. InSaccharomyces cerevisiae, the nuclearly-relocated pCLU1 replicated in a linear form (termed pTLU-type plasmid) which carried the host telomeric repeats TG_1–3 of 300–350 bp at both ends. The telomere sequences mainly consisted of a major motif TGTGTGGGTGTGG which was complementary to part of the RNA template of yeast telomerase and were directly added to the very end of the pCLU1-terminal element ITR (inverted terminal repeat), suggesting that the ITR end played a role as a substrate of telomerase. The telomere sequences varied among isolated pTLU-type plasmids, but the TG_1–3 organization was symmetrically identical on both ends of any one plasmid. During cell growth under non-selective condition, the telomeric repeat sequences were progressively rearranged on one side, but not on the opposite side of pTLU plasmid ends. This indicates that the mode of telomeric DNA replication or repair differed between both ends. Clonal analysis showed that the intense rearrangement of telomeric DNA was closely associated with extreme instability of pTLU plasmids. Published: February 17, 2003     

Linear Streptomyces plasmids form superhelical circles through interactions between their terminal proteins

Linear chromosomes and linear plasmids of Streptomyces possess covalently bound terminal proteins (TPs) at the 5' ends of their telomeres. These TPs are proposed to act as primers for DNA synthesis that patches the single-stranded gaps at the 3' ends during replication. Most ('archetypal') Streptomyces TPs (designated Tpg) are highly conserved in size and sequence. In addition, there are a number of atypical TPs with heterologous sequences and sizes, one of which is Tpc that caps SCP1 plasmid of Streptomyces coelicolor. Interactions between the TPs on the linear Streptomyces replicons have been suggested by electrophoretic behaviors of TP-capped DNA and circular genetic maps of Streptomyces chromosomes. Using chemical cross-linking, we demonstrated intramolecular and intermolecular interactions in vivo between Tpgs, between Tpcs and between Tpg and Tpc. Interactions between the chromosomal and plasmid telomeres were also detected in vivo. The intramolecular telomere interactions produced negative superhelicity in the linear DNA, which was relaxed by topoisomerase I. Such intramolecular association between the TPs poses a post-replicational complication in the formation of a pseudo-dimeric structure that requires resolution by exchanging TPs or DNA.     

Inverted terminal repeats and terminal proteins of the genomes of pneumococcal phages

The nucleotide (nt) sequence at the ends of the genomes of the Streptococcus pneumoniae phages Cp-5 and Cp-7 has been determined and compared with the corresponding sequence of phage Cp-1. The genomes of phages Cp-5 and Cp-7 have inverted terminal repeats (ITRs) 343 and 347 bp long, respectively. In Cp-1 DNA the ITR is 236 bp long and the following 116 bp are 93% homologous. Some regions within the ITRs are conserved in the three genomes although the complete sequence of the ITRs is no more conserved than the rest of their genomes. The chromatographic behavior of their tryptic peptides suggests that the terminal proteins (TPs) of at least two of the phages are similar and that the TPs of the three pneumococcal phages differ markedly from that of the Bacillus subtilis phage psi 29.     

Progressive alteration of telomeric sequences at one end of a yeast linear plasmid and its possible association with reduced plasmid stability

After selection for migration into the nucleus, a cytoplasmic yeast linear plasmid bearing an inverted terminal repeat (ITRs) at each end replicates in Saccharomyces cerevisiae in a linear form, called pTLU, which carries host telomeric repeats (TG(1-3))(n) of about 300-350 bp added to the ITR ends. We previously showed that the nucleotide composition of the added telomeric sequences varied among individual pTLU isolates, while those on the two ends of any given pTLU were always identical. The telomeric sequences of pTLU remained unchanged over numbers of cell generations when cells were selected for expression of the plasmid-borne nuclear marker. We report here that progressive alterations in telomeric sequences can be detected in cells which are grown under non-selective conditions. Surprisingly, in any given molecule, the telomeric alterations occur exclusively on one side, either the left or the right end, while the sequence at the opposite end remained identical to the original, suggesting a difference in the mode of DNA replication between the plasmid ends. These alterations occur over a broad area extending from the termini of telomeres to nucleotides near the junction between the telomeric sequences and the pTLU-ITR, implying that the plasmid ends undergo successive rounds of extension and contraction. Clonal analysis under non-selective conditions indicated that the alterations in telomeric sequences are generally associated with extreme instability of the pTLU plasmid.     

