Abstract
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.
Highlights
Soil bacteria of the genus Streptomyces possess linear chromosomes and linear plasmids that are capped by terminal proteins (TPs) covalently bound to the 59 ends of the DNA [1,2]
Replication of these linear replicons is accomplished in two steps: (i) bidirectional replication initiated from an internal origin, which results in single-stranded gaps at the 39 end; and (ii) patching of the single-stranded gaps by TP-primed DNA synthesis [3]
In vitro deoxynucleotidylation of TPs typically selects the nucleotide corresponding to the first nucleotide at the 59 end of the replicons, such as dCMP for adenovirus-2 [35], dAMP for phage ø29 [21], dCMP for linear plasmid pAL1of Arthrobacter nitroguajacolicus [36], dGMP for linear plasmid SCP1 of Streptomyces (Tsai, H.-H, unpublished data), and dCMP for the S. lividans chromosome [19]
Summary
Soil bacteria of the genus Streptomyces possess linear chromosomes and linear plasmids that are capped by terminal proteins (TPs) covalently bound to the 59 ends of the DNA [1,2]. In the case of S. lividans chromosome, dCMP was shown to attach to a Thr residue of its TP (designated TpgSli) in in vitro deoxynucleotidylation [19].
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