Telomere associated primase Tap repairs truncated telomeres of Streptomyces.

Replication of the linear chromosomes of soil bacteria Streptomyces proceeds from an internal origin towards the telomeres, followed by patching of the resulting terminal single-strand overhangs by DNA synthesis using terminal proteins as the primer, which remains covalently bound to the 5΄ ends of the DNA. In most Streptomyces chromosomes, the end patching requires the single-strand overhangs, terminal protein Tpg, and terminal associated protein Tap. The telomere overhangs contain several palindromic sequences capable of forming stable hairpins. Previous in vitro deoxynucleotidylation studies indicated that Tap adds the Palindrome I sequence to Tpg, which is extended by a polymerase to fill the gap. In this study, the stringency of Palindrome I sequence was examined by an in vitro deoxynucleotidylation system and in vivo replication. Several nt in Palindrome I were identified to be critical for priming. While the first 3 G on the template were required for deoxynucleotidylation in vitro, deletions of them could be suppressed by the presence of dGTP. In vivo, deletions of these G were also tolerated, and the telomere sequence was restored in the linear plasmid DNA. Our results indicated that the truncated telomeres were repaired by extension synthesis by Tap on the foldback Palindrome I sequence.

Telomere-associated proteins add deoxynucleotides to terminal proteins during replication of the telomeres of linear chromosomes and plasmids in Streptomyces.

Typical telomeres of linear chromosomes and plasmids of soil bacteria Streptomyces consist of tightly packed palindromic sequences with a terminal protein ('TP') covalently attached to the 5' end of the DNA. Replication of these linear replicons is initiated internally and proceeds bidirectionally toward the telomeres, which leaves single-strand overhangs at the 3' ends. These overhangs are filled by DNA synthesis using the TPs as the primers ('end patching'). The gene encoding for typical TP, tpg, forms an operon with tap, encoding an essential telomere-associated protein, which binds TP and the secondary structures formed by the 3' overhangs. Previously one of the two translesion synthesis DNA polymerases, DinB1 or DinB2, was proposed to catalyze the protein-primed synthesis. However, using an in vitro end-patching system, we discovered that Tpg and Tap alone could carry out the protein-primed synthesis to a length of 13 nt. Similarly, an 'atypical' terminal protein, Tpc, and its cognate telomere-associated protein, Tac, of SCP1 plasmid, were sufficient to achieve protein-primed synthesis in the absence of additional polymerase. These results indicate that these two telomere-associated proteins possess polymerase activities alone or in complex with the cognate TPs.

Mutational analysis of the terminal protein Tpg of Streptomyces chromosomes: identification of the deoxynucleotidylation site.

The linear chromosomes and linear plasmids of Streptomyces are capped by terminal proteins (TPs) covalently bound to the 5' ends of the DNA. The TPs serve as primers for DNA synthesis that patches in the single-stranded gaps at the telomeres resulting from the bi-directional replication ('end patching'). Typical Streptomyces TPs, designated Tpgs, are conserved in sequence and size (about 185 amino acids), and contain a predicted helix-turn-helix domain and a functional nuclear localization signal. The Tpg-encoding gene (tpg) is often accompanied by an upstream gene tap that encodes an essential telomere-associating protein. Five lone tpg variants (not accompanied by tap) from various Streptomyces species were tested, and three were found to be pseudogenes. The lone tpg variant on the SLP2 plasmid, although functional, still requires the presence of tap on the chromosome for end patching. Using a combination of in vitro deoxynucleotidylation, physical localization, and genetic analysis, we identified the threonine at position 114 (T114) in Tpg of Streptomyces lividans chromosome as the deoxynucleotidylated site. Interestingly, T114 could be substituted by a serine without destroying the priming activity of Tpg in vitro and in vivo. Such T114S substitution is seen in and a number of pseudogenes as well as functional Tpgs. T114 lies in a predicted coil flanked by two short helixes in a highly hydrophilic region. The location and structural arrangement of the deoxynucleotidylated site in Tpg is similar to those in the TPs of phage ø 29 and adenoviruses. However, these TPs are distinct in their sequences and sizes, indicating that they have evolved independently during evolution. Using naturally occurring and artificially created tpg variants, we further identified several amino acid residues in the N-terminus and the helix-turn-helix domain that were important for functionality.

