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.
Soil bacteria Streptomyces are the most important producers of secondary metabolites, including most known antibiotics. These bacteria and their close relatives are unique in possessing linear chromosomes, which typically harbor 20 to 30 biosynthetic gene clusters of tens to hundreds of kb in length. Many Streptomyces chromosomes are accompanied by linear plasmids with sizes ranging from several to several hundred kb. The large linear plasmids also often contain biosynthetic gene clusters. We have developed a targeted recombination procedure for arm exchanges between a linear plasmid and a linear chromosome. A chromosomal segment inserted in an artificially constructed plasmid allows homologous recombination between the two replicons at the homology. Depending on the design, the recombination may result in two recombinant replicons or a single recombinant chromosome with the loss of the recombinant plasmid that lacks a replication origin. The efficiency of such targeted recombination ranges from 9 to 83% depending on the locations of the homology (and thus the size of the chromosomal arm exchanged), essentially eliminating the necessity of selection. The targeted recombination is useful for the efficient engineering of the Streptomyces genome for large-scale deletion, addition, and shuffling.
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