(Received for publication, February 10, 1997, and in revised form, April 16, 1997)
From the Unit of Biochemistry, The Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, P. O. Box 9649, Haifa 31096, Israel
A rat liver nuclear protein, unimolecular
quadruplex telomere-binding protein 25, (uqTBP25) is described
that binds tightly and specifically single-stranded and unimolecular
tetraplex forms of the vertebrate telomeric DNA sequence
5-d(TTAGGG)n-3
. A near homogeneous uqTBP25 was purified by
ammonium sulfate precipitation, chromatographic separation from other
DNA binding proteins, and three steps of column chromatography.
SDS-polyacrylamide gel electrophoresis and Superdex© 200 gel
filtration disclosed for uqTBP25 subunit and native
Mr values of 25.4 ± 0.5 and 25.0 kDa,
respectively. Sequences of uqTBP25 tryptic peptides were closely
homologous, but not identical, to heterogeneous nuclear
ribonucleoprotein A1, heterogeneous nuclear ribonucleoprotein A2/B1,
and single-stranded DNA-binding proteins UP1 and HDP-1. Complexes of
uqTBP25 with single-stranded or unimolecular quadruplex
5
-d(TTAGGG)4-3
, respectively, had dissociation constants,
Kd, of 2.2 or 13.4 nM. Relative to
d(TTAGGG)4, complexes with 5
-r(UUAGGG)4-3
,
blunt-ended duplex telomeric DNA, or quadruplex telomeric DNA had >10
to >250-fold higher Kd values. Single base
alterations within the d(TTAGGG) repeat increased the
Kd of complexes with uqTBP25 by 9-215-fold.
Association with uqTBP25 protected d(TTAGGG)4 against nuclease digestion, suggesting a potential role for the protein in
telomeric DNA transactions.
Linear eukaryotic chromosomes end with a specialized DNA-protein
structure termed the telomere that guards the chromosome terminus
against degradative attack or fusion with ends of other chromosomes
(1-4). Telomeric DNA consists of evolutionarily conserved short,
tandemly repeated nucleotide sequences. The telomeric DNA strand,
oriented 5 to 3
toward the chromosome end ("G-strand") in all
vertebrates, slime molds, filamentous fungi, and
Trypanosoma, is a repeated 5
-d(TTAGGG)-3
sequence paired
to a complementary 5
-d(CCCTAA)-3
strand. At their 3
-end, vertebrate
telomeres terminate with a 12-16-nucleotide-long unpaired overhang of
the G-strand (1-5). This single-stranded tract was shown to be capable of forming in vitro under physiological conditions a hairpin
or unimolecular or bimolecular tetrahelical structures (5-14), which may have a role in telomere transactions (5).
The telomere hypothesis of cellular aging and tumorigenesis claims that progressive shortening of telomeres during the life span of somatic cells leads to the cessation of cell division and to cellular senescence (15). In contrast, by maintaining a stable length of either long or short telomeres, respectively, germ line cells and tumor cells avoid division cycle exit and continue to divide infinitely (15). This hypothesis is sustained by data showing that whereas telomeric DNA is progressively trimmed in the course of the aging of diverse somatic cells, its length persists in immortal germ line and cancer cells (Refs. 16-19; reviewed in Ref. 15). The progressive shortening of telomeres in somatic cells and the contrasting maintenance of their length in indefinitely dividing cells is partly accounted for by their different levels of activity of telomerase, the telomeric G-strand-extending enzyme. Whereas telomerase activity is undetectable in numerous somatic tissues and in dividing primary cells, many cancer cells retain an active enzyme (Refs. 17-21; reviewed in Refs. 22 and 23). That telomerase does not exclusively determine telomere length is inferred, however, by the observation that some tumor cells whose telomere length remains stable have no measurable telomerase activity (24), and conversely, an active telomerase is detected in normal bone marrow cells and blood cells (25). Further, the loss of 50-200-nucleotide-long segments of telomeric DNA with each round of replication of somatic cells (26-28) suggests that exonucleolytic degradation of the terminus of telomeric DNA may also contribute to its progressive trimming. It has been suggested, therefore, that telomeric DNA binding proteins that were identified in diverse species may participate in the complex dynamics of elongation and shortening of telomeric DNA (4, 29). Some such proteins from different species bind tightly single-stranded telomeric DNA (30-33). Other proteins bind to or mediate the formation of tetraplex forms of telomeric DNA (33-42). Proteins of a third category selectively associate with the duplex region of telomeric DNA (43-46).
In this work we describe the purification from rat hepatocytes and
characterization of a 25-kDa monomeric protein, termed uqTBP25, that
binds tightly and in a sequence-specific fashion single-stranded and
unimolecular tetraplex forms of the G-strand of telomeric DNA. A
partial amino acid sequence of uqTBP25 is closely homologous but not
identical with sequences of hnRNP1 A1 and
hnRNP A2/B1 and their respective derivative single-stranded DNA-binding
proteins UP1 and HDP-1. Protein uqTBP25 is distinguished from hnRNP A1
and A2/B1 by its molecular size, preferential binding to DNA over
RNA, and sequence-specific binding to the telomeric DNA G-strand.
This protein also differs from UP1 and HDP-1 by its selective binding
to the G-strand of telomeric DNA and by its failure to
significantly stimulate the activity of DNA polymerase .
