Biochemistry 1989, 28. 769-774 Chang, D. D., & Clayton, D. A. (1984) Cell (Cambridge, Mass.) 36, 635-643. Chang, D. D., & Clayton, D. A. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 351-355. Chang, D. D., & Clayton, D. A. (1986) Mol. Cell. Biol. 6, 1446-1453. Chang, D. D., Hauswirth, W. W., & Clayton, D. A. (1985) EMBO J. 4, 1559-1567. Chang, D. D., Hixson, J. E., & Clayton, D. A. (1986) Mol. Cell. Biol. 6, 294-301. Christianson, T., & Rabinowitz, M. (1983) J. Biol. Chem. 258, 14025-1 4033. Clayton, D. A. (1982) Cell (Cambridge, Mass.) 28, 693-705. Clayton, D. A. (1984) Annu. Rev. Biochem. 53, 573-594. Dawid, I. B. (1972) Dev. Biol. 29, 139-151. Hu, N., & Messing, J. (1982) Gene 17, 271-277. Kafatos, F. C., Jones, C. W., & Efstratiadis, A. (1979) Nucleic Acids Res. 7 , 1541-1552. Kantharaj, G. R., Bhat, K. S., & Avadhani, N . G. (1983) Biochemistry 22, 3 151-3 156. King, T. C., & Low, R. L. (1987) J. Biol. Chem. 262, 621 4-6220.
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Lanave, C., Preparata, G., Saccone, C., & Serio, G. (1984) J . Mol. E d . 20, 86-93. Lehrach, H., Diamond, J., Wozney, J., & Boedtker, H. (1977) Biochemistry 16, 4743-475 1. Maxam, A. M., & Gilbert, W. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 560-564. Montoya, J., Christianson, T., Levens, D., Rabinowitz, M., & Attardi, G. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 7195-7199. Ojala, D., Crews, S., Gelfand, R., & Attardi, G. (1981) J. Mol. Biol. 150, 303-314. Ricca, G., Hamilton, R., McLean, J., Conn, A., Kalinyak, J., & Taylor, J. (1981) J. Biol. Chem. 256, 10362-10368. Roe, B. A., Ma, D.-P., Wilson, R. K., & Wong, J. F.-H. (1985) J. Biol. Chem. 260, 9759-9774. Upholt, W. B., & Dawid, I. B. (1977) Cell (Cambridge, Mass.) 1 1 , 571-583. Walberg, M. W., & Clayton, D. A. (1983) J. Biol. Chem. 258, 1268-1275. Yoza, B. K., & Bogenhagen, D. F. (1984) J. Biol. Chem. 259, 3909-391 5.
Properties of the Telomeric DNA-Binding Protein from Oxytricha novat Carolyn M. Price$ and Thomas R. Cech* Department of Chemistry and Biochemistry and the Howard Hughes Medical Institute, University of Colorado, Boulder, Colorado 80309 Received June 21, 1988: Revised Manuscript Received September 14, 1988
Oxytricha macronuclear DNA exist as discrete DNA-protein complexes. Different regions of each complex display characteristic DNA-protein interactions. In the most terminal region, binding of a 43- and a 55-kDa protein to the telomeric DNA appears to account for all the DNA-protein interactions that can be detected by chemical and nuclease footprinting. We have used gradient sedimentation and protein-protein cross-linking to establish that the 43- and 55-kDa proteins are subunits of a heterodimer. Both subunits are very basic, which is unexpected considering the resistance of the DNA-protein interaction to high concentrations of salt. It is extremely difficult to dissociate the two subunits either from telomeric D N A or from each other. Even after extensive treatment of protein preparations with nuclease, a fragment of the 3’ tail from macronuclear DNA remains bound to the protein. A wide range of conditions was screened for dissociation of the subunits from the D N A and/or from each other. Dissociation was only obtained by using conditions that caused some inactivation of the DNA-binding capacity of the protein. The use of reagents that covalently modify sulfydryl groups during the purification procedure facilitates preparation of telomere protein with full DNA-binding activity.
