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Biology of Reproduction 62, 340-346 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Articles

Identification and Characterization of a Bovine Sperm Protein That Binds Specifically to Single-Stranded Telomeric Deoxyribonucleic Acid1

Alexander Kozika, E. Morton Bradburya,b, and Andrei O. Zalensky2,a

a Department of Biological Chemistry, School of Medicine, University of California at Davis, Davis, California 95616 b Los Alamos National Laboratory, Los Alamos, New Mexico 87545


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Telomere DNA at the physical termini of chromosomes forms a single-stranded 3' overhang. In lower eukaryotes, e.g., ciliated protozoa, this DNA extension is capped by specific proteins that have been structurally and functionally characterized. Much less is known about single-stranded telomere DNA-binding proteins in vertebrates. Here we describe a new protein from bovine sperm designated bsSSTBP that specifically interacts with single-stranded (TTAGGG)N DNA. The bsSSTBP was extracted from nuclei by 0.6 M KCl. The native size of this protein, estimated by gel filtration, was 20–40 kDa. SDS-PAGE of the UV cross-linked complex between bsSSTBP and telomere DNA indicated that several polypeptides are involved in complex formation. Bovine sSSTB had high specificity toward nucleotide sequence, since single nucleotide substitutions in the (TTAGGG)4 substrate suppressed binding. The minimal number of (TTAGGG) repeats required for binding of bsSSTBP was 3, and the protein recognized linear but not folded DNA structures. We propose that the bsSSTBP participates in telomere-telomere interactions and the telomere membrane localization observed in mature sperm. In mammals, somatic telomere-binding proteins are apparently substituted by sperm-specific ones that may lead to a structural reorganization of telomere domains to fulfill functions important during meiosis and fertilization.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The physical termini of chromosomes in eukaryotes are composed of a specific DNA-protein complex called telomeres. Telomeres have evolved to fulfill several essential functions: 1) they protect chromosomes from end degradation and fusion; 2) they facilitate complete replication of chromosomal DNA molecules; and 3) they participate in chromosome positioning and proper segregation during mitosis and meiosis (see [1, 2] for recent reviews). Telomere DNA in vertebrates is represented by a tandemly repeated double-stranded hexanucleotide [(TTAGGG)(AATCCC)]N [3] and a single-stranded overhang of the G-strand at the very end [4]. Both double-stranded and single-stranded telomere DNAs are associated with various proteins; double-stranded telomere-binding proteins (dsTBP) and single-stranded telomere-binding proteins (ssTBP) have been identified. Telomere-binding proteins were first discovered and described in detail in ciliated protozoa and Saccharomyces cerevisiae (see [5–8] for recent reviews). In vertebrates, the first double-stranded (ds)TBP characterized at the molecular and functional levels was human telomere repeat factor (hTRF) [9, 10]. The 60-kDa hTRF1 was isolated from HeLa cell nuclear extracts, and the corresponding cDNA was cloned [11]. TRF1 activity has been identified in nuclear extracts from monkey, rodent, and chicken cells, whereas TRF1 mRNA has been detected in all human tissues so far analyzed [9, 12]. Therefore, all vertebrates have similar dsTBP, and the corresponding gene is expressed in every somatic tissue. Recently, a second human dsTBP—hTRF2—has been isolated [13–14]. Sequence analysis indicated that TRF1 and TRF2 are distinct proteins sharing a common DNA-binding domain that has strong sequence homology to the DNA-binding domains of Myb protooncogenes [11, 14, 15]. Important telomere functions have been associated with TRF1/2 proteins; TRF1 indirectly regulates telomerase activity [16]; and hTRF2 prevents chromosome end-to-end fusion [17]. So far, few ssTBP have been identified in vertebrates. Among these are XTEF in Xenopus laevis eggs and ovaries [18]; qTBP42—single-stranded and quadruplex telomeric DNA-binding protein from rat hepatocytes [19]; and avian cells protein recognizing hairpin DNA structures [20]. Nuclear proteins interacting with TTAGGG repeats have been identified in mouse liver [21, 22] and HeLa cells [23]. Peptide sequencing and mobility shift experiments using (UUAGGG) as a substrate showed that these proteins are highly homologous to heterogeneous nuclear ribonucleoproteins. Their possible telomere-related functions and association with telomere DNA in vivo are unknown. A 50-kDa protein active in binding to the C-strand of telomere DNA has been identified in HeLa nuclear extracts [24], but its function is also unknown.

