The terminal proteins of linear Streptomyces chromosomes and plasmids: a novel class of replication priming proteins tides are similar in length (184-185 amino acids) and sequences, which include a putative helix domain that is homologous to part of the DNA-binding 'thumb' domain of HIV reverse transcriptase, and a putative amphiphilic beta-sheet that may be involved in the observed self-aggregation of the TP and/or the proposed membrane binding. IntroductionSoil bacteria of the genus Streptomyces are unusual among bacteria in having protein-capped linear chromosomes . The terminal sequences of these chromosomes consist of inverted repeats (TIR) of variable lengths ranging from 20 to 550 kb. The nucleotide sequences of these TIR are generally not conserved except for the first ª 200 bp, which are packed with palindromic sequences (Huang et al., 1998a). Streptomyces species also harbour many linear plasmids of a wide range of sizes (tens to hundreds of kilobases). Like the Streptomyces chromosomes, these linear plasmids contain TIR and covalently bound terminal proteins (TPs).Both linear chromosomes and linear plasmids of Streptomyces are replicated from internal origins (Chang and Cohen, 1994;Musialowski et al., 1994). This would leave single-stranded gaps at the 3¢ ends of the replicons (Chang and Cohen, 1994), which are patched by an as yet undefined mechanism presumably involving the TPs (Chen, 1996).The presence of the covalently linked TPs at the telomeres has been indicated by various lines of experimental evidence, including immobilization or retardation of the telomere DNA during electrophoresis in the absence of proteolytic or denaturing treatments (Ito et al., 1978), protection against exonuclease digestion and binding to silica (Coombs and Pearson, 1978;Thomas et al., 1979). The TPs may be readily removed when subjected to mild alkaline conditions, suggesting that the DNA-protein phosphodiester linkage is at a serine residue on the TP. That the TP is linked to the 5¢ end of the DNA has been suggested by protection of the terminal DNA from 5¢ exonucleases but not 3¢ exonucleases, although in at least one case, both 5¢ and 3¢ ends are protected .Linear DNA molecules with 5¢ covalently bound TPs were discovered previously in many eukaryotic and prokaryotic viruses and plasmids, the best studied of which are adenoviruses and bacillus phage f29. These viral genomes have relatively very short (as short as 6 bp in adenoviruses) TIR and are replicated from end to end using the TP as the primer. The TP-primed replication initiation of these viruses contrasts with the proposed TPprimed patching at the Streptomyces telomeres, in that the substrate for the former is double stranded, whereas that for the latter is single stranded. This difference may explain the need for extensive palindromes in the latter, which supposedly fold into elaborate secondary structures containing many hairpin loops and 'bulges' closed by purine:purine sheared pairs resembling those found in the genomes of autonomous parvoviruses (Huang...

SummaryLinear plasmids and chromosomes of Streptomyces carry terminal proteins (TPs) covalently attached to the 5Ј 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.

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.