Relocation of a cytoplasmic yeast linear plasmid to the nucleus is associated with circularization via nonhomologous recombination involving inverted terminal repeats

The linear plasmid pCLU1 from the yeast Kluyveromyces lactis normally replicates in the cytoplasm, with the aid of the helper linear plasmid pGKL2, using terminal protein (TP) as a primer. However, it relocates to the nucleus when selection is applied for the expression of a plasmid-borne nuclear marker. Migration to the nucleus occurred in K. lactis at a frequency of about 10⁻³/cell ten or more times higher than the rate observed in Saccharomyces cerevisiae. The nuclear plasmids existed only in a circularized form in K. lactis, while in S. cerevisiae a telomere-associated linear form is also found. Sequence analysis showed that circularization in K. lactis was caused by non-homologous recombination between the inverted terminal repeat (ITR) at the ends of the linear form and non-specific internal target sites in pCLU1. No sequence similarity existed among the junction sites, indicating that the free ITR end plays a crucial role in circularization. In S. cerevisiae, circular plasmids were generated not only by non-homologous recombination, but also by homologous recombination between short direct repeats within pCLU1. Circularization via the ITR end was observed independently of RAD52 activity. Sequences highly homologous to ARS core elements, 5′-ATTTATTGTTTT-3′ for K. lactis and 5′-(A/T)TTTAT(T/G)TTT(A/T)-3′ for S. cerevisiae, were detected at multiple sites in the nuclear forms of the plasmids. Received: 25 October 1999 / Accepted: 13 March 2000     

Uncapping and deregulation of telomeres lead to detrimental cellular consequences in yeast

Telomeres are the protein-nucleic acid structures at the ends of eukaryote chromosomes. Tandem repeats of telomeric DNA are templated by the RNA component (TER1) of the ribonucleoprotein telomerase. These repeats are bound by telomere binding proteins, which are thought to interact with other factors to create a higher-order cap complex that stabilizes the chromosome end. In the budding yeast Kluyveromyces lactis, the incorporation of certain mutant DNA sequences into telomeres leads to uncapping of telomeres, manifested by dramatic telomere elongation and increased length heterogeneity (telomere deregulation). Here we show that telomere deregulation leads to enlarged, misshapen "monster" cells with increased DNA content and apparent defects in cell division. However, such deregulated telomeres became stabilized at their elongated lengths upon addition of only a few functionally wild-type telomeric repeats to their ends, after which the frequency of monster cells decreased to wild-type levels. These results provide evidence for the importance of the most terminal repeats at the telomere in maintaining the cap complex essential for normal telomere function. Analysis of uncapped and capped telomeres also show that it is the deregulation resulting from telomere uncapping, rather than excessive telomere length per se, that is associated with DNA aberrations and morphological defects.     

The telomere system of the Streptomyces linear plasmid SCP1 represents a novel class

Linear plasmids and chromosomes of Streptomyces carry terminal proteins (TPs) covalently attached to the 5' ends of the DNA. Most known telomeres are conserved in primary sequence and in the potential secondary structures formed during replication. The TP that caps these telomeres is also highly conserved and its coding gene, tpg, is present in all Streptomyces chromosomes and some linear plasmids. Linear plasmid SCP1 contains atypical telomere sequences and no tpg homologue, and can replicate in the absence of tpg, suggesting that it carries a novel TP gene. To isolate the TP on the SCP1 telomeres, we constructed a multicopy mini-SCP1 plasmid. The TP capping the plasmid was isolated and subjected to tryptic digestion and mass spectrometric analysis, and the results indicated that the TP was encoded by an open reading frame (ORF), SCP1.127 (tpc), on SCP1. Of the two ORFs upstream of tpc, SCP1.125 (tac) but not SCP1.126 was essential for replication of mini-SCP1. The Tac-Tpc system of SCP1 represents a convergently evolved novel telomere-capping system of Streptomyces linear replicons.     