Radioactively 5-labeled
[
-32P]ATP (~3000 Ci/mmol),
[
-32P]dGTP (~3000 Ci/mmol), Klenow fragment of
Escherichia coli polymerase I, and molecular mass RainbowTM
marker proteins were products of Amersham Corp. Synthetic DNA
oligomers, listed in Table I, were purchased from Operon Technologies.
The HPLC-purified RNA oligomer r(UUAGGG)4 (Table
I) was a product of Midland Reagent. Boric acid,
-mercaptoethanol, dithiothreitol (DTT), N-ethylmaleimide (MalNEt), poly(dG)·poly(dC), thymidine 3
,5
-diphosphate, dimethyl sulfate, leupeptin, aprotinin, benzamidine, phenylmethylsulfonyl fluoride, Nonidet P-40, Sephadex G-50, phenyl-Sepharose, proteinase K,
salmon sperm DNA, soybean trypsin inhibitor, and micrococcal nuclease
were supplied by Sigma. DEAE-cellulose (DE-52) and DE-81 filter paper
were the products of Whatman. Total RNA from yeast was supplied by
Boehringer Mannheim. Bacteriophage T4 polynucleotide kinase and RNasin
were provided by Promega. Acryl/bisacrylamide (19:1 or 30:1.2) was
purchased from Amresco. Bacteriophage T4 gene 32 protein was purchased
from Boehringer Mannheim. Immunoaffinity-purified calf thymus DNA
polymerase
was the gift of Dr. L. A. Loeb (University of
Washington). Kodak XAR5 and Biomax MR-1 autoradiographic film, urea,
TEMED, bromphenol blue, and xylene cyanol FF were supplied by IBI.
HiTrap Blue HPLC column and Superdex© 200 HPLC gel filtration column
were provided by Pharmacia Biotech Inc. Econo-Pac S HPLC cartridge,
reagents for sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE), and molecular weight protein standards were the products of
Bio-Rad. Novex provided MultimarkTM molecular size protein standards.
Biotrace polyvinylidene difluoride binding matrix membranes were
supplied by Gelman Sciences.
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Full-length DNA oligomers were purified by
electrophoresis through a 8 M urea, 15% polyacrylamide
denaturing gel (acryl/bisacrylamide, 19:1) as we described (47). The
purified DNA or RNA oligomers were labeled at their 5-end with
32P in a bacteriophage T4 polynucleotide kinase-catalyzed
reaction (48). Oligomers were maintained in their single-stranded
conformation as a 0.25-0.70 µM solution in 1.0 mM EDTA, 10 mM Tris-HCl buffer, pH 8.0 (TE
buffer), and were boiled immediately prior to use. Double-stranded
telomeric DNA was prepared by annealing a 1.25-fold molar excess of a
cytosine-rich sequence with a complementary guanine-rich oligomer, and
duplex DNA molecules were electrophoretically resolved from residual
DNA single strands as we described (33). Labeling of a protruding end
of the annealed duplex DNA was catalyzed by the Klenow fragment of
E. coli polymerase I using 5
-[
-32P]dGTP as
we described previously (47). Unimolecular (G
4) and bimolecular (G
2)
tetraplex forms of TeR DNA were prepared, their stoichiometry was
verified, and their stabilization by Hoogsteen bonds was demonstrated,
as we described in detail elsewhere (33). Parallel G4 quadruplex forms
of oligomers Q and single Q were prepared according to Sen and Gilbert
(48), and their stoichiometry was shown to be tetramolecular as
recently described (33). The parallel quadruplex form of
d(CGG)8 was prepared, and its structure was verified as
previously detailed (49).
The DNA binding activity of uqTBP25 was monitored by
electrophoretic mobility shift assay as we described previously (33, 50). In a typical assay for the binding of single-stranded TeR-4 or
TeR-2 DNA, 5.0-15.0 ng of 32P-5-labeled TeR-2 or TeR-4
DNA was incubated at 4 °C for 20 min with 30-3000 ng of purified or
crude protein fraction in a 15-µl final volume of buffer D (0.5 mM EDTA, 20% glycerol in 25 mM Tris-HCl buffer, pH 7.5). The binding mixture was electrophoresed in a Mini
PROTEAN II electrophoresis system (Bio-Rad) at 4 °C under 10 V/cm
through a nondenaturing 6% polyacrylamide gel (acryl/bisacrylamide, 30:1.2) in 0.6 × TBE buffer (1.2 mM EDTA in 0.54 M Tris borate buffer, pH 8.3) until a bromphenol blue
tracking dye migrated 2.5-4.0 cm into the gel. The gels were dried on
DE-81 filter paper and exposed to x-ray film or to a phosphor imaging
plate (Fuji). The proportion of free and uqTBP25-bound TeR DNA was
determined by phosphor imaging, and their amounts were deduced from the
known specific activity of the labeled DNA probe. One unit of uqTBP25 DNA binding activity was defined as the amount of uqTBP25 that bound 66 pmol of single-stranded TeR-2 DNA under the described standard
conditions. Standard assay conditions were also employed for the
binding of tetramolecular G4 quadruplex DNA and of double-stranded DNA.