The sperm cell is a highly differentiated cell type that results from a specialized genetic and morphological process termed spermatogenesis. During spermatogenesis, the somatic histones are gradually replaced with protamines or protamine-like proteins [25]. As a result, chromatin structure is reorganized, DNA becomes supercondensed, and genetic activity is completely shut down. Specific and nonrandom nuclear architecture has been demonstrated for mammalian sperm cells [26–28]. In particular, this architecture is characterized by telomere localization at the extreme nuclear periphery where telomeres interact in dimers and tetramers [27]. Telomere localization and interactions observed in mature mammalian sperm are different from those in somatic nuclei (reviewed in [30]). Distinctive sperm-specific features of telomeres are established early during spermatogenesis [28, 29, 31]. In spermatogonia, telomeres are randomly scattered throughout the nucleoplasm; in spermatocytes, all telomeres relocalize toward the nuclear membrane and form associations [28, 29]. Telomere associations during meiosis have been described in a variety of organisms [32, 33], and it has been suggested that they are involved in initiation of synapsis or crossing over [34].

Recently we proposed that the ability of telomeres in sperm to associate and interact with nuclear membrane is a protein-dependent feature, and protein binding to double-stranded telomere DNA has been identified in human sperm and partially characterized [28]. Here we describe a novel telomere-binding protein activity from bovine sperm that specifically binds to single-stranded (TTAGGG)N. We have performed partial purification of this protein and provided its detailed biochemical characterization.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Oligonucleotides and DNA probes

HPLC-purified DNA oligonucleotides were purchased from Operon Technologies (Alameda, CA). These were 5'-(TTAGGG)N-3' (ssTEL-N, where n = 2, 3, 4, 6, or 7) imitating the 3'-telomere overhang, and telomere-related mutant oligonucleotides: 5'-(TTAGCG)4-3', 5'-(TTAGGC)4-3', 5'-(TTGGGG)4-3', and 5'-(ATTGGG)4-3'. Single-stranded DNA was labeled with [{gamma}-32P]ATP (DuPont, Wilmington, DE) and T4 polynucleotide kinase (New England Biolabs, Beverly, MA). DNA probes were purified from unincorporated nucleotides by passing through Chroma Spin G-10 columns (Clontech, Palo Alto, CA). A double-stranded human telomere DNA probe containing 12 repeats (dsTEL-12) was isolated from the pTH12 plasmid kindly provided by Dr. T. de Lange (The Rockefeller University, New York, NY). We used the 160-base pair (bp) EcoRV/HindIII fragment, which contains 72 bp of telomere insert, and 88 bp of nontelomeric sequence that remained from the host plasmid. The isolated fragment was labeled by filling the 3' end gap with [{alpha}-32P]dATP (Du Pont) and Klenow enzyme (New England Biolabs) and purified using Chroma Spin G-10 columns (Clontech).

Isolation of Sperm Nuclei

All procedures for isolation of nuclei protein and extraction were carried out at 4°C. Preparation of sperm cells from bulk bovine semen was essentially as described earlier [35]. PBS-washed sperm cells were resuspended in buffer A (100 mM KCl; 10 mM 3-[N-morpholino]-propanesulphonic acid], i.e., MOPS, pH 7.0; 0.25 M sucrose; 0.2 mM PMSF; 0.2 µg/ml N{alpha}-p-tosyl-L-lysine chloromethyl ketone, i.e., TLCK) and centrifuged at 2500 rpm for 15 min (Sorvall, Newtown, CT; HS-4 rotor). The pellet was resuspended in buffer A and sonicated using XL-2020 (Ultrasonic Processor, Heat Systems, Farmingdale, NY) for 30 sec, 3 times, to cleave tails from heads. Then the suspension was diluted with 3 volumes of buffer A, and sperm heads were pelleted by centrifugation (2000 rpm x 15 min). This procedure was repeated 1–2 times and resulted in about 80% purification of sperm heads from tails as judged by phase-contrast microscopy. Finally, pellet consisting of purified sperm heads was resuspended in buffer A, and Triton X-100 was added to 0.5%. Nuclear membranes were partially removed from sperm heads by mild homogenization (Potter homogenizer) followed by a 15-min incubation. Demembranated nuclei were pelleted by centrifugation (2200 rpm, 15 min). The nuclear pellet was resuspended and washed once in buffer A to remove Triton X-100.

