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.     

In Vitro Deoxynucleotidylation of the Terminal Protein of Streptomyces Linear Chromosomes

Single-stranded gaps at the 3′ ends of Streptomyces linear replicons are patched by DNA synthesis primed by terminal proteins (TP) during replication. We devised an in vitro system that specifically incorporated dCMP, the first nucleotide at the 5′ ends, onto a threonine residue of the TP of Streptomyces coelicolor.     

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.     

The telomeres of Streptomyces chromosomes contain conserved palindromic sequences with potential to form complex secondary structures

The chromosomes of the Gram-positive soil bacteria Streptomyces are linear DNA molecules, usually of about 8 Mb, containing a centrally located origin of replication and covalently bound terminal proteins (which are presumably involved in the completion of replication of the telomeres). The ends of the chromosomes contain inverted repeats of variable lengths. The terminal segments of five Streptomyces chromosomes and plasmids were cloned and sequenced. The sequences showed a high degree of conservation in the first 166–168 bp. Beyond the terminal homology, the sequences diverged and did not generally cross-hybridize. The homologous regions contained seven palindromes with a few nucleotide differences. Many of these differences occur in complementary pairs, such that the palindromicity is preserved. Energy-optimized modelling predicted that the 3' strand of the terminal palindromes can form extensive hairpin structures that are similar to the 3' ends of autonomous parvovirus genomes. Most of the putative hairpins have a GCGCAGC sequence at the loop, with the potential to form a stable single C-residue loop closed by a sheared G:A pairing. The similarity between the terminal structures of the Streptomyces replicons and the autonomous parvoviral genomes suggests that they may share some structural and/or replication features.     

Specific recognition of parental terminal protein by DNA polymerase for initiation of protein-primed DNA replication

The linear genome of Bacillus subtilis phage phi29 has a protein covalently linked to the 5' ends, called parental terminal protein (TP), and is replicated using a free TP as primer. The initiation of phage phi29 DNA replication requires the formation of a DNA polymerase/TP complex that recognizes the replication origins located at the genome ends. The DNA polymerase catalyzes the formation of the initiation complex TP-dAMP, and elongation proceeds coupled to strand displacement. The same mechanism is used by the related phage Nf. However, DNA polymerase and TP from phi29 do not initiate the replication of Nf TP-DNA. To address the question of the specificity of origin recognition, we took advantage of the initiation reaction enhancement in the presence of Mn(2+), allowing us to detect initiation activity in heterologous systems in which DNA polymerase, TP, and template TP-DNA are not from the same phage. Initiation was selectively stimulated when DNA polymerase and TP-DNA were from the same phage, strongly suggesting that specific recognition of origins is brought through an interaction between DNA polymerase and parental TP.     

The putative coiled coil domain of the phi 29 terminal protein is a major determinant involved in recognition of the origin of replication

The linear double-stranded genome of phage phi29 contains a terminal protein (TP) covalently linked at each 5' DNA end, called parental TP. Initiation of phi29 DNA replication starts with the recognition of the origins of replication, constituted by the parental TP-containing DNA ends, by a heterodimer containing phi29 DNA polymerase and primer TP. It has been argued that origin recognition involves protein-protein interactions between parental and primer TP. Analysis of the TP sequence revealed that the region between amino acids 84 and 118 has a high probability to form an amphipatic alpha-helix that could be involved in the interaction between parental and primer TP. Therefore, this TP region may be important for origin recognition. To test this hypothesis we introduced various mutations in the predicted amphipatic alpha-helix and analyzed the functionality of the corresponding purified TP mutants. The results obtained show that the identified putative amphipatic alpha-helix of TP is an important determinant involved in origin recognition.     