Binding of tetraplex G
2 TeR DNA or G
4 TeR DNA was assayed as
described above except that 50 mM KCl or 50 mM
NaCl, respectively, was added to the DNA binding mixture to preserve
the quadruplex structure of the DNA, the 0.6 × TBE gel running
buffer contained 50 mM KCl or 50 mM NaCl as
necessary, and electrophoresis was performed at 4 °C.
SDS-PAGE and silver or Coomassie Blue staining of resolved protein bands was carried out as we described previously (50). Molecular size protein markers were the Amersham RainbowTM, Novex MultimarkTM, or Bio-Rad prestained or unstained molecular weight standards.
Southwestern analysis was conducted according to Petracek et
al. (31) with the minor modifications that we recently introduced (33). TeR-4 DNA binding activity was detected in nuclear extracts by
exposing the electrophoretically resolved proteins to 0.85 µg of
32P-5-labeled TeR-4 DNA in the presence of 50 mM NaCl.
In a typical preparation, protein
uqTBP25 was purified to near homogeneity from ~700 g of liver tissue
from adult rats. Salt extracts of nonhistone nuclear proteins were
prepared from isolated hepatocyte nuclei as we described elsewhere
(52), except that the composition of the extraction buffer was 0.4 M NaCl, 1 mM phenylmethylsulfonyl fluoride, 0.1 mM benzamidine, and 10 µg/ml each of the protease
inhibitors soybean trypsin inhibitor, leupeptin, and aprotinin in
buffer D. The preparation of protein extracts and all of the subsequent
steps of uqTBP25 purification were conducted at 4 °C.
Electrophoretic mobility shift assays and Southwestern analysis showed
that rat liver nuclear extracts contained G4 TeR-4 DNA binding
activity with an approximate molecular size of 24 kDa (see
"Results"). This protein was further purified by successive steps
of ammonium sulfate precipitation and column chromatography. Elution
profiles of the DNA binding activity from the various columns, as
assessed by electrophoretic mobility shift analysis were identical when
32P-5
-labeled single-stranded TeR-2 or TeR-4 DNA or
unimolecular tetraplex G
4 TeR-4 DNA were used as probes. In an initial
purification step, extract proteins were precipitated by 50% ammonium
sulfate and removed by centrifugation. The majority of the TeR DNA
binding activity that remained in the supernatant was subsequently
precipitated by 70% ammonium sulfate. The 70% ammonium sulfate
precipitate was resuspended in buffer D and was dialyzed overnight at
4 °C against ~200 volumes of the same buffer. After adding NaCl to the dialyzed protein fraction to a final concentration of 50 mM, it was mixed with heat-denatured salmon sperm DNA and
incubated for 20 min at 4 °C at a protein:DNA ratio of 30:1 (w/w).
Whereas the major TeR-binding protein qTBP42 (33) and additional
single-stranded DNA-binding proteins associated tightly with the
denatured DNA, uqTBP25 did not bind detectably to it. The strong
binding of DNA and DNA-protein complexes to DEAE-cellulose (53) was
subsequently utilized to separate uqTBP25 from qTBP42 (33) and from
additional proteins that associated with denatured DNA. The protein-DNA
mixture was loaded at a ratio of 4.0 mg of protein/ml of packed resin, onto a DE-52 column equilibrated in buffer D containing 50 mM NaCl. The protein-loaded column was washed with 0.5 and
subsequently with 1.5 packed column volumes of the equilibration
buffer, and bound proteins were eluted by two packed column volumes
each of 100 and 225 mM NaCl in buffer D. Electrophoretic
mobility shift analysis and SDS-PAGE separation of UV cross-linked
TeR-4 DNA-protein complexes, respectively, revealed TeR-4 DNA binding
activity and UV cross-linked TeR-4 DNA-protein complexes of 34 kDa in
the 50 mM NaCl wash fractions. Similar analysis did not
reveal DNA-protein complexes in the 100 mM NaCl fraction;
instead, multiple complex bands, the dominant of which was qTBP42 (33),
were present in the 225 mM NaCl eluate. The 50 mM NaCl eluate fractions were pooled together and dialyzed
overnight against ~50 volumes of buffer P (0.5 mM EDTA,
20% glycerol in 25 mM NaPO4 buffer, pH 7.0).