Protein Extraction

Sperm nuclei were resuspended in a small volume of buffer A at DNA concentration of 8 mg/ml. An equal volume of double-strength extraction buffer (1.2 M KCl, 20 mM EDTA, 20 mM MOPS, pH 7.0, 20 mM dithiothreitol [DTT], 0.2 mM PMSF, 0.2 mM TLCK) supplemented with the Complete Protease Inhibitors (Boehringer-Mannheim, Indianapolis, IN) was added, and the extraction was performed overnight with shaking. The extract was separated from the nuclear pellet by centrifugation at 10 000 rpm for 20 min (Sorvall, SS-34 rotor). Clarified protein extract was used immediately or stored in aliquots at -80°C after quick freezing in liquid nitrogen. Concentration of total protein in the extract was determined by the Bradford method (Bio-Rad, Richmond, CA).

Purification of bsSSTBP

The clarified extract was dialyzed against 100 mM KCl, 10 mM MOPS, pH 7.0, 2 mM DTT, 0.2 mM PMSF overnight. Pellets that formed during dialysis were removed by centrifugation and discarded; the supernatant was used for further protein purification. The supernatant was loaded on the Superdex 200 HR 10/30 Fast Protein Liquid Chromatography (FPLC) gel-filtration column (Amersham Pharmacia Biotech, Piscataway, NJ) equilibrated with 100 mM KCl, 10 mM MOPS, pH 7.0, and 2 mM DTT. Proteins were eluted with the same buffer at a flow rate 0.3 ml/min. Fractions were assayed for the DNA-binding activity using electrophoretic mobility shift assay (EMSA; see below). Active fractions were stored at -80°C for further purification steps. To estimate protein molecular weights, the column was calibrated using the low Molecular Weight Calibration Kit (Pharmacia).

DNA-Protein Binding and EMSA

Unless otherwise indicated, the labeled 5'-(TTAGGG)4-3' probe (0.5–1 ng) was incubated with extracts of sperm nuclei containing 1–3 µg of total protein in EMSA binding buffer (10 mM MOPS, pH 7.0, 50 mM NaCl, 300 mM KCl, 1 mM EDTA, 1 mM DTT, 0.1 mg/ml BSA, 5% glycerol). We used denatured HaeIII digest of Escherichia coli DNA as a nonspecific DNA competitor in the amount 100–200 ng/reaction. In some experiments, specific telomere-related competitor oligonucleotides were added to the binding mixture in the amounts indicated in the figure legends. After incubation for 15–30 min at room temperature, probes were loaded on a 5% PAG prepared on single-strength TBE buffer (89 mM Tris base, 89 mM boric acid, 2 mM EDTA). All gels were subject to pre-electrophoresis for about 1 h. Electrophoresis was carried out for 2–3 h at 15–25 mA/150–175 V. The gels were then dried and visualized by autoradiography.

SDS-PAGE

SDS-PAGE was performed according to method of Laemmli [36]. Gels were stained with Coomassie after fixation. Gel images were taken using computer scanner. Apparent molecular weights of protein bands were determined using a BenchMark protein ladder (11 to 221-kDa range; Gibco BRL, Oakville, ON, Canada) as molecular weight marker.

G-Quartet Formation by ss-Telomeric Oligonucleotides

Identification of the G-quartet formation by ss-telomeric oligonucleotides was performed as described in Zahler et al. [37] with minor modifications. Briefly, 32P-radiolabeled single-stranded oligonucleotides corresponding to human telomeric sequence (TTAGGG)N (where n = 2 to 7), the Tetrahymena repeat (TTGGGG)4, and unstructured oligonucleotide dT27 were subjected to gel electrophoresis in nondenaturing 12% TBE-PAGE with Na+ and K+ added to the gel and running buffer at concentrations of 20 mM each. Prior to loading, oligonucleotides were incubated for 10 min in EMSA binding buffer. The same oligonucleotides were dissolved in 50% formamide, boiled for 3 min, and then separated using denaturing 12% PAGE containing 6 M urea.

