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. When indicated, an additional fresh aliquot of mi- crococcal nuclease was added. To inactivate the nuclease, 15 A ,, co mM EGTA was added. We estimate that telomere protein -CO C,o co 0 was in a 10- to 100-fold excess over telomerase in our assays; C. t X- Transferase, the 0 Klenow Fragment, or Reverse Transcriptase. One microliter 0 of telomere protein (2-15 pmol of tail fragment) was incu- bated with 6 micromolar units of micrococcal nuclease for 30 0 min at 37°C and then added to the labeling reaction. Labeling with terminal transferase and cordycepin [32P]triphosphate was performed as described (3, 4). When required, 3 pmol of TG4T4G4 synthetic tail oligonucleotide or a nontelomeric oligonucleotide, 5'-CTCCTGCAGGATTTTCTCAATG- GCGTGTGGGGAT-3' (a portion of the Euplotes crassus t3,?K actin gene), was added to the reaction. *-input 13-mer The Klenow fragment (United States Biochemical, EC 2.7.7.7) and reverse transcriptase (Promega, EC 2.7.7.49) 1 2 3 4 5 6 7 8 9 10 1112 13 1 2 3 4 reactions were performed as described by the manufacturers FIG. 1. Extension ofthe telomere protein-bound tail fragment by except that 15 mM EGTA was added. Three picomoles of telomerase. (A) Reactions with the Oxytricha telomerase. In dupli- template oligonucleotide (C4A4)4 was added per reaction cate reactions, macronuclear extracts were incubated with 2 pmol of when required. Control reactions lacking telomere protein telomeric primer or 6 pmol of telomere protein-bound tail fragment. contained 3 pmol of TG4T4G4 synthetic tail oligonucleotide. Lanes: 1 and 2, (T4G4)3; 3 and 4, (T4G4)3 plus telomere protein; 5 and 6, (T2AG3)3; 7 and 8, (T2AG3)3 plus telomere protein; 9 and 10, telomere protein only; 11 and 12, synthetic tail fragment TG4T4G4; RESULTS 13, 32P-labeled synthetic tail fragment (arrow). (B) Telomere protein- Elongation ofthe Telomere Protein-Bound Tail Fragment by bound tail-fragment elongation by Oxytricha and Tetrahymena telo- merases. Telomere protein was preincubated with an additional Telomerase. Native telomere protein-DNA complexes iso- aliquot of micrococcal nuclease just prior to addition to telomerase lated from Oxytricha were digested with micrococcal nucle- reactions. Assays were carried out with 0.5 pmol of telomere protein ase to remove any DNA not tightly bound by the telomere in the absence of exogenous telomeric primer. Lanes: 1, 32P-labeled protein. After nuclease treatment, telomere protein was synthetic telomeric tail fragment; 2, Oxytricha telomerase reaction; added to telomerase reactions containing Oxytricha macro- 3, Tetrahymena telomerase reaction; and 4, Oxytricha telomerase nuclear extract and dNTPs. When the products were re- reaction without added telomere protein. Downloaded by guest on September 28, 2021 Biochemistry: Shippen et al. Proc. Natl. Acad. Sci. USA 91 (1994) 407 coccal nuclease treatment. Telomere protein samples incu- Table 1. Labeling of the telomere protein-bound tail fragment by bated with a fresh aliquot of micrococcal nuclease, in addi- terminal deoxynucleotidyltransferase tion to the nuclease carried over from the isolation proce- Cordycepin dure, efficiently primed telomere repeat addition by Mnase [32P]triphosphate telomerase (Fig. 1B, lanes 2 and 3). Moreover, the 3' termi- Reaction components treatment incorporated, cpm nus of the telomeric tail is not accessible to terminal deoxy- nucleotidyltransferase (see below). Synthetic tail - 12,860 To gauge how efficiently DNA bound by the telomere + 120 protein is elongated by telomerase, we assayed tail fragment +* 6,000 extension in the presence of exogenous telomeric oligonu- Telomere proteint + 360 cleotide primers. Telomere protein (carrying =6 pmol of + oligot +§ 8,410 tail-fragment DNA) was added to telomerase reactions that Oligot - 12,150 contained 2 pmol of either the Oxytricha telomeric sequence Synthetic tail, telomere protein-bound tail fragment, control oli- (T4G4)3 or the human sequence (T2AG3)3. In both cases, gonucleotide (oligo), or telomere protein and oligo, were incubated telomerase added telomeric repeats to the protein-bound tail with terminal deoxynucleotidyltransferase and 32P-labeled nucleo- fragment (Fig. 1A, lanes 3 and 4, 7 and 8) instead of the tide. Mnase, micrococcal nuclease. oligonucleotide primers (Fig. 1A, lanes 1 and 2, 5 and 6). *Sample was treated with inactivated micrococcal nuclease. tFive different telomere protein