Translesion-synthesis DNA polymerases participate in replication of the telomeres in Streptomyces.

Linear chromosomes and linear plasmids of Streptomyces are capped by terminal proteins that are covalently bound to the 5'-ends of DNA. Replication is initiated from an internal origin, which leaves single-stranded gaps at the 3'-ends. These gaps are patched by terminal protein-primed DNA synthesis. Streptomyces contain five DNA polymerases: one DNA polymerase I (Pol I), two DNA polymerases III (Pol III) and two DNA polymerases IV (Pol IV). Of these, one Pol III, DnaE1, is essential for replication, and Pol I is not required for end patching. In this study, we found the two Pol IVs (DinB1 and DinB2) to be involved in end patching. dinB1 and dinB2 could not be co-deleted from wild-type strains containing a linear chromosome, but could be co-deleted from mutant strains containing a circular chromosome. The resulting ΔdinB1 ΔdinB2 mutants supported replication of circular but not linear plasmids, and exhibited increased ultraviolet sensitivity and ultraviolet-induced mutagenesis. In contrast, the second Pol III, DnaE2, was not required for replication, end patching, or ultraviolet resistance and mutagenesis. All five polymerase genes are relatively syntenous in the Streptomyces chromosomes, including a 4-bp overlap between dnaE2 and dinB2. Phylogenetic analysis showed that the dinB1-dinB2 duplication occurred in a common actinobacterial ancestor.

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.

The terminal proteins of linear Streptomyces chromosomes and plasmids: a novel class of replication priming proteins.

The chromosomes of the soil bacteria Streptomyces, unlike those of most other bacteria, are linear DNA molecules. Their telomeres contain long-terminal inverted repeats and covalently bound terminal proteins (TPs). These bacteria also harbour linear plasmids that share the same structural features. In this study, we demonstrated that the TP was covalently bound to the 5' ends as proposed previously. A linear plasmid with chromosomal telomeres was constructed and used to purify the TPs of the Streptomyces coelicolor A3(2) chromosome. A 20 kDa protein and its 10 kDa degradation product were isolated and their sequences determined by mass spectrometry. The coding sequence (tpgC) was about 100 kb from the right end of the chromosome. Two tpg homologues were identified by sequencing the 50kb linear plasmid SLP2 of Streptomyces lividans: tpgSLP2 at 6 kb from the left end and a putative tpg pseudogene at 8 kb from the right. The latter was in a terminal repeat shared by the right end of SLP2 and both ends of the S. lividans chromosome. The lack of the typical Streptomyces codon preference in this open reading frame suggests that it is a pseudogene. The close physical linkage between the tpg genes and their cognate telomeres would favour their cosegregation and co-evolution. All the Tpg polypeptides 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.

The homologous terminal sequence of the Streptomyces lividans chromosome and SLP2 plasmid.

The chromosome of Streptomyces lividans shares 15.4 kb homology with one end of the linear plasmid SLP2, consisting of a 10.1 kb terminal sequence followed by the 5.3 kb transposable element Tn4811. The 10.1 kb terminal sequence was determined. The mean G+C content of this sequence is 67.9 mol% with a striking G vs C bias in the last kb. The terminal 232 nt contained 10 palindromic sequences with potential to form complex secondary structures. One typical Streptomyces coding sequence (designated ORF1) of 2643 bp was predicted in the determined sequence. The amino acid sequence of the ORF1 product contained a DEAH helicase motif, and exhibited similarity to type I restriction enzyme HsdR subunits in the database, suggesting a possible role in replication of the telomeres. However, all the ORF1 sequences on the chromosome and SLP2 could be simultaneously knocked out by targeted recombination without affecting the viability of the cells and the linearity of the chromosome and SLP2. This ruled out ORF1 as an essential component in the maintenance of the linear chromosome and plasmids.