Replication at the telomeres of the Streptomyces linear plasmid pSLA2

The Streptomyces linear plasmid pSLA2 initiates DNA replication bidirectionally towards its telomeres from a site located near the centre of the molecule; at the telomeres, the recessed ends of lagging strands are filled in by non-displacing DNA synthesis. Here, we report experiments that test three proposed mechanisms for lagging-strand fill-in. We present data inconsistent with recombinational or terminal hairpin models for the formation of full-length duplex pSLA2 DNA. Instead, we find that deletions in short, distantly separated homologous palindromes in the leading-strand 3' overhang prevent propagation of linear pSLA2 DNA, implicating a mechanism of palindrome-mediated leading-strand fold-back in telomere replication. We further show that circularized pSLA2 DNA molecules are opened in vivo precisely at the terminal nucleotides of telomeres, generating functional linear replicons containing native telomeres covalently bound to a protein at their 5' DNA termini. Together, our results support a model in which pairing of multiple widely separated pSLA2 palindromes anchors the 3' end of the leading-strand overhang to a site near the overhang's base — providing a recognition site for terminal-protein-primed DNA synthesis and subsequent endonucleolytic processing. Thus, the replication of Streptomyces plasmid telomeres may have features in common with the mechanism proposed for telomere replication in autonomous parvoviruses.     

Characterization of Streptomyces plasmid-phage pFP4 and its evolutionary implications

Autonomous-replicating plasmid pFP4 of Streptomyces sp. FR1 isolated from a heavy metal-contaminated land was cloned and sequenced. Surprisingly, the 40,949-bp pFP4 contains a cluster of 20 genes, resembling these chromosome-integrated prophages of Streptomyces sp. SPB78 and Streptomyces scabiei 87.22. Plasmid pFP4 could transfer by conjugation and a replication locus, iteron/repA/repB, was identified. The filtered FR1 culture could infect both FR1 and FR1 cured of pFP4 to form plaques, and also six out of 13 strains from the same land, but failed to form plaques on other seven strains from same source and all ten Streptomyces species from different sources. pFP4 phage particles were observed by transmission electron microscopy. Major structural proteins (capsid, portal and tail, etc.) of pFP4 virions were encoded by twelve pFP4 genes. pFP4 phage DNA contained 3' protruding cohesive ends of 9-nt. Streptomyces pFP4 represents a novel plasmid-phage.     

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.     

Physical characterization of SCP1, a giant linear plasmid from Streptomyces coelicolor.

SCP1, coding for the methylenomycin biosynthesis genes in Streptomyces coelicolor, was shown to be a giant linear plasmid of 350 kb with a copy number of about four by analysis with pulsed-field gel electrophoresis. A detailed physical map of SCP1 was constructed by extensive digestion with six restriction endonucleases, by DNA hybridization experiments, and finally by cloning experiments. SCP1 has unusually long terminal inverted repeats of 80 kb on both ends and an insertion sequence at the end of the right terminal inverted repeat. Analysis by pulsed-field gel electrophoresis in agarose containing sodium dodecyl sulfate revealed that a protein is bound to the terminal 4.1-kb SpeI fragments derived from both ends of SCP1. Treatment with lambda exonuclease or exonuclease III and SpeI digestion also indicated that the 5' ends of SCP1 are attached to a protein.     

Chromosomal circularization in Streptomyces griseus by nonhomologous recombination of deletion ends

Streptomyces linear chromosomes frequently cause deletions at both ends spontaneously or by various mutagenic treatments, and concomitantly display dynamic structural changes such as circularization and arm replacement. We have cloned and sequenced the fusion junctions of circularized chromosomes in two deletion mutants of Streptomyces griseus. No homology and a 1-bp overlap were found between the deletion ends of the mutant chromosomes. Taking this together with previous results, we concluded that chromosomal circularization in Streptomyces occurs by nonhomologous recombination between deletion ends.     

Genetic instability of the Streptomyces chromosome

The Streptomyces wild-type chromosome is linear in all examples studied. The ends of the chromosome or telomeres consist of terminal inverted repeats of various sizes with proteins covalently bound to their 5' ends. The chromosome is very unstable and undergoes very large deletions spontaneously at rates higher than 0.1% of spores. Frequently, the telomeres are included in the deletions. Loss of both telomeres leads to circularization of the chromosome. The wild-type chromosome can also be circularized artificially by targeted recombination. Spontaneously or artificially circularized chromosomes are even more unstable than the linear ones. High-copy-number tandem amplifications of specific chromosomal regions are frequently associated with the deletions. RecA seems to be involved in the amplification mechanism and control of genetic instability.     

The adenovirus terminal protein influences binding of replication proteins and changes the origin structure.