preparations were tested, and the From these findings we conclude that DNA bound by the mean level of labeling was determined. telomere protein can compete effectively with naked DNA *Oligonucleotide was derived from Euplotes crassus actin gene for telomerase. sequence. Recognition and priming ofthe Oxytricha telomere protein- §Telomere protein was predigested with micrococcal nuclease before bound tail fragment was not specific for the Oxytricha the oligonucleotide was added to the reaction. telomerase. The Tetrahymena enzyme also extended the protein-bound tail (Fig. 1B, lane 3), generating even longer protein was efficiently labeled by terminal transferase (Table products than the Oxytricha enzyme (Fig. 1B, lane 2). The 1). These results show that DNA bound by the telomere ability of the Tetrahymena telomerase to extend the Ox- protein is not accessible to all DNA polymerase activities. ytricha telomere protein-bound tail fragment demonstrates Assays with the Klenow fragment and reverse transcrip- that the interaction between telomerase and the telomere tase were carried out with the same telomere protein prep- protein is not species-specific. Rather, DNA bound by the arations used in the terminal deoxynucleotidyltransferase telomere protein appears to be generally accessible to telo- reactions. Telomere protein samples containing -3-5 pmol merases. of tail fragment were incubated with 3 pmol of (C4A4)4 Extension of the Telomere Protein-Bound Tail Fragment by template oligonucleotide, dTTP, [32P]dGTP, and either the Other DNA Polymerases. Because the telomere protein pro- Klenow fragment or reverse transcriptase. Reactions con- tects the tail fragment from nuclease digestion (4), it has been taining the telomere protein-bound tail fragment exhibited assumed that this piece ofDNA is buried within the telomere significant incorporation of [32P]dGTP compared with con- protein molecule. In the light of this assumption, our obser- trols containing 3 pmol of the 13-nucleotide synthetic tail vation that the tail fragment is accessible to telomerase was (Table 2). This incorporation was dependent upon the addi- unexpected. There are two possible explanations for our tion of C4A4 template. Digestion of the synthetic tail with finding. First, a specific protein-protein interaction between micrococcal nuclease prior to adding the other reaction telomerase and the telomere protein could cause the tail components abolished [32P]dGTP incorporation. Thus, the fragment to become accessible. Alternatively, although the activity of the micrococcal nuclease was confirmed and, phosphate backbone of the DNA is protected from nuclease hence, its efficacy at removing any free tail fragment. When digestion, the bases and 3' terminus might be exposed so that examined by gel electrophoresis, reactions with the telomere the tail fragment could be extended by many different DNA protein and the synthetic tail fragment yielded similar prod- polymerases. ucts (data not shown). The ability of the Klenow fragment We examined these possibilities by exploring the accessi- and reverse transcriptase as well as telomerase to elongate bility of the telomere protein-bound tail fragment to three DNA bound by the telomere protein argues strongly that the DNA polymerase activities: calf thymus terminal deoxynu- DNA bases are exposed in the protein complex and available cleotidyltransferase, the Klenow fragment of E. coli DNA to form Watson-Crick base pairs with a complementary DNA polymerase I, and AMV reverse transcriptase. Like telo- or RNA template molecule. merase, the Klenow fragment and AMV reverse transcrip- tase extend primers that are base-paired to a complementary Table 2. Klenow fragment and AMV reverse transcriptase template molecule. Thus, extension of the tail fragment by extension of telomere protein-bound DNA either of these enzymes would occur only if the 3' terminus [32P]dGTP incorporated, of the tail fragment were exposed and some ofthe bases were able to pair with bases of the C4A4 template molecule. In Mnase mean cpm contrast, terminal transferase is a nontemplate-directed poly- Reaction components treatment Klenow fragment AMV RT merase that appears to interact with the sugar phosphate Synthetic tail - 150 170 backbone of the primer DNA (21). + (C4A4)4 - 16,850 20,210 Fortheterminaldeoxynucleotidyltransferase reaction, telo- + 270 110 mere protein was digested heavily with micrococcal nuclease +* 14,210 19,700 (to remove