Reverse transcriptase activity innate to DNA polymerase I and DNA topoisomerase I proteins of Streptomyces telomere complex

Abstract

Replication of Streptomyces linear chromosomes and plasmids proceeds bidirectionally from a central origin, leaving recessed 5' termini that are extended by a telomere binding complex. This complex contains both a telomere-protecting terminal protein (Tpg) and a telomere-associated protein that interacts with Tpg and the DNA ends of linear Streptomyces replicons. By using histidine-tagged telomere-associated protein (Tap) as a scaffold, we identified DNA polymerase (PolA) and topoisomerase I (TopA) proteins as other components of the Streptomyces telomere complex. Biochemical characterization of these proteins indicated that both PolA and TopA exhibit highly efficient reverse transcriptase (RT) activity in addition to their predicted functions. Although RT activity innate to other DNA-dependent DNA polymerases has been observed previously, its occurrence in a topoisomerase is unprecedented. Deletion mapping and sequence analysis showed that the RT activity of Streptomcyces TopA resides in a peptide region containing motifs that are absent from most bacterial topoisomerases but are highly conserved in a novel subfamily of eubacterial topoisomerases found largely in Actinobacteria. Within one of these motifs, and essential to the RT function of Streptomyces TopA, is an Asp-Asp doublet sequence required also for the RT activities of human immunodeficiency virus and eukaryotic cell telomerases.

Fig. 1.

RT activity of components of the telomere replication complex of Streptomyces linear replicon. (A) SDS/PAGE analysis of components with RT activity in extracts of S. lividans transfected with pBC174 expressing His-6-tagged Tap protein. Samples were precipitated with 0.015% deoxycholate/5% trichloroacetic acid. Proteins were visualized by Coomassie blue staining. m, protein molecular mass marker (Bio-Rad) shown at left; the approximate mass of marker bands is indicated. Samples isolated at disparate steps in purification are shown. Lanes a and b, corresponding chromatography column fractions eluted from Ni-NTA resin that had been loaded with lysate containing only the expression vector (a) or the same vector expressing Tap. Fractions containing separate protein bands purified to apparent homogeneity by using RT activity as an assay are shown in lanes c and d. The positions of the two purified protein bands and of the 80-kDa Tap protein are indicated at right. (B) Lanes a-d show RT activity analyses of the fractions in A by using polyA as the template. ss, DNA synthesized on polyA template by SuperScript II reverse transcriptase (Invitrogen) was used as a positive control. Lanes e and f show the RT activity of proteins renatured from SDS/PAGE gel corresponding to the single protein band in lanes c and d of A. (C) RT activity analysis by using murine fibroblast cell 3T3 mRNA as template. Lane M, 1-kb DNA ladder labeled with ³²P by using KinaseMax 5′ End-Labeling Kit (Ambion, Austin, TX). Lanes ss, c, and d show the DNA synthesized on 3T3 mRNA template by SuperScript II reverse transcriptase (Invitrogen) by using protein fractions c and d of A. Lane e, negative control in which no protein was added to the reaction mixture.

Fig. 2.

Analysis of catalytic activity of DNA topoisomerase I. Proteins are used for analyzed as follows: TopA_E, E. coli TopA; TopA_S, S. coelicolor TopA; N^-, truncated S. coelicolor TopA proteins lacking the NH₂-terminal 140 aa; C^-, truncated S. coelicolor TopA proteins lacking the COOH-terminal 353 aa; N^-C^-, truncated S. coelicolor TopA proteins lacking both NH₂-terminal and COOH-terminal segments; DD→AA, mutated S. coelicolor TopA protein containing amino acids substitutions of Asp-Asp with Ala-Ala doublet. (A) Assay of DNA topoisomerase activity. Negatively supercoiled plasmid pSP72 DNA (lane plasmid, isolated from E. coli by Qiagen miniprep kit) and the same DNAs relaxed by treatment with above proteins were resolved in a 0.7% agarose gel and stained with ethidium bromide. The relative positions of the negatively supercoiled DNA, and the relaxed plasmid DNA topoisomers ladder are as indicated. (B) RT activity analysis of proteins as indicated above by using a poly(A) template. Lane ss, SuperScript II RT (Invitrogen) used as a positive control.

Fig. 3.

Schematic diagram showing Streptomyces TopA regions identified by blast protein sequence search. An ≈600-aa segment corresponding to the TopA domain of multiple bacterial proteins annotated as topoisomerases contains two short segments (amino acid residues 221-264 and 441-498 of the S. coelicolor TopA protein) that are absent in most of these proteins. These segments are conserved in proteins annotated as topoisomerases in the subclass of enzymes shown in B. As shown, the 441-498 segment contains two Asp-Asp doublets. (B) Alignment of the conserved motif regions of the subfamily bacterial topoisomerases. The bold DD in S. coelicolor are the DD doublets that were mutated to Ala doublets. (C) Phylogenetic analysis of proteins annotated from DNA sequences of certain bacterial genomes as topoisomerase I enzymes. The subfamily identified by the analysis is discussed in the text. The Calculation Phylogeny Unweighted Pair Group Method with Arithmetic Mean Analysis (UPGMA) Tree program was used for analysis. The numbers indicate tree distance proportional to the amount of inferred evolutionary change.