ABSTRACT: Telomeres of
Telomeres, the natural ends of chromosomes, have several properties that make them essential for proper cell growth and development [reviewed in Blackburn and Szostak (1984)l. They stabilize chromosomes by preventing end-to-end joining reactions and degradation by exonucleases (McClintock, 1941; Muller & Herskowitz, 1954), and they make possible the complete replication of linear DNA molecules so that no gap is left at the 5’ end of the daughter strand (Cavalier-Smith, 1974; Bateman, 1975; Holmquist & Dancis, 1979). They may ‘This work was supported in part by NIH Grant GM25273 to T.R.C. and by Damon Runyon-Walter Winchell Cancer Research Fund Fellowship DRG 875 to C.M.P. T.R.C. is an American Cancer Society Professor. * Author to whom correspondence should be addressed. *Presentaddress: Department of Chemistry, University of Nebraska, Lincoln. NE 68588.
0006-2960/89/0428-0769$01 .SO10
also help to position chromosomes within cells (Dancis & Holmquist, 1979). Telomeres from humans, plants, and unicellular eukaryotes seem to be fundamentally similar both in function and in DNA composition (Blackburn, 1984; Richards & Ausubel, 1988; Allshire et al., 1988). Nuclear telomeric DNAs from these phylogenetically diverse organisms all contain tandem repeats of simple sequences with a C-rich strand at the 5’ end and a G-rich strand at the 3’ end. Although the telomeric repeated sequences are necessary to make the end of a chromosome act as a telomere (Pluta et al., 1984), it appears that telomeric DNA-binding proteins are also required to form a functional telomere. Telomeric DNA-binding proteins have been detected in a few organisms including yeast, Physarum, Tetrahymena, and Oxytricha (Berman et al., 1986; Cheung et al., 1981; Black-
0 1989 American Chemical Society
770 Biochemistry, Vol. 28, No. 2, I989 burn & Chiou, 1981; Gottschling & Zakian, 1986). In the ciliates these proteins form part of a telomeric nucleoprotein complex (Blackburn & Chiou, 1981; Gottschling & Cech, 1984). However, only the telomeric complexes from Oxytricha nova have been well characterized. The macronucleus of Oxytricha contains around 24 X lo6 linear gene-sized DNA molecules that have telomeres with the sequence (Swanton et al., 1980b; Prescott, 1983; Klobutcher et al., 1981) 5'
CCCCAAAACCCCAAAACCCCnnnnnnn
3' HO-GGGGTTTTGGGGTTTTGGGGTTTTGGGGTTTTGGGGnnnnnnnn
The DNA adjacent to the C4A4.T4G4repeats is unique in sequence. In macronuclei, the terminal 100-150 bp of each DNA molecule are part of the telomeric nucleoprotein complex (Gottschling & Cech, 1984). Different regions of the telomeric complex are characterized by very distinct DNA-protein interactions (Price & Cech, 1987). In the most terminal region of the complex the DNA-protein interactions are sequence specific and remarkably salt stable. These interactions cause methylation protection of specific G residues in the 3' terminal T4G4tail when macronuclei or cells are treated with dimethyl sulfate. The interactions between protein and DNA within the region 45-135 base pairs from the 5' terminus are neither sequence specific nor salt stable. Nuclease footprinting with DNase I indicates that DNA in this more internal region of the complex lies on the outside surface of protein but is not part of a nucleosome. Two protein components of the telomeric complex have been identified, a 43- and a 55-kDa protein (Lipps et al., 1982; Gottschling & Zakian, 1986; Price & Cech, 1987). These proteins are the first truly specific telomere proteins to be characterized. The 43- and 55-kDa proteins remain bound to macronuclear DNA in 2 M NaC1, suggesting that there is a strong nonelectrostatic component to their binding. The binding is not covalent, as both proteins can be dissociated with SDS, urea, or phenol (Gottschling & Zakian, 1986; Price & Cech, 1987). The 43-kDa protein is very sensitive to proteolysis, and degradation products of around 26-kDa accumulate. When the two proteins are incubated with purified macronuclear DNA, a nucleoprotein complex is formed that displays some of the DNA-protein interactions observed in the native telomeric complex (Gottschling & Zakian, 1986; Price & Cech, 1987). The interactions that give rise to methylation protection of G residues in the 3' tail are reconstituted accurately; however, the DNA-protein interactions in the more internal region of the complex are not reconstituted. In the present study we have examined the biochemistry of the 43and 55-kDa telomere proteins in more detail. EXPERIMENTAL PROCEDURES Culturing of 0.nova and Isolation of Macronuclei. 