The dialyzed fractions were loaded at a ratio of 24.0 mg of protein/ml of packed resin onto a 5.0-ml Econo-Pac S column equilibrated in buffer
P and mounted on a GradiFrac low pressure chromatography device
(Pharmacia). The loaded column was washed with 7.5 packed resin volumes
of buffer P, and bound proteins were eluted from the column by a linear
gradient of 19.5 column volumes of 0.0-1.0 M NaCl in
buffer P. Fifty fractions were collected, and as done in every
subsequent chromatography, aliquots were dialyzed overnight against 150 volumes of buffer D and then assayed for G
4 TeR-4 DNA binding. G
4
TeR-4 DNA binding activity of uqTBP25 was detected in the 150-320
mM NaCl eluate both by electrophoretic mobility shift
analysis and by the identification in SDS-PAGE of a ~34-kDa UV-cross-linked protein-TeR-4 DNA complex. The active fractions were
pooled together, dialyzed overnight against 150 volumes of P2 buffer
(0.5 mM EDTA, 20% glycerol in 10 mM
NaPO4, pH 7.0), and loaded at a ratio of 8.5 mg of
protein/ml of packed resin onto a 1.0-ml HiTrap Blue HPLC column
equilibrated in P2 buffer and mounted on a GradiFrac device. The loaded
column was washed by six column volumes of the equilibration buffer,
and adsorbed proteins were eluted by a 21-packed column volume linear
gradient of 0.0-4.0 M NaCl in P2 buffer. Fifty fractions
were collected, aliquots were dialyzed, and uqTBP25 binding activity
was detected by electrophoretic mobility shift analysis and SDS-PAGE of
UV cross-linked protein-TeR-4 DNA complexes in fractions that were eluted from HiTrap Blue by 2.5-3.5 M NaCl. Fractions
containing the binding activity were pooled together and dialyzed
overnight against ~50 volumes of 4.0 M NaCl in buffer S
(1.0 mM EDTA in 25 mM Tris-HCl buffer, pH 7.5)
and loaded at a ratio of 1.0 mg of protein:1.0 ml of packed resin onto
a phenyl-Sepharose column equilibrated in buffer S. The loaded column
was washed with two packed column volumes of the equilibration buffer,
and bound proteins were eluted by a stepwise gradient of 4.0-0.0
M NaCl in buffer S followed by a 40% ethylene glycol wash
to elute proteins that remained adsorbed to phenyl-Sepharose at 0.0 M NaCl. Fractions were collected into Nonidet P-40 (0.05%
final concentration), and the activity of uqTBP25 was detected in
fractions that were eluted from the phenyl-Sepharose column by 1.0-0.5
M NaCl. Silver and Coomassie Blue staining of the eluted
proteins indicated that a 25-kDa species was the major protein eluted
by 1.0-0.5 M NaCl (Fig. 2C), whereas the
majority of the proteins that were loaded onto the column were eluted
by 40% ethylene glycol. Determination of the protein content of the
collected fractions and silver or Coomassie Blue staining of
SDS-PAGE-resolved protein bands was directly performed on fractions
that were dialyzed against water. Fractions that were used for the
assay of DNA binding activity were stabilized by the immediate addition
soybean trypsin inhibitor protein (200 µg/ml final concentration),
and following their dialysis overnight against ~200 volumes of buffer
D, they were stored in aliquots at
80 °C. Under these storage
conditions, the DNA binding activity was fully preserved for at least 4 months.
Measurement of the Effect of uqTBP25 on Polymerase
DNA synthesis was conducted for 30 min at 37 °C
in a reaction mixture that contained in a final volume of 25 µl: 20 mM Tris-HCl buffer, pH 7.5, 3.0 mM
MgCl2, 1.0 mM DTT, 2.1 units of
immunoaffinity-purified calf thymus DNA polymerase . The DNA
primer-templates and dNTP substrates were either 1.0 µg of
poly(dG)·poly(dC) and 25 µM [
-32P]dGTP
(specific activity 5,450 cpm/pmol) or 0.5 µg of bacteriophage M13mp2
single-stranded DNA primed by a 17-mer universal primer and 25 µM concentration of each of the four dNTPs and
[
-32P]dGTP (specific activity 1260 cpm/pmol).
Increasing amounts of phenyl-Sepharose-purified uqTBP25 were added to
the reaction mixtures as detailed under "Results." DNA synthesis
was terminated, acid-insoluble DNA was precipitated, and incorporation
of [32P]dGMP into DNA was measured as described
previously (53).
The Bio-Rad protein assay kit was used to determine amounts of protein.
Activities that bind single-stranded and tetraplex forms
of the vertebrate telomeric sequence TeR-4,
5-d(TTAGGG)4-3
, were detected by electrophoretic mobility
shift analysis in extracts of nonhistone nuclear proteins from rat
hepatocyte. Southwestern analysis of the unimolecular tetraplex G
4
TeR-4 DNA binding activity detected in replicate nuclear extracts a
major protein-G
4 TeR-4 DNA complex band of ~24 kDa and two minor
bands of 31 and 33 kDa, respectively (for a typical analysis, see Fig.
1A). To isolate the ~24-kDa binding
activity, the nuclear extract was initially fractionated by ammonium
sulfate precipitation. The major portion of an activity that bound
single-stranded TeR-4 or unimolecular tetraplex G
4 TeR-4 DNA was
detected by electrophoretic mobility shift analysis in the 50-70%
(NH4)2SO4 precipitate. To resolve TeR DNA-specific binding activity from other proteins that bind nonspecifically to single-stranded DNA, the resuspended and dialyzed 70% ammonium sulfate precipitate was incubated with denatured salmon
sperm DNA and than chromatographed on a DE-52 column. DNA and
DNA-protein complexes strongly adsorb to the anion exchanger, whereas
some proteins that do not bind to denatured DNA adsorb weakly to DE-52
(53). Electrophoretic mobility shift analysis of G
4 TeR-4 DNA binding
activity and SDS-PAGE resolution of UV-cross-linked protein-G
4 TeR-4
DNA complexes revealed an electrophoretically retarded 34-kDa complex
band in fractions that were eluted from DE-52 by 50 mM NaCl
(see "Experimental Procedures"). Several additional proteins,
including qTBP42 (33), that formed complexes with denatured
DNA and tightly adsorbed to DE-52 were eluted from the column by 225 mM NaCl (see "Experimental Procedures"). The TeR-4 DNA
binding activity that was eluted from DE-52 by 50 mM NaCl was further purified by successive steps of chromatography on columns
of Econo-Pac S, HiTrap Blue and phenyl-Sepharose. Elution profiles of
the TeR DNA binding activity from the different columns, as revealed by
electrophoretic mobility shift analysis were identical when
32P-5
-labeled single-stranded TeR-2 or unimolecular
tetraplex G
4 TeR-4 DNA were used as probes. SDS-PAGE resolution of
proteins in the different fractions, and their silver staining
demonstrated a progressive depletion of proteins in the course of
uqTBP25 purification (Fig. 1B). Note a 25-kDa protein band
that became discernible in the HiTrap Blue fraction of uqTBP25 (Fig.