The adenovirus terminal protein (TP) is covalently linked to the 5' ends of the adenovirus genome and enhances DNA replication in vitro by increasing template activity. To study the effect of TP in more detail we isolated short origin fragments containing functional TP using anion exchange chromatography. These fragments were highly active as templates for DNA replication in a reconstituted system. Employing band-shift assays we found that the affinity of the precursor terminal protein-DNA polymerase complex for the TP-containing origin was increased 2 to 3-fold. Binding affinities of two other replication stimulating proteins, NFI and Oct-1, were not influenced by the terminal protein. Upon DNaseI footprinting we observed, unexpectedly, that the breakdown pattern had changed at various positions in the origin, notably in the area 3-6 and 41-51 by the presence of TP. Some differences in the footprint pattern of NFI and Oct-1 were also found. Our results indicate that TP induces subtle changes in the origin structure that influence the interaction of other replication proteins.     

A linear DNA plasmid from Streptomyces rochei with an inverted terminal repetition of 614 base pairs.

The terminal structure of a linear plasmid pSLA2 , which was isolated from Streptomyces rochei , was analysed. The 5' ends of pSLA2 DNA were blocked by the association of a protein probably covalently bonded with the DNA. This block is removed by alkali treatment and blunt ends with 5'-phosphate and 3'-hydroxy termini were released. The two terminal fragments of pSLA2 were cloned and the nucleotide sequence was determined. An inverted terminal repetition of 614 bp was found along with the presence of further interrupted homologous sequences beyond this area up to 800 bp. These are the first inverted terminal repeat sequences found in microbial linear plasmids.     

Transposition of Mu DNA: joining of Mu to target DNA can be uncoupled from cleavage at the ends of Mu

Transposition of Mu involves transfer of the 3' ends of Mu DNA to the 5' ends of a staggered cut in the target DNA. We find that cleavage at the 3' ends of Mu DNA precedes cutting of the target DNA. The resulting nicked species exists as a noncovalent nucleoprotein complex in which the two Mu ends are held together. This cleaved donor complex completes strand transfer when a target DNA, Mu B protein, and ATP are provided. Mu end DNA sequences that have been precisely cut at their 3' ends by a restriction endonuclease, instead of by Mu A protein and HU, are efficiently transferred to a target DNA upon subsequent incubation with Mu A protein, Mu B protein, and ATP. Cleavage of the Mu ends therefore cannot be energetically coupled with joining these ends to a target DNA. We discuss the DNA strand transfer mechanism in view of these results, and propose a model involving direct transfer of the 5' ends of the cut target DNA, from their original partners, to the 3' ends of Mu.     

Telomere sequences attached to nuclearly migrated yeast linear plasmid

The yeast linear plasmid pCLU1, derived from pGKL1, has terminal proteins (TPs) covalently attached at the 5' ends of inverted terminal repeats (ITRs) and replicates in the cytoplasm, presumably using the TP as a primer for DNA synthesis. In Saccharomyces cerevisiae, under certain conditions, pCLU1 migrated into the nucleus and replicated in either linear or circular form. The linear-form plasmid lacked TPs; instead it carried host-telomere repeats at the ITR ends. The present study showed that (1) the added telomere was primarily composed of the repeated tracts of TGTGTGGGTGTGG, which was complementary to the RNA template of yeast telomerase, (2) the telomeric addition occurred at the very end of the ITRs, and (3) the sequence composition of the added telomeres was diverse among individual plasmids, but symmetrically identical at both ends of each plasmid. A similar mode of telomere addition was also observed in cells defective in the RAD52 gene.     

A novel mechanism for the initiation of Tacaribe arenavirus genome replication.

The ends of arenavirus genome and antigenome RNAs are highly conserved and where determined directly, always contain a 3' G (referred to as position +1). However, primers extended to the 5' ends of Tacaribe virus genomes and antigenomes extend to position -1. When genomes and antigenomes are annealed either inter or intramolecularly and treated with RNase A or T1, there appears to be a single unpaired G at the 5' ends of the hybrids. A single extra G is also found by cloning the 5' ends of S antigenomes, and studies with capping enzyme detect (p)ppG at the 5' ends of genome and antigenome chains. A model is proposed in which genome replication initiates with pppGpC to create the nontemplated extra G. In contrast, the nontemplated bases at the 5' ends of the N mRNAs, which extend to positions -1 to -5, were found to be capped and also heterogeneous in sequence.