0.nova were grown in nonsterile culture using live Chlorogonium as the food source (Swanton et al., 1980a). Macronuclei were isolated essentially as described by Swanton et al. (1980a). The following protease inhibitors were included in the isolation buffers: 0.1 mM phenylmethanesulfonyl fluoride (PMSF), 0.1 mM tosylphenylalanine chloromethyl ketone (TPCK), and 1.O mM p-(ch1oromercuri)benzenesulfonic acid (PCMBS). Preparation of Telomere Proteins. Telomere proteins were isolated as described by Price and Cech (1987). Macronuclei were lysed in TE (10 mM Tris, pH 8.0, 1 mM EDTA), 2 M NaC1, 0.1 mM PMSF, and 0.1 mM TPCK at 4 "C for 2 h. The supernatant was applied to a Bio-Gel A 15M column equilibrated with TE 2 M NaC1, and DNA-containing fractions were collected. The salt was removed by dialysis against TE containing 0.01 mM PMSF and TPCK.
+
Price and Cech When the DNA was removed by digestion with micrococcal nuclease, CaC1, was added to a concentration of 2.5 mM and the enzyme to a concentration of 0.2 units/mL of protein preparation. When DNase I was used, MgClz was added to 2.5 mM and 25 Kunitz units of enzyme were used per milliliter of protein preparation. The preparations were digested at 37 "C for 1 h. The nuclease was inactivated by addition of EGTA or EDTA to a final concentration of 15 mM. Preparations of telomere protein were concentrated by using an Amicon stirred cell. Chemical Cross-Linking of Telomere Proteins. Samples of telomere protein from the Bio-Gel A 15M column were dialyzed against 70 mM NaCl, 1 mM EDTA, and 10 mM HEPES, pH 7.5. The final protein concentration was 0.5-1 pg/mL. Macronuclear DNA was removed either before or after cross-linking by adding CaCl, to 2.5 mM and digesting with micrococcal nuclease. Dithiobis(succinimidy1propionate) (DSP) was added to the telomere proteins to a final concentration of 0-100 pg/mL. Cross-linking was allowed to proceed for 20 min at 23 "C (Lomant & Fairbanks, 1976). The reaction was quenched by adding a molar excess of lysine. Reconstitution of Telomeric Nucleoprotein Complexes. Telomeric DNA-protein complexes were formed by incubating telomere proteins with DNA at room temperature for 1 h. The reconstitution mixture consisted of TE, 15 mM EGTA, 1 pg of DNA (1.4 pmol of ends), and 0-4 pmol of each telomere protein per 8-12-~Lsample (Price & Cech, 1987). Nitrocellulose Filter Binding Assay. Telomeric nucleoprotein complexes were reconstituted by using macronuclear DNA that had been 3' end labeled with 32P-labeledcordycepin. Aliquots of the complexes were applied to nitrocellulose filters, and the filters were washed with 2.5 mL of TE followed by 2.5 mL of TE plus 200 mM NaC1. The filters were then counted in a scintillation counter to determine how much of the DNA remained bound to the filter. The filter binding assay was used to examine the DNA-binding activity of telomere protein preparations under standard and dissociating conditions. Gel Electrophoresis. Electrophoresis of proteins on SDSpolyacrylamide gels was performed as described by Laemmli (1970). Isoelectric focusing gels were prepared and run as described by O'Farrell(l975, 1977). The nonequilibrium gels were run for 1500 Vh; this gave sufficient separation of the telomere proteins without allowing the pH gradient to collapse significantly. Electrophoresis of DNA on sequencing gels was performed according to standard procedures (Maniatis et al., 1982).