The number of vertebrate repeats can be regulated at yeast telomeres by Rap1-independent mechanisms

The number of telomeric DNA repeats at chromosome ends is maintained around a mean value by a dynamic balance between elongation and shortening. In particular, proteins binding along the duplex part of telomeric DNA set the number of repeats by progressively limiting telomere growth. The paradigm of this counting mechanism is the Rap1 protein in Saccharomyces cerevisiae. We demonstrate here that a Rap1-independent mechanism regulates the number of yeast telomeric repeats (TG(1-3)) and of vertebrate repeats (T(2)AG(3)) when TEL1, a yeast ortholog of the human gene encoding the ATM kinase, is inactivated. In addition, we show that a T(2)AG(3)-only telomere can be formed and maintained in humanized yeast cells carrying a template mutation of the gene encoding the telomerase RNA, which leads to the synthesis of vertebrate instead of yeast repeats. Genetic and biochemical evidences indicate that this telomere is regulated in a Rap1-independent manner, both in TEL1 and in tel1Delta humanized yeast cells. Altogether, these findings shed light on multiple repeat-counting mechanisms, which may share critical features between lower and higher eukaryotes.     

Protein-primed DNA replication: role of inverted terminal repeats in the Escherichia coli bacteriophage PRD1 life cycle.

Escherichia coli bacteriophage PRD1 and its relatives contain linear double-stranded DNA genomes, the replication of which proceeds via a protein-primed mechanism. Characteristically, these molecules contain 5'-covalently bound terminal proteins and inverted terminal nucleotide sequences (inverted terminal repeats [ITRs]). The ITRs of each PRD1 phage species have evolved in parallel, suggesting communication between the molecule ends during the life cycle of these viruses. This process was studied by constructing chimeric PRD1 phage DNA molecules with dissimilar end sequences. These molecules were created by combining two closely related phage genomes (i) in vivo by homologous recombination and (ii) in vitro by ligation of appropriate DNA restriction fragments. The fate of the ITRs after propagation of single genomes was monitored by DNA sequence analysis. Recombinants created in vivo showed that phages with nonidentical genome termini are viable and relatively stable, and hybrid phages made in vitro verified this observation. However, genomes in which the dissimilar DNA termini had regained identical sequences were also detected. These observations are explained by a DNA replication model involving two not mutually exclusive pathways. The generality of this model in protein-primed DNA replication is discussed.     

Two chimeric chromosomes of Streptomyces coelicolor A3(2) generated by single crossover of the wild-type chromosome and linear plasmid scp1

Streptomyces coelicolor A3(2) strain 2106 carries a 1.85-Mb linear plasmid, SCP1'-cysD, in addition to a 7.2-Mb linear chromosome. Macrorestriction analysis indicated that both linear DNAs are hybrids of the wild-type chromosome and the linear plasmid SCP1 on each side. Nucleotide sequencing of the fusion junctions revealed no homology between the recombination regions. SCP1'-cysD contains an SCP1 telomere and a chromosomal telomere at each end and therefore does not have terminal inverted repeats. In addition, SCP1'-cysD could not be eliminated from strain 2106 by various mutagenic treatments. Thus, we concluded that both the 7.2-Mb chromosome and SCP1'-cysD are chimeric chromosomes generated by a single crossover of the wild-type chromosome and SCP1. This may be regarded as a model of chromosomal duplication in genome evolution.     

Linear DNA replication: inverted terminal repeats of five closely related Escherichia coli bacteriophages

The closely related lipid-containing bacteriophages PRD1, PR4, PR5, PR722 and L17 isolated from different parts of the world have double-stranded DNA genomes which replicate in a linear form. The nucleotide (nt) sequences of the genome termini of these viruses reveal 110-111-bp-long inverted terminal repeats (ITRs). Both ends of the viral DNA are identical. The first 18 bp and the last 35 bp of the ITRs are totally conserved in all viruses. Between these conserved nt sequences there is a variable sequence, which enables us to divide the phages into two groups. Comparison of the virus ITRs led also to the identification of a 10-bp-long A + T stretch, where the only changes observed were transversions between A and T. The termini of the PRD1 virus family genomes exhibit sequence similarities to those of phi 29 and Cp-1 families.     

Telomeres, telomerase and senescence

Eukaryotic chromosomes end with tandem repeats of simple sequences. These GC rich repeats allow telomere replication and stabilize chromosome ends. Telomere replication involves an equilibrium of sequence loss and addition at the ends of chromosomes. Repeats are added de novo by telomerase, an unusual DNA polymerase. Telomerase is an RNP in which an essential RNA component provides the template for the added telomere repeats. Telomere length maintenance plays an essential role in cell viability.     

Cloning yeast telomeres on linear plasmid vectors

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.     