any free tail fragment) and incubated with termi- Telomere protein + 180 190 nal transferase and cordycepin [32P]triphosphate. The reac- + (C4A4)4 + 9,400 13,740 tion products were then examined by a DE 81 filter-binding Synthetic tail fragment or telomere protein-bound tail fragment assay to determine the amount of 32p incorporated. No was incubated with Klenow fragment or AMV reverse transcriptase labeling ofthe tail fragment was observed with any ofthe five (RT) and 32P-labeled nucleotide. Mnase, micrococcal nuclease. Four telomere protein preparations tested (Table 1). The terminal (Klenow fragment) or five (AMV RT) different telomere protein transferase was not inactivated by a contaminant in the preparations were tested and the mean label incorporated was telomere protein preparation because an exogenous oligonu- determined. cleotide added to one of the reactions containing telomere *Sample was treated with inactivated micrococcal nuclease. Downloaded by guest on September 28, 2021 408 Biochemistry: Shippen et al. Proc. Natl. Acad Sci. USA 91 (1994) Accessibility of the Telomere Protein-Bound Tail Fragment. Table 3. Klenow fragment extension of the telomeric tail in Previous estimates indicate that -60% of telomere protein boiled and native telomere protein preparations molecules isolated from Oxytricha contain a telomeric tail Telomere protein [32P]dGTP fragment (5). To examine whether all of these protein mol- ecules leave the tail accessible, we compared extension ofthe Sample Preparation incorporated, cpm tail fragment in reactions containing either native or boiled 1 Boiled 42,800 telomere protein. Boiling causes denaturation of the protein Native 9,520 and hence release of the tail fragment (ref. 2; C.M.P., 2 Boiled 40,780 unpublished results). A sample of telomere protein was Native 23,150 predigested with micrococcal nuclease and then split into two 3 Boiled 48,340 equal aliquots. One aliquot was added directly to a polymer- Native 8,590 ase reaction containing either telomerase or the Klenow One microliter of boiled or unboiled telomere protein from three fragment, while the second aliquot was first boiled for 2 min different samples was added to a Klenow reaction mix containing 3 and then added to the reaction. Telomerase reaction products pmol of (C4A4)4 template oligonucleotide. The telomere protein were examined on a sequencing gel (Fig. 2), and the extent of sample contained -3-4 pmol of tail fragment per gl (sample 1), 2 [32P]dGTP incorporation in the Klenow reaction was moni- pmol (sample 2), or 15 pmol (sample 3). tored by DE 81 assay (Table 3). For both polymerase reactions, a striking increase in extension ofthe tail fragment increase the amount of the extension reaction. However, if was observed with boiled telomere protein samples. In telo- the tail-fragment DNA were completely sequestered by the merase reactions, the abundance of tail elongation products telomere protein, addition ofexcess template oligonucleotide was -5-fold greater with boiled telomere protein than native should not alter 32p incorporation. protein (Fig. 2, compare lanes 3 and 4). Similarly, reactions When increasing amounts of (C4A4)4 template were added with the Klenow fragment using boiled telomere protein to Klenow reactions containing a constant amount of native incorporated 2- to 6-fold more 32p than assays conducted with telomere protein, [32P]dGTP incorporation increased gradu- native telomere protein (Table 3). These results suggest that ally until a plateau was reached (Fig. 3). In contrast, exper- the telomere protein-bound tail fragment is significantly less iments using the same concentration of boiled telomere accessible to telomerase and the Klenow fragment than DNA protein reached a plateau much more rapidly, and saturating free in solution. amounts oftemplate resulted in roughly double the amount of It could be that all of the telomere protein molecules bind [32P]dGTP incorporation. These results suggest that the tail the tail fragment in such a way as to leave the DNA generally fragment in -50% of the complexes is accessible for base less accessible than DNA free in solution. Alternatively, pairing and extension. However, the large excess oftemplate there may be different populations oftelomere protein-DNA required to drive the reaction to completion indicates that complexes in which the DNA is completely buried in some DNA bound by the telomere protein is generally less acces- complexes