RESULTS The Oxytricha Telomere Protein Is a Heterodimer. The 55- and 43-kDa telomere proteins were isolated as a mixture still bound to macronuclear DNA (described under Experimental Procedures). The proteins were then digested extensively with micrococcal nuclease to degrade the DNA and analyzed by glycerol gradient sedimentation. As shown in Figure 1, both proteins sedimented faster than bovine serum albumin (67 kDa) but more slowly than alcohol dehydrogenase (150 kDa). Moreover, the 43-kDa protein sedimented at the same rate as the 55-kDa protein. The observed sedimentation rates suggested that both telomere proteins have masses of around 100 kDa. The high rate of sedimentation of the 43- and 55-kDa proteins suggested that they might be part of a multisubunit protein, possibly a heterodimer. To test this possibility we attempted to cross-link the two proteins using a protein-protein cross-linking reagent that has a short span. Preparations of
Biochemistry, Vol. 28. No. 2, 1989 77 1
Ciliate Telomere Protein
94-
67-
la a
43-
7
30-
*
20-
.
4
e ' *
14-4
+
1
' d
TOP
BOTTOM
~ O U R E1: Sedimentation of the 43- and 55-kDa telomere proteins relative to marker proteins. Telomere proteins and the marker proteins bovine serum albumin (BSA, 67 kDa), carbonic anhydrase (CA, 29 kDa), and micrococcal nuclease (MN, 17 kDa) were loaded on a 2 . 5 1 5 % glycerol gradient containing 70 mM NaCI, 1 mM EDTA, and 10 mM Tris, pH 8.0. After spinning the gradients at 170000gfor 16 h, fractions were collected, precipitated with trichloroacetic acid, and analyzed on a 12%polyacrylamide SDS gel. The gel was then silver stained. The arrows above the gel indicate the peak fractions for the marker proteins. Alcohol dehydrogenase (AD, 150 kDa) was loaded on a separate but identical gradient. An open circle marks the peak fractions for the 55-kDa protein and its position in the gel. A filled circle marks the peak fractions for the 43-kDa protein and its position in the gel. A filled triangle marks the position of proteolytic degradation products of the 43-kDa protein. (Lane M) Marker proteins; (lane CA) carbonic anhydrase; (lane TP) the two telomere proteins.
the telomere proteins were treated with the reversible crosslinker dithiobis(succinimidy1 propionate) (DSP) (Lomant & Fairbanks, 1976) either before or after removal of the bound DNA by digestion with micrococcal nuclease. The crosslinking conditions (i.e., the concentrations of telomere proteins and DSP) were chosen so that intramolecular but not intermolecular cross-linking was obtained in comparable control experiments using bovine serum albumin (data not shown). As shown in Figure 2A, when telomere protein preparations were treated with increasing concentrations of DSP, the 43and 55-kDa bands disappeared concomitantly and one band of 100-120 kDa appeared. This was particularly clear in experiments where the protein preparations were not treated with nuclease prior to cross-linking (Figure 2A). Similar results were obtained when the telomere protein preparations were treated with nuclease prior to cross-linking. However, during nuclease digestion the 43-kDa and, to a lesser extent, the 55-kDa protein undergo some proteolysis. This proteolysis resulted in broadening of the cross-linked band (data not shown). However, the top of this broad band was still about 120 kD, and no larger bands appeared. That the 100-120-kDa band was composed of only the 43and 55-kDa proteins was shown by 2-dimensional gel electrophoresis. Lanes containing cross-linked telomere proteins were excised from gels similar to the one shown in Figure 2A. The gel slices were incubated in dithiothreitol to reverse the cross-linking; they were then loaded on the top of a second SDS-polyacrylamide gel. In the second dimension the 100120-kDa band disappeared and the 43- and 55-kDa bands reappeared (Figure 28). As expected, the telomere proteins (and their degradation products) that had not been cross-linked by DSP ran on the diagonal in the second dimension, while the proteins that had been cross-linked ran off the diagonal. The low molecular weight band in lanes 0-100 of Figure 2A is micrococcal nuclease. The nuclease was added to the samples after cross-linking with DSP to partially degrade the DNA. As the nuclease digestion step was brief, only a little proteolysis of the telomere proteins occurred. The proteolytic fragments are apparent in Figure 2B. The data shown in Figures 1 and 2 indicate that, under the experimental conditions described, the telomere protein is a
A
UGNL DSP M
0
6
1 2 25 100
200976743
-
26-
..