UV Cross-linking

Cross-linking of proteins to DNA was performed as described in Lu et al. [38]. Briefly, the wet EMSA gel was exposed to 254 nm UV light source at a distance of about 5 cm for 1 to 10 min. Then the position of the complex in the gel was determined by autoradiography. The piece of gel containing the complex was cut out, soaked in SDS sample buffer for 5 min, and put onto the stacking gel of 12% SDS PAGE. After electrophoresis, the gel was dried, and cross-linked bands were visualized using autoradiography.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of ssTBP in Bovine Sperm Extracts

The terminal structure of the vertebrate telomere consists of the double-stranded (TTAGGG)(CCCTAA) repeats and the single-stranded 3' TTAGGG overhang. Labeled (TTAGGG)4 oligonucleotide (ssTEL-4) was tested in an EMSA for the formation of complexes with components of the 0.6 M KCl nuclear extract prepared from bovine sperm nuclei. One major shifted band accumulated after incubation of the ssTEL-4 with the crude sperm extract (Fig. 1a). The retarded complex was formed in the presence of a 200 times excess of nonspecific single-stranded DNA and was out-competed by increasing concentrations of unlabeled ssTEL-4. Addition to the reaction mixture of SDS to 0.01%, treatment with pepsin, or 3 min boiling completely eliminated binding (Fig. 1b). Treatment with RNase did not influence complex formation (Fig. 1b). Thus, the observed band shift can be attributed to the formation of a complex of ssTEL-4 with a protein. This DNA-protein complex was resistant to salt treatment (up to 1 M KCl in binding buffer); the optimal salt concentration for bovine protein ssTEL-4 binding was 0.3 M KCl (data not shown).



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  		FIG. 1. Identification of a telomere-binding protein activity in bovine sperm using EMSA. All binding mixtures contained 1 ng of 32P-labeled (TTAGGG)4, 200 ng of unlabeled E. coli DNA (nonspecific competitor), and 1 µg of total protein (0.6 M KCl sperm nuclear extract). DNA and DNA-protein complexes were fractionated on 6% polyacrylamide gels prepared on single-strength TBE. a) Formation of the retarded complex was inhibited by single-stranded telomere DNA. Amounts of unlabeled competitor (nanograms per binding reaction) are indicated above lanes. b) Protein nature of telomere-binding activity. Crude nuclear extract was treated either by 3 min boiling, or with 100 µg/ml RNase, or with 100 µg/ml pepsin prior to binding reaction; alternatively, SDS was added to binding mixture up to 0.01%. Only treatment with RNase did not affect complex formation

Double-stranded TEL-12 competed with the complex formation by labeled ssTEL-4 substrate (Fig. 2a), although much less efficiently than ssTEL-4 (compare EMSA in Figures 1a and 2a, performed in identical conditions). In experiments in which labeled dsTEL-4 was used as a substrate in EMSA, we did not observe any DNA-protein complex (data not shown). Slight competition between ssTEL-4 and dsTEL-12 (Fig. 2a) is explained by the invasion of ss-telomeric oligonucleotides into double-stranded telomere DNA resulting in the formation of an ssDNA-dsDNA triplex, thus sequestering ssTEL substrate and making it unavailable for binding with proteins. The data in Figure 2b illustrate that this reaction was efficient at room temperature and moderate ionic strength. In this particular experiment, labeled ssTEL-4 was incubated with unlabeled dsTEL-12 for 10 min in EMSA binding mixture without protein and then separated using nondenaturing PAGE. Formation of the complex between single- and double-stranded telomere sequences is evidenced by the transition of 32P label to a DNA band migrating just above dsTEL-12 (Fig. 2b, two right lanes). Therefore, dsTEL DNA did not compete with ssTEL DNA for binding with bovine sperm protein, but rather sequestered the ssTEL DNA from the reaction.



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  		FIG. 2. Double-stranded telomere DNA competed with single-stranded DNA in binding with bsSSTBP due to a complex formation between dsTEL and ssTEL oligonucleotides. a) Double-stranded TEL-12 partially inhibited formation of the complex between bsSSTBP and ssTEL-4. EMSA was performed as described for Figure 1; amounts of unlabeled competitor dsTEL-12 (nanograms per binding reaction) are indicated above lanes. b) Invasion of single-stranded telomere oligonucleotide into double-stranded telomere DNA. Two left lanes indicate positions (unfilled arrows) of labeled ssTEL-4 and dsTEL-12 in the gel. Right lane shows formation of complex ssTEL-4-dsTEL-12 (shaded arrow); 1 ng of labeled ssTEL-4 was added to 100 ng unlabeled dsTEL-12, incubated at room temperature in the EMSA binding buffer that was used for identification of bsSSTBP, and then separated in native 6% PAGE

In summary, the data of figures 1 and 2 show that bovine sperm nuclei possess a protein activity that specifically interacts with the single-stranded TTAGGG DNA. We named this protein bsSSTBP (bovine sperm single-stranded telomere-binding protein).

