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DNA Bound by the Oxytricha Telomere Protein Is Accessible to Telomerase and Other DNA Polymerases DOROTHY E

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DNA Bound by the Oxytricha Telomere Protein Is Accessible to Telomerase and Other DNA Polymerases DOROTHY E Proc. Natl. Acad. Sci. USA Vol. 91, pp. 405-409, January 1994 Biochemistry DNA bound by the Oxytricha telomere protein is accessible to telomerase and other DNA polymerases DOROTHY E. SHIPPEN*, ELIZABETH H. BLACKBURNt, AND CAROLYN M. PRICE0§ tDepartment of Microbiology and Immunology, University of California, San Francisco, CA 94143; and tDepartment of Chemistry, University of Nebraska, Lincoln, NB 68588 Contributed by Elizabeth H. Blackburn, August 25, 1993 ABSTRACT Macronuclear telomeres in Oxytricha exist as oftelomere protein in these two populations is not altered by DNA-protein complexes in which the termini of the G-rich additional nuclease treatment. strands are bound by a 97-kDa telomere protein. During The fragment of DNA bound by the majority of telomere telome'ic DNA replication, the replication machinery must protein molecules corresponds to the most terminal 13 or 14 have access to the G-rich strand. However, given the stability nucleotides of the T4G4T4G4 overhang (4). Dimethyl sulfate of telomere protein binding, it has been unclear how this is footprinting demonstrated that the complex formed between accomplished. In this study we investigated the ability of the telomere protein and the residual DNA fragment retains several different DNA polymerases to access telomeric DNA in the same DNA-protein contacts present at native telomeres Oxytricha telomere protein-DNA complexes. Although DNA (4). Thus, these telomeric DNA-protein complexes are useful bound by the telomere protein is not degraded by micrococcal substrates for in vitro investigations of telomere structure nuclease or labeled by terminal deoxynucleotidyltrnsferase, (10). In this study we have employed the DNA-protein this DNA serves as an efficient primer for the addition of complexes to analyze the interaction of protein-bound telo- telomeric repeats by telomerase, a specialized RNA-dependent meric DNA with components of the DNA replication ma- DNA polymerase (ribonucleoprotein reverse tanscriptase), chinery. EC 2.7.7.49. Moreover, in the presence of a suitable comple- During DNA replication the ribonucleoprotein reverse mentary C-rich DNA template, AMV reverse transcriptase and transcriptase called telomerase-a specialized RNA- the E. cofi Klenow fragment will also elongate DNA bound by dependent DNA polymerase, EC 2.7.7.49-compensates for the telomere protein. These rmdings indicate that the 3' the inability of conventional DNA polymerases to replicate terminus and the Watson-Crick base pairing positions are the extreme terminus of a linear DNA molecule (11, 12). In exposed in the protein complex. We propose that the telomere Oxytricha, as in other organisms, telomerase polymerizes protein can serve a dual role at the telomere by protecting the G-rich repeats onto the 3' terminus of telomeric DNA using DNA phosphate backbone from degradation while simulta- a C-rich telomeric sequence in the RNA subunit as a template neously exposing the DNA bases for replication. (13-15). While much less is known about the replication of the C-rich telomeric strand, it is generally believed that Macronuclear telomeres ofthe ciliate Oxytricha nova exist as synthesis is initiated by a DNA primase that lays down a nonnucleosomal DNA-protein complexes that contain a primer at or near the 3' end ofthe G-rich template strand (11, short stretch of repeated sequence DNA and a 97-kDa 16-19). telomere protein (1-3). The DNA comprises 36 nucleotides of To replicate the two strands of the telomere completely, T4G4 sequence; 20 of the nucleotides are part of a C4A4T4G4 both telomerase and primase must have access to the termi- duplex, while the remaining 16 form a 3' single-stranded nus of the G-rich strand. However, given the very stable overhang. The telomere protein recognizes both the se- interaction between the telomere protein and telomeric quence and structure of the 3' G-rich overhang and, upon DNA, the mechanism by which the replication machinery binding, protects the DNA from nuclease digestion (4, 5). gains access to this DNA has been enigmatic. Here we show Because of these properties, the telomere protein is thought that although DNA bound by the telomere protein is not to form a protective cap-like structure over the terminus of accessible to all DNA-modifying activities, it is accessible to the DNA. The telomere protein is a heterodimer with a telomerase, the Klenow fragment of Escherichia coli DNA 56-kDa a subunit and a 41-kDa ,8 subunit (4). Although the a polymerase I, and avian myeloblastosis virus (AMV) reverse subunit is responsible for the sequence-specific DNA binding transcriptase. These findings suggest that the telomere pro- (6, 7), the ,B subunit stabilizes the DNA-protein complex (7, tein not only can form a protective cap over the end of the 8). chromosome but also can allow simultaneously