9767-
43-
30-
20-
Protein-protein cross-linking of telomere protein preparations. (A) Samples of telomere proteins were treated with 0-100 & n L of the cross-linker DSP as indicated at the top of the gel. The samples were precipitated with trichloroacetic acid, the proteins were separated on a 5515% polyacrylamide gradient gel, and the gel was stained with Coomassie Blue. Lane M shows marker proteins; the molecular masses (kDa) are indicated at the left. (B)An unstained lane of a gel similar to that shown in (A) was excised. This lane contained telomere proteins that had been cross-linked with 12 , & n L DSP. The excised lane was incubated with 50 mM dithiothreitol to reverse the cross-linking and then loaded on the top of a 12%p l y acrylamide gel. The arrow shows the position of the top of the gel slice. Lane TP contains the 43- and 55-kDa telomere proteins. Lane M contains marker proteins; their molecular masses (kDa) are indicated at the left. FIGURE 2
772 Biochemistry, Vol. 28, No. 2. 1989
Price and Cech
heterodimer composed of subunits of 43 and 55 kDa. If the protein contained more subunits, one would expect to see additional bands upon DSP cross-linking (e.g., dimers and trimers), and the multisuhunit protein should sediment faster in 2 5 1 5 % glycerol gradients. In many experiments, the Coomassie Blue stained 43- and 55-kDa hands appear to he present at a ratio of 1 2 or 1:3 (Price & Cech, 1987). This apparently uneven ratio of the two subunits seems to arise because the 43-kDa subunit stains p r l y with Coomassie Blue and is preferentially proteolysed. When gels are silver stained, the ratio of the two subunits appears closer to one, provided the proteins are relatively undegraded. The Telomere Protein Is Very Basic. When samples of the telomere protein were electrophoresed in equilibrium isoelectric focusing gels (O'Farrell, 1975) with pH gradients from 4.5 to 8.5, both subunits ran off the basic end of the gel (data not shown). When the protein was run in basic nonequilibrium isoelectric focusing gels (OFarrell et al., 1977), both subunits had electrophoretic mobilities that reflected an isoelectric point greater than 8.5 (data not shown). Telomere Protein Isolated by using Nuclease Digestion Retains a Small Fragment of Telomeric DNA. Samples of telomere protein were extensively digested with either micrococcal nuclease or DNase I in the presence of 2.5 mM CaCI, or MgCI,, respectively. The nuclease activity was then inhibited by addition of EGTA or EDTA to 15 mM. The telomere protein was removed from the samples by treatment with proteinase K in the presence of 0.5% SDS, followed by extraction with phenol and chloroform. Any residual DNA fragments in the samples were then 3' end labeled with I2Plabeled cordycepin triphosphate and terminal deoxynucleotidyl transferase (Tu & Cohen, 1980). When the samples were electrophoresed in sequencing gels, it was apparent that despite the extensive nuclease digestion, several DNA fragments of discrete size had been left in the telomere protein preparation (Figure 3, lanes MNase and DNase I). DNase I treatment yielded fragments of 19-20 bases, while micrococcal nuclease yielded fragments of 13-14 bases. In Figure 3, the fragments resulting from micrococcal nuclease digestion appear to be 14-15 bases in length; this is because they lack a 5' phosphate and so are electrophoretically retarded compared to fragments generated by DNase I digestion or MaxamGilbert sequencing, which do have a 5' phosphate (Maxam & Gilbert, 1977; data not shown). To determine which portion of the macronuclear DNA was so strongly protected by the telomere protein, preparations of the protein containing the DNA fragments were subjected to the dimethyl sulfate sequencing reaction for G residues. As shown in lanes MNase DMS and DNase I DMS of Figure 3, the DNA fragments have the same repeating G4N4 sequence as 3' end labeled