Partial Purification of the bsSSTBP and Estimation of Its Molecular Weight

The activity was initially purified from 0.6 M KCl nuclear extract by dialysis in a buffer containing 0.1 M KCl. During dialysis to lower ionic strength, significant amounts of proteins that did not bind telomere DNA (EMSA data not shown) precipitated. All the activity was retained in the 0.1 M KCl supernatant, and this was further fractionated on a FPLC Superdex 200 HR 10/30 sizing column. During the gel filtration, the bsSSTBP activity was recovered in fractions 13 and 14 (Fig. 3a). We do not know the nature of the lower molecular shifted bands (fractions 10–16); it is likely that they represent nonspecific complexes.



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  		FIG. 3. Partial purification of a bsSSTBP. Crude 0.6 M KCl protein extract was dialyzed against 100 mM KCl; soluble proteins were then separated on Superdex 200 HR sizing column. Numbers above gel indicate eluted fractions. a) Determination of binding activity in fractions collected from the gel-filtration column using EMSA with ssTEL-4. Bovine sSSTBP activity was isolated in fractions 13 and 14 corresponding to a native size of 20–40 kDa. b) Concentrated proteins from fractions 11 through 17 obtained by gel filtration were separated in 12% SDS-PAGE; Coomassie staining. Molecular weight markers (x 10-3) are on the right

Fractions 13 and 14 corresponded to a molecular size range of 20–40 kDa as determined by column calibration using globular protein markers. SDS-PAGE analysis of proteins concentrated by lyophilization (Fig. 3b) demonstrated that fraction 14 (manifesting the highest TTAGGG-binding activity) consisted of four major polypeptides with apparent molecular sizes of 14, 22, 31, and 42 kDa. Most probably the 22 and 42 kDa bands were contaminants, since they were more abundant in the less active fraction 13.

To estimate the upper limit of the bsSSTBP molecular weight, we performed in situ DNA-protein crosslinking in complexes identified by EMSA. After crosslinking, the nucleoprotein band was cut out, soaked in the SDS-PAGE sample buffer, and subjected to electrophoresis in the second dimension (Fig. 4). Since SDS treatment dissociated the complex (Fig. 1b), the 32P-labeled band in Figure 4 corresponds to the bsSSTBP crosslinked to ssTEL-4 DNA. The apparent molecular size of this complex was 200 kDa. The molecular size of ssTEL-4 was 12 kDa. Therefore, if a single polypeptide complexed with a single ssTEL-4 molecule, the bsSSTBP should have a molecular size of 188 kDa. This value represents an upper limit of bsSSTBP molecular weight. Comparison of this value with the estimates provided by gel filtration (Fig. 3) indicated that several DNA molecules and/or polypeptides may be involved in complex formation.



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  		FIG. 4. Bovine sSSTBP-TEL DNA complex most probably consists of several polypeptides and several DNA molecules. Separation of the cross-linked bsSSTBP-ssTEL-4 complex using SDS-PAGE. UV cross-linking was performed in situ by radiation of the shifted band in EMSA gel with 254 nM UV light. Afterward the band was cut out and subjected to electrophoresis in the second dimension. Note the absence of cross-linking in cases in which no protein was added or in the absence of UV radiation. The amount of cross-linked product with apparent molecular size of about 200 kDa, indicated by shaded arrow, increased with the time of UV radiation

Furthermore, our attempts to assign TTAGGG-binding activity using South-Western hybridization with proteins separated by SDS-PAGE were unsuccessful. This negative result indicated that bsSSTBP activity could not be renatured after SDS-PAGE separation. Once again, this might be due to a complex polypeptide composition of the bsSSTBP. Alternatively, the active form of bsSSTBP is an oligomere.