1B). The intensity of Coomassie Blue staining of this 25-kDa
protein band was directly proportional to the level of TeR-4 DNA
binding activity in the HiTrap Blue fractions (results not shown). This
protein became highly enriched after phenyl-Sepharose purification
(Fig. 1B).
Results shown in Fig. 2 indicated that the 25-kDa
protein band, which was purified to near homogeneity by
phenyl-Sepharose chromatography, corresponded to the TeR DNA binding
activity. As seen in Fig. 2A, TeR-4 DNA binding activity was
detected by mobility shift electrophoresis in phenyl-Sepharose
fractions 10-14 (1.0-0.5 M NaCl eluate). Covalent UV
cross-linking of labeled DNA to the phenyl-Sepharose-resolved proteins
revealed in fractions 10-14 a 34-kDa protein-TeR-4 DNA complex (Fig.
2B), whose amount corresponded to the TeR DNA binding
activity (Fig. 2A). Finally, SDS-PAGE resolution of the
phenyl-Sepharose protein fractions showed that the intensity of a
Coomassie Blue-stained 25-kDa band, which constituted >80% of the
protein content of fractions 11-13 (Fig. 2C) was well
correlated with the level of TeR-4 DNA binding activity in fractions
10-14 (Fig. 2A) and with the amount of the UV-cross-linked
TeR-4-protein complex in these fractions (Fig. 2B). The
~25-kDa size of the unimolecular tetraplex G4 TeR-4 DNA binding
activity as detected in nuclear extracts by Southwestern analysis (Fig.
1A) as well as the 25-kDa molecular mass of the highly
purified active protein (Fig. 2C) and the 34-kDa size of its
complex with TeR-4 DNA (Fig. 2B) strongly suggested that the 25-kDa protein represented uqTBP25. Details of a typical purification scheme summarized in Table II indicated that, relative
to crude nuclear extract, the phenyl-Sepharose fraction of uqTBP25 was purified more than 1000-fold with a final yield of 2.2%.
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Some properties of
uqTBP25 are presented in Table III. That uqTBP25 was
proteinaceous was demonstrated by its heat lability, inactivation by
SDS, and sensitivity to proteinase K. The resistance of uqTBP25 to
digestion by micrococcal nuclease (Table III) suggested that it did not
require an essential nucleic acid component. Binding of TeR-4 DNA by
uqTBP25 was not affected by exposure to 8.5 mM MalNEt
(Table III), indicating that reduced protein sulfhydryl groups were not
directly involved in the protein interaction with DNA. This was also
corroborated by the equally efficient renaturation of the 25-kDa
protein in Southwestern blotting with or without the presence of
-mercaptoethanol (Fig. 1A).
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The highly purified uqTBP25 migrated in SDS-PAGE as a 25.4 ± 0.4-kDa polypeptide (n = 6). An apparent molecular size of 25.0 kDa (n = 2), which was found for native uqTBP25 by Superdex 200© gel filtration, suggested that uqTBP25 was a 25-kDa monomeric protein.
Sequence Homology among uqTBP25, Two hnRNP Species, and Their Derivative Single-stranded DNA-binding ProteinsTo find out whether or not uqTBP25 represented a known protein, sequences of five tryptic peptides of uqTBP25 were determined. A computerized search through GenBankTM revealed all five uqTBP25 peptide sequences to be closely homologous, although not identical, to conserved amino acid sequences in hnRNP A1 and A2/B1 and in their derivative amino-terminal proteolytic fragments, calf thymus single-stranded DNA-binding proteins UP1 (58, 61, 64) and mouse HDP-1 (58, 64), respectively. The sequence of hnRNP A1 that remained identical in four mammalian species (Table IV) and of its cognate single-stranded binding protein UP1 (58, 61, 64) differed from the corresponding partial sequence of uqTBP25 by 4 amino acids out of 43 sequenced residues. One dissimilar amino acid represented a nonconservative G to Q substitution in uqTBP25 peptide IV (Table IV). Most notable, amino acid sequences of uqTBP25 were clearly distinct from those of cloned rat hnRNP A1 and of rat UP1 (Table IV; Refs. 61 and 62). The dissimilarity between uqTBP25 and rat hnRNP A1 extended to their different molecular sizes of 25 and 34.2 kDa, respectively (see above, under "Results"; Ref. 61), and uqTBP25 differed from both hnRNP A1 and UP1 by its nucleic acid binding preferences (see below, under "Results" and "Discussion"). Hence, despite their close sequence similarity, uqTBP25, hnRNP A1, and UP1 was each a distinct protein. An extensive sequence similarity was also found for uqTBP25 and human hnRNP A2/B1 (Table IV) and its derivative fragment, the DNA-binding protein HDP-1 (58, 64). However, six amino acid alterations were noted among the 43 sequenced uqTBP25 residues, two of which, E to A and N to A in uqTBP25 peptide V, constituted nonconservative substitutions (Table IV). Differences between rat uqTBP25 and human hnRNP A2/B1 also extended to their different molecular sizes of 25 and 36-37.4 kDa, respectively (Table IV; Ref. 54), and both hnRNP A2/B1 and HDP-1 differed from uqTBP25 by their nucleic acid binding specificity (see below, under "Results" and "Discussion").