macronuclear DNA (macro DNA lane). Thus, the protected fragment must arise from the region of the telomere that contains the G4T4repeats. In fact, the protected fragment seems to arise from the 3' tail. As seen in the DNase I + DMS lane, the G cleavage pattern reflects the methylation protection characteristic of telomeric complexes [seethe reconst. DNA lane, and also Price and Cech (1987)l. Because treatment with dimethyl sulfate preceded deproteinization, it is reasonable that the 3'-terminal DNA fragment would be protected by the isolated protein in essentially the same manner as in the intact telomeric complex. It is not surprising that the length of the protected fragment varies depending on which nuclease is used. Single-stranded DNA is digested well hy micrococcal nuclease, whereas DNase
+
+
I T4
7%
I
DOUBLE STFWND
L
3' TAIL
-
-. --
iz 1..
I T 4
~ G U R E3: Binding of the telomere protein protects the 3' tail from nuclease digestion. Telomere protein preparations were treated extensively with either micrococcal nuclease or DNase 1. Any residual DNA fragments left in the protein preparations were isolated, 3' end labeled, and electrophoresed on a 20% polyacrylamide gel. (Lane MNase) DNA fragments remaining after micrococcal nuclease digestion: (lane DNase I) DNA fragments remaining after DNase I digestion. In lanes MNase + DMS and DNase I + DMS, the nuclease-digested protein preparations were treated with 10 mM dimethyl sulfate for IO min at 25 OC prior to isolating and end labeling the DNA fragments. The DNA was cleaved at methylated G ' s by treatment with piperidine and loaded on the sequencing gel. Lanes macro DNA and reconst. DNA show the cleavage patterns resulting from dimethyl sulfate treatment of purified macronuclear DNA and reconstituted telomeric complexes, respectively.
I prefers double-stranded DNA (Drew, 1984). Dissociationof the Telomere Proteinfrom Telomeric DNA. A wide range of conditions was screened in an attempt to dissociate the telomere protein from telomeric DNA (Table I). Nitrocellulose filter binding was used to assess the degree of dissociation. The most effective condition is 100 mM MgCI, at a pH of 5 or lower. If less MgCI, is used, the amount of dissociation falls off rapidly, but increased concentrations do not increase dissociation significantly (data not shown). Unfortunately, the low pH and magnesium is of limited value for isolating DNA-free telomere protein with full DNAbinding activity. If treatment is for 15-30 min, not all of the DNA is dissociated. However, if the treatment is prolonged, the DNA-binding activity of the protein decreases. Other conditions tested that had little effect on binding of the telomere protein to telomeric DNA include 10% glycerol, 10% ethanol, 0.1 M cobalt bexaammine chloride, and nonionic detergents (data not shown). Dissociation of the Telomere Protein Subunits. When
Ciliate Telomere Protein Table I: Dissociation of the Telomere Protein from Telomeric DNA‘ dissociation conditions % boundb Tris, pH 8 100 citrate, pH 6 94 94 citrate, pH 5 2.5 M NaCl 78 100 mM MgC12 95 2.5 M NaC1, pH 5 66 2.5 M NaCI, pH 5, 100 mM MgC12 35 100 mM MRCI,. DH 5 36 Telomeric complexes were reconstituted by using purified telomere protein and 3’ end labeled macronuclear DNA. Aliquots of the reconstituted complexes were added to an equal volume of 2 X concentrated dissociation buffer, and the samples were left for 15 min. The samples were then diluted 100-fold into TE and applied to nitrocellulose filters. The dilution step was to prevent the dissociation buffers from affecting binding to the filter; this step had no effect on the binding of control samples. The filters were washed and counted in a liquid scintillation counter. bThe percent of counts that remained bound to nitrocellulose filters as compared to control samples of reconstituted telomeric complex that were not subjected to dissociation conditions.