How telomerase reaches its end: mechanism of telomerase regulation by the telomeric complex

The telomerase enzyme, which synthesizes telomeric DNA repeats, is regulated in cis at individual chromosome ends by the telomeric protein/DNA complex in a manner dependent on telomere repeat-array length. A dynamic interplay between telomerase-inhibiting factors bound at duplex DNA repeats and telomerase-promoting ones bound at single-stranded terminal DNA overhangs appears to modulate telomerase activity and to be directly related to the transient deprotection of telomeres. We discuss recent advances on the mechanism of telomerase regulation at chromosome ends in both yeast and mammalian systems.     

Terminal proteins of Streptomyces chromosome can target DNA into eukaryotic nuclei

Streptomyces species are highly abundant soil bacteria that possess linear chromosomes (and linear plasmids). The 5' ends of these molecules are covalently bound by terminal proteins (TPs), that are important for integrity and replication of the telomeres. There are at least two types of TPs, both of which contain a DNA-binding domain and a classical eukaryotic nuclear localization signal (NLS). Here we show that the NLS motifs on these TPs are highly efficient in targeting the proteins along with covalently bound plasmid DNA into the nuclei of human cells. The TP-mediated nuclear targeting resembles the inter-kingdom gene transfer mediated by Ti plasmids of Agrobacterium tumefaciens, in which a piece of the Ti plasmid DNA is targeted to the plant nuclei by a covalently bound NLS-containing protein. The discovery of the nuclear localization functions of the Streptomyces TPs not only suggests possible inter-kingdom gene exchanges between Streptomyces and eukaryotes in soil but also provides a novel strategy for gene delivery in humans and other eukaryotes.     

The structure of a subterminal repeated sequence present on many human chromosomes.

All telomeres which have been studied consist of an array of simple G/C rich repeats. Human telomeres were shown to share sequence similarity with those of lower eukaryotes by cross-hybridization and human telomeric sequences have been cloned by complementation of telomere function in yeast. Analysis of human telomeric sequences cloned in this way is described here. The terminal part of the cloned human telomeric DNA consists of an array of simple repeats, principally of the sequence TTAGGG and derivatives. The very terminal part consists of yeast-type telomeric repeats which suggests that the human telomeric sequences have acted as a primer for the addition of additional telomeric repeats in the yeast. Subterminal sequences are shared between a number of clones and in situ data shows that these subterminal sequences are present at several different chromosomal ends. Related sequences are present at internal as well as telomeric positions. Differences in the hybridization patterns of subterminal sequences in somatic compared to germ-line tissues are described which indicate differential modification of these sequences during development.     

Cloning and sequence analysis of linear plasmid telomeres of the bacterium Borrelia burgdorferi

Borrelia burgdorferi, the Lyme disease agent, has double-stranded linear plasmids with covalently closed ends. DNA at the ends, or telomeres, of two linear plasmids of B. burgdorferi strain B31 was examined. Telomeric sequences from both ends of a 16 kb linear plasmid and from one end of a 49 kb linear plasmid were cloned and sequenced. An 18 bp AT-rich inverted repeat was found at each end of the 16 kb linear plasmid. The sequences of the two ends of this plasmid were different beyond these short inverted terminal repeats. The cloned end of the 49 kb linear plasmid had sequence identity with one end of the 16 kb linear plasmid. The end sequence common to both plasmids contained a series of phased, short direct repeats and a 52 bp palindrome adjacent to a highly AT-rich region. These findings indicate that Borrelia linear plasmid telomeres have structural features different from those of other known replicons.     

Nucleotide sequence analysis of the unusually long terminal inverted repeats of a giant linear plasmid, SCP1.

SCP1 is a giant linear plasmid of 350 kb coding for the methylenomycin biosynthetic genes in Streptomyces coelicolor. The unusually long terminal inverted repeats present on both ends of SCP1 were analyzed on the nucleotide sequence level. Analysis of six clones containing the terminal 0.35-kb XbaI fragment revealed a slight heterogeneity in the nucleotide sequences of the SCP1 ends. Moreover, it was indicated that this fragment contained seven palindromic inverted repeats and a GT-rich region in the 5'-end strand. The size of the terminal inverted repeats was determined to be 81 kb by the cloning and sequencing of their end-points. An insertion sequence, IS466 was shown to be present just at the end of the right terminal inverted repeat.