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Reverse Transcriptase Activity Innate to DNA Polymerase I and DNA

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Reverse Transcriptase Activity Innate to DNA Polymerase I and DNA Reverse transcriptase activity innate to DNA SEE COMMENTARY polymerase I and DNA topoisomerase I proteins of Streptomyces telomere complex Kai Bao*† and Stanley N. Cohen*‡§ Departments of *Genetics and ‡Medicine, Stanford University School of Medicine, Stanford, CA 94305-5120 Edited by Nicholas R. Cozzarelli, University of California, Berkeley, CA, and approved August 10, 2004 (received for review June 18, 2004) Replication of Streptomyces linear chromosomes and plasmids gation and, remarkably, that both of these eubacterial enzymes -proceeds bidirectionally from a central origin, leaving recessed 5؅ can function efficiently as RTs in addition to having the bio termini that are extended by a telomere binding complex. This chemical properties predicted from their sequences. We further complex contains both a telomere-protecting terminal protein show that the Streptomyces coelicolor and Streptomyces lividans (Tpg) and a telomere-associated protein that interacts with Tpg TopA proteins are prototypes for a subfamily of bacterial and the DNA ends of linear Streptomyces replicons. By using topoisomerases whose catalytic domains contain a unique Asp– histidine-tagged telomere-associated protein (Tap) as a scaffold, Asp doublet motif that is required for their RT activity and which we identified DNA polymerase (PolA) and topoisomerase I (TopA) is essential also to the RNA-dependent DNA polymerase func- proteins as other components of the Streptomyces telomere com- tions of HIV RT and eukaryotic cell telomerases (16, 17). plex. Biochemical characterization of these proteins indicated that both PolA and TopA exhibit highly efficient reverse transcriptase Materials and Methods (RT) activity in addition to their predicted functions. Although RT Plasmid and Bacterial Strains. S. coelicolor strains M145 and S. activity innate to other DNA-dependent DNA polymerases has lividans 1326 (18) were kindly provided by D. A. Hopwood (John been observed previously, its occurrence in a topoisomerase is Innes Center, Norwich, U.K.). The Streptomyces–Escherichia coli unprecedented. Deletion mapping and sequence analysis showed shuttle plasmid pHZ1272, which replicates in both hosts and was that the RT activity of Streptomcyces TopA resides in a peptide used for gene expression in Streptomyces, was kindly provided by region containing motifs that are absent from most bacterial Z. Deng (Shanghai Jiaotong University, Shanghai, China) in a topoisomerases but are highly conserved in a novel subfamily of personal communication. S. lividans strain BKKO19, in which eubacterial topoisomerases found largely in Actinobacteria. Within the originally linear chromosome had circularized (15), was used one of these motifs, and essential to the RT function of Strepto- to generate deletions of the topAorpolA gene as described in myces TopA, is an Asp–Asp doublet sequence required also for the ref. 15. RT activities of human immunodeficiency virus and eukaryotic cell telomerases. Expression and Purification of Tap. The tap gene of S. coelicolor (15) was amplified by using a pair of primers: 5Ј-AAACATAT- telomere replication ͉ TopA ͉ PolA GCATCATCATCATCATCATGTGTCCGGTAGAGGAGC- GCAG-3Ј and 5Ј-GCCGTTGCCGAACAGGCTCAT-3Ј, and ecause DNA replication requires both a primer and template the amplicon was inserted into the NdeI͞EcoRI-digested Band can proceed only in the 5Ј to 3Ј direction, replication at pHZ1272 vector (Z. Deng, personal communication), generat- the telomeric ends of linear plasmids and chromosomes requires ing plasmid pBC174. The correctness of the construct was a specialized mechanism to ensure that full-length replicas of confirmed by DNA sequence analysis. pBC174 was introduced both DNA strands are produced (1). In eukaryotes, the preser- by transformation into S. lividans 1326 for expressing Tap vation of telomere length involves the synthesis of tandem DNA containing an NH2-terminal his6 tag encoded by the primer. repeats on an RNA template; telomerase, a ribonucleoprotein Bacterial cultures were grown in yeast extract͞malt extract that functions as an RNA-dependent DNA polymerase (i.e., (YEME) medium (19) for 12 h, and after treatment with 10 reverse transcriptase, RT), plays a crucial role in this process ␮g͞ml thiostrepton at 30°C for 12 h, cells were harvested and ͞ (2–5). resuspended in L buffer (50 mM NaH2PO4,pH7.5300 mM Streptomyces are eubacteria that contain linear plasmids and NaCl͞10 mM imidazole͞10% glycerol͞0.2% Triton X-100͞5 chromosomes, as well as circular replicons (6, 7). In contrast to mM 2-mercaptoethanol͞1 mM PMSF) before lysis by a French eukaryotic cell adenoviruses and bacteriophage Ø29 of Bacillus press (pressure at 