Terminal, and telomere-associated proteins of pAL1, a linear plasmid from Arthrobacter nitroguajacolicus Rü61a
Zusammenfassung der Projektergebnisse
The plasmid pAL1 of Arthrobacter nitroguajacolicus Rü61a, which codes for the degradation of 2-methylquinoline, is so far the only described linear replicon within the genus Arthrobacter. Like other linear replicons of actinomycetes, it is characterized by inverted terminal repeats and terminal proteins (TP) covalently bound to its 5´-ends. Based on sequence analyses, we had proposed that DNA replication at the telomere of pAL1 besides TP involves an unprecedented putative telomere associated protein (Tap). The objectives of this first project on an Arthrobacter linear plasmid were (i) to characterize the termini of pAL1, (ii) to identify the proteins bound to the termini and involved in replication at the telomere, and (iii) to characterize the function of these proteins. Assuming that replication of pAL1 – analogous to replication of Streptomyces linear genetic elements – is initated at an internal origin and involves formation of replicative intermediates with recessed 5´-ends, possible secondary structures of terminal 3´-overhangs of pAL1 were predicted. Since the predictions suggested significant differences of the `left´ and `right´ terminus of pAL1, we hypothesized that each terminus might be recognized by a specific protein. However, the gene product of the pAL1.102 locus was found to represent the terminal protein TPpAL1 attached to both ends of the linear plasmid. To address the question of whether TappAL1 (encoded by pAL1.101) together with the terminal protein TPpAL1 and/or other proteins is part of a telomeric complex, octahistidinetagged TPpAL1 was synthesized in the homologous host, and interacting proteins were captured by in vivo formaldehyde crosslinking and affinity chromatography. In tryptic digests of the protein complex, fragments of TPpAL1 and of TappAL1 were identified by LC-MS/MS analysis, suggesting specific interaction of these proteins in vivo. TappAL1 comprises 1707 amino acids and in its C-terminal region shows some sequence similarity to part of the Tap proteins of Streptomycetes. A search for conserved domains revealed putative zinc finger and topoisomerase-primase domains, and part of a superfamily 2 helicase domain, in the N-terminal and central region of TappAL1, respectively. There was no significant similarity to known conserved domains in the C-terminal region of TappAL1, however, sequence alignments tentatively suggested possible partial conservation of motifs that are characteristic of the polymerization domain of family B DNA polymerases. Both recombinant TPpAL1 as well as TappAL were purified as fusions with the maltosebinding protein, which confers in vitro solubility. All functional studies were performed with the fusion proteins. In vitro assays indicated that TappAL1 in the presence of DNA template catalyzes the deoxycytidylation of TPpAL1. TappAL1 moreover exhibits topoisomerase, DNA helicase, and DNA- and TP-primed DNA polymerase activities. Due to its combined helicase and polymerase activity, it mediates isothermal amplification of double-stranded DNA. TappAL1 represents a novel type of replicative enzyme which in principle provides all the catalytic activities necessary for protein-primed continuous synthesis of single-stranded DNA at a parental duplex DNA. However, it is an open question whether pAL1 is replicated from the telomeres in a protein-primed strand displacement reaction, or from an internal origin, resulting in replicative intermediates with recessed 5´-ends that need to be filled in. TappAL1 might either catalyze full-length polymerization of pAL1, or it might act as combined helicase and polymerase in a telomere patching reaction.
Projektbezogene Publikationen (Auswahl)
- (2007) Catabolic linear plasmids. pp 63-98, in: Meinhardt F, Klassen R (eds.) Microbiology Monographs, vol 7: Microbial linear plasmids. Springer-Verlag, Berlin Heidelberg 2007
Fetzner S, Kolkenbrock S, Parschat K
- (2007) Complete nucleotide sequence of the 113-kilobase linear catabolic plasmid pAL1 of Arthrobacter nitroguajacolicus Rü61a and transcriptional analysis of genes involved in quinaldine degradation. J. Bacteriol. 189: 3855-3867
Parschat K, Overhage J, Strittmatter AW, Henne A, Gottschalk G, Fetzner S
- (2007) Terminal protein of the linear plasmid pAL1 of Arthrobacter nitroguajacolicus Rü61a. BIOspektrum Sonderausgabe zur VAAM-Jahrestagung 2007, S. 159, PL011
Kolkenbrock S, Tomm M, Parschat K, Fetzner S
- (2007) The linear plasmid pAL1 of Arthrobacter nitroguajacolicus Rü61a – termini, transcriptional analyses, and copy number. BIOspektrum Sonderausgabe zur VAAM-Jahrestagung 2007, S. 157, PL003
Wagenknecht M, Parschat K, Fetzner S, Klassen R, Meinhardt F
- (2008) Linear plasmid pAL1 of Arthrobacter nitroguajacolicus Rü61a: Terminal protein, and transcriptional analysis of genes potentially involved in replication. BIOspektrum Sonderausgabe zur VAAM-Jahrestagung 2008, S. 176, PN05
Wagenknecht M, Kolkenbrock S, Meinhardt F, Fetzner S