and is accessible in others. We investigated these sible than DNA that is free in solution. possibilities by assaying the ability ofthe Klenow fragment to extend the telomere protein-bound DNA fragment in the presence of increasing concentrations of a complementary DISCUSSION C4A4 template oligonucleotide. We reasoned that if the tail Historically, the telomere protein has been viewed as a fragment were only partially accessible, addition of excess protective cap that shields the 3' terminus of the telomere template might drive the formation of duplexes and hence from attack by nucleases (2, 3). In this paper we provide evidence that the telomere protein surrounds telomeric DNA less fully than was previously thought. Our data suggest that +w~\°~~iS,.A000\Q the telomere protein-DNA complex is not only unusual from a structural point of view, but may also provide a solution to 10 0I -8 - x Q 7- C 6- 0 -i, 0 5 Q-

FIG. 2. Increased elongation °._ 4-~ of the telomere protein-bound tail CLI- 3 -4 fragment by telomerase after de- /A naturation. Oxytricha telomerase v 2- activity was assayed with an equal I amount of native telomere protein c2-.1cmJ ~I (lane 1) or boiled telomere protein rsn j (lane 2) containing 2.5 pmol of tail v Ip II . II I .I rI fragment. In a separate experi- 0 20 40 60 80 100 120 ment, Oxytricha telomerase reac- (C4A4)4, pmol tions were carried out with native telomere protein containing 2.5 FIG. 3. Effect of increasing C4A4 template concentration on tail pmol of tail fragment (lane 3) or fragment extension. Increasing amounts of (C4A4)4 were added to boiled telomere protein carrying reactions containing constant amounts of the Klenow fragment 0.5 pmol oftail fragment (lane 4) or and boiled (-) or native telomere protein (---). The extent of 1 2 3 4 5 2.5 pmol of tail fragment (lane 5). [32P]dGTP incorporation was measured by DE 81 assay. Downloaded by guest on September 28, 2021 Biochemistry: Shippen et al. Proc. Natl. Acad. Sci. USA 91 (1994) 409 the problem of accessibility faced by the DNA replication exposure of the DNA bases for activity. Once the Oxytricha machinery at the telomere. primase has been isolated, it will be feasible to test this Structure of the Telomere Protein-DNA Complex. As the possibility directly. sugar phosphate backbone ofthe telomeric DNA is protected Elongation of the G-rich strand of the telomere by telo- from nuclease digestion, the DNA-binding site of the telo- merase does not necessarily require that telomerase interact mere protein appears to be tailored so that the phosphate with DNA bound by the telomere protein. Copying of the backbone is surrounded by protein. In addition, the telomere parent strands by DNA polymerase is thought to result in protein presumably contacts the bases since previous studies production of a molecule that is blunt on one end (11), and it have shown that telomere protein binding is highly sequence- is here that telomerase is needed to regenerate the 3' over- specific and the majority of the guanine residues are pro- hang. However, since neither telomerase nor telomere pro- tected from dimethyl sulfate modification (3, 5, 6, 10). tein bind blunt-ended DNA (5, 12), the mechanism by which Although the N-7 positions may be in close contact with the G strand extension occurs remains unclear. For elongation, protein, our findings indicate that at least some of the the double-stranded DNA must somehow be opened. This Watson-Crick base pairing positions are accessible in the could be accomplished by the action of a helicase with the DNA-protein complex. In fact, probably only a subset ofthe separated strands being stabilized by binding of a single- 13 residues of the tail-fragment DNA are exposed. The stranded binding protein, possibly the telomere protein. requirement for high concentrations of complementary oli- Currently, we are investigating whether the telomere protein gonucleotide to drive the Klenow reaction to completion performs additional roles in telomere replication. suggests that the tail fragment forms a relatively unstable We thank D. Prescott for providing Oxytricha nuclei at an early duplex with a templating molecule. stage in this work. We are also grateful to D. Williams for help with The possibility that some fraction of the telomere protein- growing Oxytricha, M. Lee for providing Tetrahymena telomerase, bound tail fragment could pair with a complementary C-rich M. Melek and R. White for assistance with