macronuclei were treated with NaCl concentrations ranging from 0.5 to 3 M and the resulting extract was fractionated on Bio-Gel A 15M, both subunits remained bound to macronuclear DNA. However, when the pH was raised to 10 (in the presence of 2 M NaCl), both subunits dissociated from the DNA. In addition, separation of the subunits at pH 10 and also at pH 5 in the presence of 100 mM MgC12 was demonstrated by glycerol gradient sedimentation. Treatment with pH 5 alone was not sufficient to cause separation. Unfortunately, the only conditions that resulted in separation of the two subunits also inactivated the DNA-binding capacity of the telomere protein. As the proteins do not regain their full DNA-binding capacity upon renaturation (Price & Cech, 1987), this poses an obstacle to studying the DNA-binding properties of the individual subunits. Preparation of Telomere Proteins with Full DNA-Binding Activity. When preparations of the telomere protein display full DNA-binding activity, the protein (i) causes retention of macronuclear DNA on nitrocellulose filters and (ii) confers a specific pattern of methylation protection on G residues in the 3’ tail (Gottschling & Zakian, 1986; Price & Cech, 1987). While almost all preparations of telomere protein cause retention of macronuclear DNA on nitrocellulose filters, it is more difficult to prepare telomere protein that confers methylation protection on G’s in the 3’ tail. As this methylation protection pattern is indicative of the in vivo DNA-protein interactions, protein that does not display this DNA-binding activity cannot be classified as fully active. Fully active telomere protein can be obtained reproducibly if a reagent that modifies sulfhydryl groups is included during isolation of macronuclei. The protease inhibitor p-(chloromercuri)benzenesulfonicacid (2 mM) is usually used; however, 10 mM iodoacetamide, iodoacetic acid, or N-ethylmaleimide are also effective. These reagents were all used at the concentrations normally designed to inhibit proteases and phosphatases rather than at concentrations designed for studies of sulfhydryl chemistry (Paulson, 1980; Gurd, 1972; Riordan & Vallee, 1972). Of 13 telomere protein preparations that were made by using sulfhydryl-modifyingreagents, 12 were capable of causing methylation protection. Only one of four protein preparations made in the absence of these reagents was capable of causing methylation protection. If the sulfhydryl groups were not modified but instead dithiothreitol was included during all steps of the telomere protein isolation, active telomere protein was obtained, but the
Biochemistry, Vol. 28, No. 2, 1989 713 activity was substantially less stable upon storage. DISCUSSION The data presented here indicate that the previously identified 43- and 55-kDa telomere polypeptides are part of a single protein. We have used two different techniques to investigate the size of this protein, and we conclude that it is a heterodimer. Glycerol gradient analysis indicates that the protein has a mass of around 100 kDa. This is too small to be a trimer or a tetramer. Following cross-linking of the telomere protein with DSP, only one higher molecular weight band appears. If the protein contained more than two subunits, additional cross-linked bands would be expected. An accurate measurement of the heterodimer mass might not be obtained from either the gradient analysis or the cross-linking experiments. The telomere protein that was loaded on the glycerol gradients had a small fragment of DNA bound to it; this could affect the sedimentation rate of the heterodimer. Cross-linking of proteins can affect their electrophoretic mobility on SDS gels (Pfeiffer et al., 1987; C. Price, unpublished observations). Depending on how two subunits are cross-linked together, the mobility could be either more or less than that of a single un-cross-linked protein of the same molecular