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The
sequence specificity of binding of DNA by uqTBP25 was first assessed by
measurements of the relative association of the protein with
32P-5-labeled TeR-2 DNA in the presence of a 50 or 75-fold
molar excess of different unlabeled competing DNA sequences. Results summarized in Table V indicated that the binding of
single-stranded TeR-2 DNA was not diminished significantly when a
50-fold molar excess of various unlabeled single-stranded DNA sequences
was present. Similar results were obtained in reactions that contained a 75-fold molar excess of the competing sequences over TeR-2 DNA (data
not shown). Notably, DNA sequences that did or did not contain guanine
clusters were similarly ineffective as competitors with TeR-2 DNA.
Hence, the guanine-rich oligomers d(G)16, the fragile X
syndrome expanded sequence d(CGG)8, Tetrahymena
telomeric TeT G-strand DNA, and the IgG switch region sequence oligomer
Q did not compete efficiently with TeR-2 DNA upon its binding to
uqTBP25 (Table V). As a result of quantitative annealing under the
standard binding conditions of TeR-4 C DNA to TeR-4 DNA (Ref. 66; our results), the labeled single-stranded TeR-4 DNA was eliminated from the
reaction, and the efficacy of TeR-4 C DNA as a competitor could not be
assessed. However, a direct binding assay failed to reveal the
formation of a detectable complex between uqTBP25 and
32P-5
-labeled TeR-4 C DNA when the labeled probe was added
at concentrations of up to 560 nM (results not shown).
Hence, unlike the recently described qTBP42 (33), uqTBP25 did not
measurably bind to the cytosine-rich telomeric DNA strand. An excess of
yeast total RNA also failed to compete with TeR-2 DNA upon binding to
uqTBP25 (Table V), and a direct binding assay did not detect complex formation between uqTBP25 and labeled yeast total RNA at up to 200 nM (results not shown).
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To assess more precisely the DNA sequence specificity and structure
specificity of DNA binding by uqTBP25, we determined values of
dissociation constants, Kd, for complexes of uqTBP25 with sequence and structure variants of telomeric DNA. Fig.
3 shows a typical steady-state binding analysis of
complex formation between uqTBP25 and TeR-2 DNA. A constant amount of
phenyl-Sepharose-purified uqTBP25 protein was incubated under standard
binding conditions with increasing amounts of
32P-5-labeled TeR-2 DNA, and formed protein-DNA complexes
were separated from unbound DNA by mobility shift electrophoresis (Fig. 3A). Amounts of protein-bound and free TeR-2 DNA were
determined by phosphor imaging measurements of the respective bands,
and the value of the dissociation constant, Kd, was
inferred from the negative reciprocal of the slope of a Scatchard plot of the results (Fig. 3B). Compiled in Table VI are
Kd values for complexes of uqTBP25 with different
structures of TeR DNA or with oligomers closely homologous to the
telomeric sequence. Apparently, complexes of uqTBP25 with
single-stranded telomeric sequences that contained two or more
d(TTAGGG) clusters had nanomolar range dissociation constants (Table
VI). However, a complex of uqTBP25 with an oligomer that
contained a single d(TTAGGG) cluster had a dissociation constant 8.5- or 12.5-fold higher than the Kd values of complexes
with oligomers that had two or four telomeric repeat units,
respectively (Table VI). As shown in Table VI, binding of TeR DNA by
uqTBP25 was highly sequence-specific, such that complexes of uqTBP25
with oligomers that contained single base substitutions within the
TeR-4 DNA repeat unit had considerably elevated Kd
values. Substituting the single adenosine residue,
d(TTAGGG), within the TeR DNA sequence with a guanine, d(TTGGGG), in TeT DNA increased the Kd
value of the protein-TeT DNA complex 215-fold relative to the
Kd of a uqTBP25-TeR-4 DNA complex (Table VI).
Similarly, altering the TeR DNA d(TTAGGG) repeat unit into
d(TTAGAG) in Mut1 TeR-4 DNA increased 85-fold the
Kd value of the uqTBP25-Mut1 TeR-4 DNA complex
(Table VI). Interestingly, an increase of only 9-fold in
Kd value, was obtained when the TeR-4 DNA sequence
d(TTAGGG) was changed into d(TAAGGG) in Mut2
TeR-4 DNA (Table VI).