figures, and P. Cohen and oligonucleotide was originally raised by Raghuraman, Cech, J. Kapler for many helpful discussions. This work was funded by the and coworkers (5) when they showed that the telomere National Institutes of Health (Grant GM41803 to C.M.P. and protein causes a mobility shift of a C4A4 oligonucleotide in a GM26259 to E.H.B.), the March ofDimes-Birth Defects Foundation nondenaturing gel. We found that only a small fraction of the (Grant 1-FY92-0616 to C.M.P.), an American Cancer Society Junior protein-bound tail fragment binds and shifts the C4A4 oligo- Faculty Research Award (to C.M.P.), and an American Cancer nucleotide even though a sizeable fraction of the tail is Society Postdoctoral Fellowship (to D.E.S.). D.E.S. and C.M.P. accessible to the Klenow fragment or reverse transcriptase made equal contributions to the research. (C.M.P., unpublished results). The low level of complex 1. Klobutcher, L. A., Swanton, M. T., Donini, P. & Prescott, formation in a standard mobility-shift assay may reflect the D. M. (1981) Proc. Natl. Acad. Sci. USA 78, 3015-3019. relatively low concentration of oligonucleotide used in the 2. Gottschling, D. E. & Zakian, V. A. (1986) Cell 47, 195-205. binding reaction (0.1-10 pmol). 3. Price, C. M. & Cech, T. R. (1987) Genes Dev. 1, 783-793. Although the structure of telomerase and its mode of 4. Price, C. M. & Cech, T. R. (1989) Biochemistry 28, 769-774. interaction with telomeric DNA are unknown, the Klenow 5. Raghuraman, M. K., Dun, C. J., Hicke, B. J. & Cech, T. R. fragment of DNA polymerase I has been cocrystallized with (1989) Nucleic Acids Res. 17, 4235-4253. duplex DNA (22). From the cocrystal, it is apparent that the 6. Gray, J. T., Celander, D. W., Price, C. M. & Cech, T. R. duplex DNA binds in a cleft on the protein surface. Part ofthe (1991) Cell 67, 807-814. 7. Fang, G., Gray, J. T. & Cech, T. R. (1993) Genes Dev. 7, DNA duplex remains exposed in the crystal; however, it is 870-882. difficult to visualize how the Klenow fragment could bind and 8. Fang, G. & Cech, T. R. (1993) Proc. Natl. Acad. Sci. USA 90, extend the telomeric DNAC4A4 duplex with the tail still 6056-6060. associated with the telomere protein. One possibility is that 9. Raghuraman, M. K. & Cech, T. R. (1990) Nucleic Acids Res. the binding of the complementary templating DNA to the 18, 4543-4551. protein-bound tail fragment causes full or partial release of 10. Raghuraman, M. K. & Cech, T. R. (1989) Cell 59, 719-728. the tail from the telomere protein, thus facilitating elongation. 11. Blackburn, E. H. (1991) Trends Biochem. Sci. 16, 378-381. Accessibility of Telomere Protein-Bound DNA to the Telo- 12. Blackburn, E. H. (1993) Annu. Rev. Biochem. 61, 113-129. mere Replication Machinery. It has been suggested that the 13. Zahler, A. M. & Prescott, D. M. (1988) Nucleic Acids Res. 16, DNA allow 6953-6972. telomere protein must be removed from the to 14. Greider, C. W. & Blackburn, E. H. (1989) Nature (London) access to the replication machinery (9, 18). This model is 337, 331-337. unsatisfactory, particularly for organisms like Oxytricha that 15. Shippen-Lentz, D. & Blackburn, E. H. (1990) Science 247, have extremely short telomeres, as the DNA would be left 546-552. unprotected and vulnerable to attack by nucleases. Our 16. Biessman, H. & Mason, J. M. (1988) EMBO J. 7, 1081-1086. findings indicate that removal ofthe telomere protein may not 17. Levis, R. W. (1989) Cell 58, 791-801. be required to initiate telomere replication. If as postulated 18. Zahler, A. M. & Prescott, D. M. (1989) Nucleic Acids Res. 17, (11, 16-19), replication of the C-rich strand begins by syn- 6299-6317. thesis of an RNA primer, our observation that the telomere 19. Biessmann, H., Carter, S. B. & Mason, J. M. (1990) Proc. means Natl. Acad. Sci. USA 87, 1758-1761. protein leaves the bases exposed that primer synthesis 20. Avilion, A. A., Harrington, L. A. & Greider, C. W. (1992) may be able to occur even in the presence of the telomere Dev. Genet. 13, 80-86. protein. Although we have not directly demonstrated that 21. Ratliff, R. L. (1981) The Enzymes, ed. Boyer, P. (Academic, telomere protein-bound DNA is accessible to primase, it is New York), Vol. 14, pp. 105-118. likely that this is the case, given the accessibility oftelomeric 22. Beese, L. S., Derbyshire, V. & Steitz, T. A. (1993) Science DNA to a diverse selection of polymerases that also require 260, 352-355. Downloaded by guest on September 28, 2021