Nucleotide Specificity of the bsSSTBP

To investigate the sequence specificity of bsSSTBP binding to TTAGGG, single-stranded mutant oligonucleotide probes were tested. In these oligonucleotides, single base substitutions were introduced into the wild-type telomere overhang sequence. Mutant oligonucleotides were used as specific competitors in EMSA experiments with the partially purified protein (the same results had been obtained using crude 0.6 M extract). Figure 5 shows that all tested substitutions strongly decreased binding with the bsSSTBP, as none of the mutant sequences competed with (TTAGGG)4 in complex formation. In reciprocal experiments we used labeled mutant oligonucleotides as substrates and unlabeled (TTAGGG)4 as a specific competitor. Formation of a shifted band had been observed only at low (50-fold) excess of the ssTEL-4, whereas higher amounts of the ssTEL-4 in binding reaction completely eliminated formation of complexes (data not shown). In summary, bsSSTBP had very high specificity toward the nucleotide sequence of 3' guanine-rich telomere overhang.



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  		FIG. 5. Nucleotide specificity of bsSSTBP binding to single-stranded telomere DNA. Each binding reaction contained 1 ng of labeled ss-(TTAGGG)4, 200 ng of unlabeled E. coli DNA, partially purified bsSSTBP (1 µg protein), and unlabeled single-stranded telomere-related competitors with the sequences and amounts (nanograms per reaction) indicated above lanes

Length of DNA Substrate and Secondary Structure Requirements for Binding

To determine a minimal DNA sequence required for binding sperm protein, we used a set of (TTAGGG)N oligonucleotides with different lengths (n = 2, 3, 4, and 7) as labeled DNA substrates in EMSA. Figure 6a shows no complex formation with two (TTAGGG) repeats, whereas three or more repeats were substrates for bsSSTBP binding. The efficiency of complex formation apparently increased with the increasing number of telomere repeats in the DNA substrate. It is known that telomere-related DNA oligonucleotides have the potential to form noncanonical secondary structures, e.g., G-quartets [39]. These folded structures are induced by physiological concentrations of monovalent cations.



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  		FIG. 6. Bovine sSSTBP recognized TTAGGG sequence of specific length, and G-quartet formation was not required for complex formation. a) Binding of bsSSTBP to TTAGGG oligonucleotides of different length. EMSA using labeled TEL-N substrates of different lengths. Standard binding and electrophoresis conditions were used; only part of gel containing bsSSTBP-ssTEL-N complex is shown. b) Under denaturing conditions, telomere oligonucleotides were linear molecules and electrophoretically separated in accordance with their molecular weights. 32P-labeled oligonucleotides were separated using denaturing 12% PAGE containing 6 M urea. c) In the presence of low concentrations of monovalent cations, telomere oligonucleotides may form folded structures. Oligonucleotides were incubated in the binding buffer that had been used for the identification of bsSSTBP activity and then separated in native 12% PAGE containing 20 mM NaCl. All (TTAGGG)N with n > 3 and (TTGGGG)4 formed folded structures as evidenced by an increased mobility. Shaded ovals indicate relative calculated positions of unfolded oligonucleotides of the same lengths

We compared the electrophoretic behavior of the (TTAGGG)N oligonucleotides, where n = 2 to 7, in denaturing (Fig. 6b) and nondenaturing (Fig. 6c) gels. In the latter experiment, oligonucleotides were preincubated in EMSA binding buffer that contained 50 mM KCl and 50 mM NaCl, and the running gel and buffer contained 20 mM KCl and 20 mM NaCl (see Materials and Methods for details). Increased electrophoretic mobility of oligonucleotides under nondenaturing conditions is indicative of secondary structure formation [39]. The data in Figure 6b and 6c show that only (TTAGGG)N with n >= 4 formed folded structures. Importantly, (TTAGGG)3, which is a substrate for bsSSTBP (Fig. 6c), did not form secondary structure. At the same time, mutant sequence (TTGGGG)4, which is not a substrate (Fig. 5), formed a folded structure. Therefore, bsSSTBP recognized linear TTAGGG sequence with three and more repeats.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
For an understanding of vertebrate telomere cellular functions, molecular characterization of the telomere-binding proteins is of primary importance. This has been clearly demonstrated by recent work from de Lange's laboratory concerning TRF1/2 proteins specifically interacting with the double-stranded telomere DNA [9, 16, 17]. Much less is known about vertebrate proteins interacting with the single-stranded telomere DNA overhang.