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That uqTBP25 bound preferentially TeR-4 DNA over the homologous rTeR-4
RNA sequence is evident by the 11.6-fold higher Kd of the protein-rTeR-4 complex (Table VI). This preferential binding of
DNA over RNA contrasts with the proclivity of several hnRNP species to
bind RNA more tightly than DNA (Ref. 64; see "Discussion"). A
preference of uqTBP25 for single-stranded over double-stranded TeR DNA
was demonstrated by the 30-fold lower Kd of its complex with single-stranded TeR-4 DNA relative to the
Kd of its complex with blunt-ended double-stranded
telomeric DNA (Table VI). However, when the double-stranded TeR DNA
ended with a d(TTAGGG)2 single-stranded overhang, its
association with uqTBP25 was as tight as that of single-stranded TeR-4
DNA (Table VI). Of the various forms of tetraplex DNA, only a
unimolecular antiparallel G4 TeR-4 DNA structure bound tightly to
uqTBP25. Although the Kd value of the complex of
uqTBP25 with G
4 TeR-4 DNA was 6-fold higher than that of a
uqTBP25-TeR-4 DNA complex (Table VI), this difference was due to the
required presence of 50 mM NaCl in the binding mixture. We
found that the dissociation constant of a complex of uqTBP25 with TeR-2
DNA, which could not form a tetraplex structure, was also increased in
the presence of 50 mM KCl from 3.2 ± 0.7 × 10
9 mol/liter (Table VI) to 28.0 ± 0.3 × 10
9 mol/liter (n = 2) and to 36.0 ± 0.9 × 10
9 mol/liter (n = 2) in the
presence of 50 mM NaCl. It was concluded, therefore, that
the single-stranded and unimolecular quadruplex forms of TeR-4 DNA were
bound by uqTBP25 with a very similar affinity. By contrast, a
bimolecular G
2 TeR-2 tetraplex DNA bound poorly to uqTBP25, forming a
complex having a Kd 38-fold higher than the
dissociation constant of a complex with TeR-2 DNA (Table VI). Parallel
tetramolecular G4 quadruplex forms of oligomer Q (33, 50), single Q
(48), or d(CGG)8 at up to 123 nM (49) did not
form detectable complexes with the protein (results not shown). Hence,
our results indicated that uqTBP25 selectively bound single-stranded
and unimolecular tetraplex forms of TeR DNA in a highly
sequence-specific fashion.
To inquire whether the stability of TeR DNA is
affected by its association with uqTBP25, we compared the rate of
digestion by micrococcal nuclease of unbound and protein-bound
single-stranded TeR-4 DNA. Unbound single-stranded TeR-4 DNA or its
complex with uqTBP25 were exposed for different lengths of time at
20 °C to 0.30 ng/µl micrococcal nuclease. The nucleolytic
digestion was terminated by adding SDS to a final concentration of
0.25%, and the DNA samples were electrophoresed through a 6%
nondenaturing polyacrylamide gel to separate the intact DNA oligomer
from its digestion products, which migrated at the front of the gel.
The kinetics of breakdown of the unbound or uqTBP25-bound
single-stranded TeR-4 DNA indicated that whereas 53 or 77% of the
unbound TeR-4 DNA was digested within 1 or 3 min, respectively, only 4 or 9% of the uqTBP25-bound TeR DNA was degraded after exposure to the nuclease for these periods of time (Fig. 4A).
To assess the specificity of protection of TeR-4 DNA by uqTBP25 against
nucleolytic attack, we examined the effect of the protein on the rate
of digestion of the poorly bound TeT-4 DNA (Table VI). As seen in Fig.
4B, 25 or 39% of the unbound TeT-4 DNA were digested after
a 1- or 3-min exposure to micrococcal nuclease, and similarly, 41 or
50% of the protein-associated TeT-4 DNA was degraded after digestion for the same periods of time. Hence, uqTBP25-associated TeT-4 DNA was
not protected against nucleolytic attack, and its rate of breakdown was
even modestly accelerated in the presence of the protein. It appeared,
therefore, that the formation of a sequence-specific tight complex
between TeR-4 DNA and uqTBP25 was responsible for the observed
resistance of the protein-bound telomeric DNA to nuclease attack.
The Effect of uqTBP25 on DNA Polymerase
The
single-stranded DNA-binding protein UP1 and uqTBP25 are distinguished
from one another by their closely homologous but nonidentical amino
acid sequence (Table IV) and by their molecular sizes of 22 (58) and 25 kDa, respectively. One characteristic property of UP1 is its capacity
to enhance the activity of calf thymus polymerase (51). To further
compare uqTBP25 with UP1, we examined the effect of uqTBP25 on
polymerase
-catalyzed DNA synthesis. Calf thymus polymerase
was
incubated under DNA synthesis reaction conditions with increasing
amounts of uqTBP25 and with either a poly(dG)· poly(dC)
primer-template or with singly primed bacteriophage M13mp2
single-stranded DNA. DNA synthesis was determined by measuring the
incorporation of 32P dGMP into acid-insoluble product DNA
(see "Experimental Procedures"). As seen in Fig. 5,
the copying of a poly(dC) template strand was inhibited by uqTBP25 by
up to ~20%, whereas the copying of M13mp2 DNA was increased by less
than 2-fold. Under the same reaction conditions, bacteriophage T4 gene
32 protein inhibited by up to 90% the copying of poly(dC) by
polymerase
and stimulated the copying of M13mp2 DNA by more than
3-fold (results not shown). In modestly inhibiting polymerase
-catalyzed copying of poly(dC) and stimulating M13mp2 DNA copying by
less than 2-fold, uqTBP25 contrasted UP1, which reportedly increased
>5- or >10-fold the copying by polymerase
of poly(dC) or of
E. coli exonuclease III-treated bacteriophage
DNA
template, respectively (51). Hence, unlike UP1, uqTBP25 did not display
a significant polymerase
-stimulatory activity.