weight. In view of these limitations, it is not surprising that the numbers obtained with the two types of analysis are similar but not identical (100 and 100-120 m a ) . Pending more accurate determination of the protein mass, we find it useful to refer to the protein as the 98-kDa telomere protein, the sum of the 43- and 55-kDa subunits. The 98-kDa telomere protein binds to telomeric DNA tenaciously. Dissociation has only been achieved by using conditions that cause some denaturation of the protein. The tight association between the protein and the DNA is reflected in the protection of the 3’4erminal T4G4T4G4tail from extensive micrococcal nuclease digestion. Following digestion, over 60% of the molecules in a telomere protein preparation are associated with a 3’-tail fragment (M. K. Raghuraman, personal communication). The presence of this DNA fragment must be taken into consideration in the studies of the DNAbinding properties of the telomere protein, as exogenous DNA must compete with the fragment for binding to the protein. The very basic isoelectric point (PI > 8.5) of both the subunits is an unexpected finding. Previous studies showed that many of the in vivo interactions between the telomere protein and telomeric DNA are maintained in 2 M NaCl. This suggested that binding of the protein to the DNA has a nonelectrostatic component that is unusually large (Price & Cech, 1987). Most known DNA-binding proteins rely heavily on electrostatic interactions both during the initial formation of a DNA-protein complex and during subsequent binding to the DNA (Ohlendorf & Matthew, 1985; Chase, 1986). These electrostatic interactions are mediated by basic regions on the surface of the protein (Ohlendorf & Matthew, 1985). Perhaps the telomere protein is like the majority of DNAbinding proteins in that electrostatic interactions are required for the initial stages in binding, and it is only subsequent interactions that are unusual. Covalent modification of sulfhydryl groups during macronuclear isolation was found to facilitate preparation of active telomere protein. At present, the reason for this effect is unclear. It is possible that the telomere protein is not directly affected by the reagents, but instead macronuclear proteins that inactivate the telomere protein are themselves inactivated by the covalent modification. Alternatively, the telomere protein might contain very active sulfhydryl groups that undergo intramolecular reactions that inactivate the protein.
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Such reactions might be prevented in vivo by the presence of other proteins. Our data are somewhat more consistent with the latter than with the former explanation. Activation of a variety of proteins by p-(ch1oromercuri)benzoate has been reported (Torchinskii, 1974). ACKNOWLEDGMENTS We thank Gregg McDonald for technical assistance in studying the dissociation of the subunits of the telomere protein and for his excellent help with growing Oxytricha and Chlorogonium. REFERENCES Allshire, R. C., Gosden, J. R., Cross, S. H., Cranston, G., Rout, D., Sugawara, N., Szostak, J. W., Fantes, P., & Hastie, N. D. (1988) Nature (London) 332, 656-659. Bateman, A. J. (1975) Nature (London) 253, 379. Berman, J., Tachibana, C. Y., & Tye, B. K. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 3713-3717. Blackburn, E. H. (1984) Cell (Cambridge, Mass.) 37, 7-8. Blackburn, E. H., & Chiou, S . A. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 2263-2267. Blackburn, E. H., & Szostak, J. W. (1984) Annu. Rev. Biochem. 53, 163-194. Cavalier-Smith, T. (1974) Nature (London) 250, 467-470. Chase, J. W. (1986) Annu. Rev. Biochem. 55, 103-136. Cheung, M. K., Drivas, D. T., Littau, V. C., & Johnson, E. M. (198 1) J . Cell Biol. 91, 309-3 14. Dancis, B. M., & Holmquist, G. P. (1979) J. Theor. Biol. 278, 21 1-224.
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