Here we have identified and characterized bsSSTBP, a DNA-binding protein from bovine (Bos taurus) sperm that specifically binds to G-strand of telomere DNA. To our knowledge, this is the first example of such proteins isolated from sperm cells of any species.

Biochemical characteristics and DNA affinity properties distinguish the bovine sperm protein from known single-stranded telomere DNA-binding proteins. At the same time, bsSSTBP shares some features with telomere terminus-specific proteins from other eukaryotes.

The bsSSTBP-(TTAGGG)N complex is resistant to high salt concentrations (up to 1.0 M KCl), and in this respect the sperm protein resembles ssTEL-binding proteins from ciliates [40] and telomere-end factor XTEF from Xenopus oocytes [18]. Similar to several ssTBPs from higher and lower eukaryotes [18, 40], bsSSTBP displays in vitro high affinity for the (TTAGGG)4 oligonucleotide; single base alterations in this sequence suppress interactions with protein (Fig. 5). Therefore, the exact nucleotide sequence is important for complex formation. Interestingly, single base substitutions in the GGG-region completely cancel bsSSTBP binding, whereas substitutions in the TTA-region diminish binding.

Bovine sSSTBP is sensitive to heat denaturation (Fig. 1), and telomere-binding activity can not be renatured after SDS-PAGE separation of proteins (negative results in South-Western assay). The latter may reflect the complex polypeptide composition of bsSSTBP. Indeed, bsSSTBP had an apparent native molecular size of 20–40 kDa as determined by gel filtration (Fig. 3), but a 180-kDa mass was revealed in UV cross-linked complexes with DNA (Fig. 4). These biochemical characteristics distinguish bsSSTBP from rat telomere-binding qTBP42, the thermostable and monomeric protein from rat hepatocytes [41].

Bovine sperm factor requires at least three TTAGGG repeats for binding (Fig. 6a), whereas telomere overhang-binding proteins from Euplotes crassus [40], Oxytricha nova [42], and Xenopus [18] interact efficiently with substrates consisting of two telomere repeats. The folded, G-quartet-like structure of DNA substrate is not important for bsSSTBP binding (Fig. 6). Therefore, in this respect bsSSTBP behaves like proteins from ciliated protozoa and Xenopus [18, 40, 42] but differs from the single-stranded and quadruplex telomere DNA-binding protein from rat hepatocytes [19, 41]. Once again, we see that bovine sperm TBP and ssTBP from other eukaryotes share some characteristics but are different in others. Thus we propose that the bsSSTBP is a novel telomere-binding factor with some functional homologies to other proteins interacting with a single-stranded telomere overhang.

Our interest in telomere-associated proteins in mammalian sperm was based on the importance of telomere-telomere interactions in maintaining the unique genome architecture in these cells [26, 43]. The pronounced feature of sperm telomeres to form dimers and tetramers located at nuclear membrane may be controlled by proteins. Recently, we have identified in human sperm protein activity specific in binding to double-stranded telomere DNA [28]. Our preliminary data indicate that somatic TRF1 is absent in mature human and bovine sperm, whereas bsSSTBP is not expressed in bovine brain tissue. Further, telomere DNA in mammalian sperm is 15–70% longer than in somatic cells of the same species [35]; this may be the result of an increased length of the double-stranded telomere DNA or telomere DNA overhang. In summary, we expect the molecular organization of the telomere chromosomal domain in somatic and sperm cells to be different. The exact stage of spermatogenesis at which the rearrangement of telomere takes place is unknown, and this question is currently under investigation.

Germ cell-specific telomere-binding proteins may participate in down-regulation of telomerase activity during spermatogenesis and in establishing telomere-telomere associations localized at nuclear membrane [26, 28, 31, 34]. It has been proposed that the latter is important for telomere functions during meiosis [32–34]; and after fertilization [43], bsSSTBP could contribute to one of these functions.


   FOOTNOTES
 
First decision: 17 August 1999.

1 This work was supported by USDA grant 9601837 (to A.O.Z.) and in part by DOE grant DEFG03-88ER-60673 (to E.M.B.). Back

2 Correspondence: Andrei Zalensky, Fax: 530 752 3516; aozalensky{at}ucdavis.edu Back

Accepted: September 23, 1999.

Received: July 19, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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