replication of Since the telomere protein binds DNA in an extremely telomeric DNA. salt-stable manner (2, 3), very pure preparations of the protein can be obtained by simply lysing Oxytricha macro- MATERIALS AND METHODS nuclei in high-salt buffer and then isolating the resulting Isolation of Oxyricha Macronuclei and Telomere Protein. macronuclear DNA-protein complexes by centrifugation Oxytricha macronuclei and telomere protein were isolated as through a CsCl gradient (2-4, 9). Subsequent digestion with described (refs. 4 and 9; see also Introduction). After diges- micrococcal nuclease generates two populations of telomere tion ofmacronuclear DNA-telomere protein complexes with protein molecules. Approximately 60%o of the protein is still micrococcal nuclease, the protein was diluted and reconcen- bound to a fragment of telomeric DNA, while the remaining trated four or five times in a Centricon 30 apparatus (Amicon) 40%o is completely free of DNA and is able to bind exoge- to remove the free nucleotides. Most of the micrococcal nously added telomeric sequences (4, 5). The relative fraction Abbreviation: AMV, avian myeloblastosis virus. The publication costs of this article were defrayed in part by page charge *Present address: Department of Biochemistry and Biophysics, payment. This article must therefore be hereby marked "advertisement" Texas A&M University, College Station, TX 77843. in accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed. 405 Downloaded by guest on September 28, 2021 406 Biochemistry: Shippen et al. Proc. Natl. Acad Sci. USA 91 (1994) nuclease was retained during the Centricon step and re- solved on a sequencing gel, an 8-base repeated pattern mained active until EGTA was added just prior to use. A characteristic of telomere elongation by the Oxytricha telo- hybridization assay was used to determine the amount of tail merase (13) was observed in the presence of the telomere fragment present in each telomere protein preparation. The protein (Fig. 1A, lanes 9-10). In contrast, no products were telomere protein was digested with proteinase K and ex- detected in the absence of the protein (Fig. 1B, lane 4), tracted with phenol/chloroform. Any residual DNA was suggesting that the residual fragment of telomeric DNA dried down and then resuspended in 10 ,l of4x SSC (600 mM bound by the telomere protein was acting as a primer for NaCl/60 mM sodium citrate) plus 15 pmol of 32P-labeled telomerase. To test this hypothesis, telomerase reactions (C4A4)2 oligonucleotide. A series ofcontrol reaction mixtures were performed with a synthetic 13-base oligonucleotide was also prepared. These mixtures contained 0-15 pmol of TG4T4G4 oligonucleotide (synthetic tail fragment), 10 ,ul of corresponding to the telomeric tail fragment (Fig. 1A, lanes 4x SSC, and 15 pmol of 32P-labeled (C4A4)2 oligonucleotide. 11-12). The natural telomeric tail fragment is predominantly The samples were boiled for 2 min, incubated at 37°C and 13 nucleotides in length, although a small proportion of 14 then at 32°C for 15 min, and separated on a 20% nondena- base molecules is also present (4). As the banding pattern turing polyacrylamide gel. The amount of tail fragment pres- produced by telomerase is dependent on the length and 3' ent in a telomere protein preparation was estimated from the sequence of the DNA primer (12), the products generated amount oftail fragment-(C4A4)2 duplex formed relative to the with the synthetic 13-base oligonucleotide were expected to control samples. closely resemble the profile obtained by extension of the Macronuclei containing active telomerase were isolated in telomere protein-bound tail fragment. Indeed, the products the absence of p-(chloromercuri)benzenesulfonic acid (PC- were very similar in these two reactions (Fig. 1A, compare MBS) and purified over a Percoll/Nycodenz gradient as lanes 9 and 10 to lanes 11 and 12); the lack of a 5' phosphate described (13). on the synthetic tail primer accounts for the slight offset ofthe Telomerase Assays. To solubilize the Oxytricha telomerase, two profiles. These results strongly suggested that DNA macronuclei were resuspended in 1 mM Tris, pH 7.0/0.1 mM bound by the telomere protein can serve as a primer for EDTA/300 mM potassium glutamate and stored on ice for 30 telomerase. min. Insoluble nuclear debris was removed by centrifugation, Two lines of evidence indicated that extension of the and the supernatant was adjusted to contain 10 mM Tris (pH telomere protein-bound tail fragment by telomerase did not 7.0) and 1 mM MgCl2. Tetrahymena telomerase consisted of occur because this DNA was free in solution. First, priming enzyme fractions purified through Sephacryl S-500 (Pharma- activity from the tail-fragment DNA was resistant to micro- cia) and heparin-agarose (Bio-Rad) (14). Twenty-microliter telomerase reactions were conducted as described (15) ex- BO cept that EGTA was added to 20 mM. xo Immediately before addition to telomerase reactions, ali- quots of telomere protein were incubated for 15 min at 37°C to activate micrococcal nuclease carried with the telomere protein.
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