1,000 kg͞cm2). DNA in the lysis mixture was subtilis, which replicate full-length linear DNA molecules by a sheared by sonication, and cell debris was removed by centrif- strand displacement mechanism (8, 9), replication of linear ugation (39,000 ϫ g for 20 min). The supernatant was incubated DNAs in Streptomyces is initiated bidirectionally from an internal at 4°C for 90 min with Ni-NTA resin (Qiagen, Valencia, CA), origin, generating 3Ј leading strand overhangs on replication which was then poured into a column and washed sequentially intermediates (10). Extension of the recessed 5Ј ends is accom- with L buffer containing increasing concentrations of imidazole. plished by a still-unknown mechanism that requires the functions BIOCHEMISTRY of (i) telomeric inverted repeats (11, 12), (ii) a 21-kDa terminal protein (Tpg) that is attached covalently to 5Ј DNA termini of This paper was submitted directly (Track II) to the PNAS office. postreplicative DNA molecules and is believed to act as a primer Freely available online through the PNAS open access option. for lagging strand DNA replication (13, 14), and (iii) Tap, an Abbreviations: RT, reverse transcriptase; Tap, telomere-associated protein. 80-kDa telomere-associated protein that interacts with both Tpg See Commentary on page 14307. and the overhanging 3Ј strand sequence of replication interme- †Present address: Department of Molecular Biology, Genencor International, Palo Alto, CA diates (15). We report here that the telomere complex of 94304. Streptomyces linear replicons also includes DNA polymerase and §To whom correspondence should be addressed. E-mail: [email protected]. DNA topoisomerase proteins essential to Streptomyces propa- © 2004 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0404386101 PNAS ͉ October 5, 2004 ͉ vol. 101 ͉ no. 40 ͉ 14361–14366 Downloaded by guest on September 30, 2021 The Tap–His-6 fusion protein, which was detected in Ni-NTA temperature for 1 h. The denatured protein was then diluted with column eluates by Western blot analysis using anti-His-tag 0.55 ml of ice-cold dilution buffer (denaturing buffer without antibody (Qiagen) was eluted from the column at Ϸ100 mM guanidine-HCl), thoroughly mixed, and diluted with another imidazole. Tap-containing fractions were dialyzed against S 0.55-ml aliquot dilution buffer. Proteins were allowed to refold buffer (50 mM Tris⅐HCl, pH 8.0͞100 mM NaCl͞0.5 mM EDTA͞ for 15 h at 4°C and then dialyzed twice in 500 vol of dilution 0.5 mM DTT͞10% glycerol) and stored at Ϫ80°C. bufferfor6hat4°C. After dialysis, the samples were concen- trated by ultrafiltration on a Centricon Plus-20 tube (Amicon) RT Activity Assays. RT assays were carried out in 20-␮l reaction and then either assayed for RT activity or aliquoted and frozen. mixtures containing 20 mM Tris⅐HCl (pH 8.0), 100 mM NaCl, 1 ͞␮ mM DTT, 1 mM MgCl2,50ng l poly(rA)-(dT)12–18 (Amer- Protein MS and Amino Acid Sequence Analysis. Protein MS and sham Pharmacia), 10 ␮Ci (1 Ci ϭ 37 GBq) of [32P]dTTP amino acid sequence analysis were performed at the Stanford (Amersham Pharmacia), and 1-␮l volumes of protein fractions. Protein and Nucleic Acid facility and the Harvard Microchem- Reaction mixtures were incubated at 37°C for 45 min, stopped by istry Facility (Boston). Proteins eluted from SDS͞PAGE gels addition of 10 ␮l of 200 mM EDTA, and purified by using were processed by proteolytic digestion, microcapillary HPLC, MicroSpin S-200 HR columns (Amersham Pharmacia). Reac- nano-Electrospray Ionization (ESI), and ion trap tandem MS tion products were analyzed by autoradiography after electro- analysis. For NH2-terminal sequence analysis, proteins were phoresis on 10% polyacrylamide, 8 M urea sequencing gels in blotted onto poly(vinylidene difluoride) membrane according to buffer containing 95% formamide, 20 mM EDTA, 0.05% bro- the guidelines of the Stanford Protein and Nucleic Acid facility. mophenol blue, and 0.05% xylene cyanol FF. RT assays using mRNA isolated from a murine fibroblast 3T3 cell line as Cloning and Expression of PolA and TopA of Streptomyces and E. coli. template were carried out as follows: poly(A)-containing RNA The polA and topA genes of S. coelicolor M145 and E. coli N3433 was isolated from cell cultures by using the FastTrack 2.0 kit were amplified by PCR and cloned into the pET28a (Novagen) (Invitrogen). RT reaction mixtures (20-␮l volumes) reaction expression vector. The His-tagged fusion proteins were purified contained 20 mM Tris⅐HCl (pH 8.0), 100 mM NaCl, 1 mM DTT, by chromatography on Ni-NTA resin (Qiagen) and confirmed by ␮ ␮ 1 mM MgCl2,50 M dATP, dGTP, and dTTP, 10 M dCTP, 10 Western blot analysis using anti-His antibody. The polyhistidine ␮Ci of [␣-32P]dCTP (Amersham Pharmacia), 0.1-␮g aliquots of tag was removed from TopA by incubation with thrombin poly(A)-mRNA, 50 ng of random (dNTP)6 primers (New
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