The new mammalian telomeric DNA binding protein uqTBP25, which we describe in this manuscript, associates tightly and in a sequence-specific manner with single-stranded and unimolecular tetraplex forms of the G-strand of vertebrate telomeric DNA. Two or more d(TTAGGG) telomeric DNA repeat units suffice for the formation of uqTBP25-DNA complexes that display nanomolar range dissociation constants (Table VI). Various single-stranded sequences, including DNA oligomers that do or do not contain guanine clusters as well as RNA sequences, fail to efficiently compete at a 50- or 75-fold molar excess with TeR-2 DNA for complex formation with uqTBP25 (Table V). The specific binding of vertebrate telomeric DNA by uqTBP25 is further underscored by the greatly reduced affinity of the protein for the DNA ligand when a single base substitution is introduced into the telomeric G-strand sequence. Thus, Kd values of complexes of uqTBP25 with d(TTGGGG)4, d(TTAGAG)4 or d(TAAGGG)4 are 215-, 85-, or 9-fold higher, respectively, than the Kd of a complex with d(TTAGGG)4 (Table VI).
The amino acid sequence of five tryptic peptides of uqTBP25 (Table IV) are closely homologous, but not identical, to sequences shared by hnRNP A1 (56-63) and hnRNP A2/B1 (54, 55) and by their respective derivative single-stranded DNA-binding proteins UP1 (58, 61, 64) and HDP-1 (58, 64). Notably, a sequence within uqTBP25 peptide I (Table IV), IFVGGI, corresponds to the consensus sequence of the RNP2 element, LFVGNL, which is common to hnRNP A1, hnRNP A2/B1, UP1, and HDP-1 (67). However, despite their close sequence similarity, uqTBP25 is disparate from hnRNP A1, hnRNP A2/B1, UP1, and HDP-1.
uqTBP25 Is Distinct from hnRNP A1 and hnRNP A2/B1Four lines of evidence distinguish uqTBP25 from hnRNP A1 and hnRNP A2/B1. (i) The 25-kDa molecular mass of uqTBP25 (Fig. 2 and "Results") differs from the 34- and 36-38-kDa molecular sizes of hnRNP A1 and hnRNP A2/B1, respectively (68). (ii) Out of 43 sequenced amino acid residues in uqTBP25, 4 or 6, respectively, are different from corresponding residues in hnRNP A1 or A2/B1 (Table IV). The amino acid sequence of hnRNP A1 is 100% conserved in human, bovine, mouse, and rat cells (Ref. 68, Table IV). The finding of different amino acids at matching positions in the rat cell-derived uqTBP25 and in the highly conserved hnRNP A1 (Table IV) indicates, therefore, that uqTBP25 does not represent rat hnRNP A1 or a derivative thereof. The six-residue difference between the amino acid sequences of rat uqTBP25 and human hnRNP A2/B1 (Table IV) strongly suggest that these two proteins are the products of distinct genes. Notably, a 100% identity exists between a partial sequence of mouse hnRNP A2/B1 and its human homologue (69). It is thus unlikely that the different amino acid sequences of uqTBP25 and of hnRNP A2/B1 are due to species diversity among homologous proteins. (iii) No detectable complex is formed between uqTBP25 and yeast total RNA (Table V; see "Results"), and a complex that does form between uqTBP25 and r(UUAGGG)4 has a Kd value 11.6-fold higher than that of a complex with d(TTAGGG)4 (Table VI). The propensity of uqTBP25 for binding single-stranded DNA over RNA contrasts the preference of hnRNP A1 and hnRNP A2/B1 for association with RNA over single-stranded DNA (64). (iv) Data presented in Tables V and VI show that uqTBP25 binds d(TTAGGG)n sequences with a high degree of sequence specificity. By contrast, evidence shows that hnRNP A1 and A2/B1 bind RNA with a low sequence specificity (70), with a notable exception of a reported selective binding of d(TTAGGG)n by mouse liver hnRNP A2/B1 (69).
uqTBP25 Is Distinct from the Single-stranded Binding Proteins UP1 and HDP-1Four lines of evidence indicate that despite their
close size and sequence similarity, uqTBP25 and UP1 or HDP-1 are
distinct proteins. (i) The amino acid sequence of the single-stranded
DNA binding proteins UP1 from calf thymus and HDP-1 from mouse myeloma (51) indicate that they are fully homologous to the amino-terminal portion of HnRNP A1 and HnRNP A2/B1, respectively (58, 64). Multiple
amino acid substitutions distinguish uqTBP25 from either UP1 or HDP-1
and from their respective progenitor proteins HnRNP A1 or A2/B1 (Table
IV). Hence, it appears that uqTBP25 is not a product of proteolytic
cleavage of either HnRNP A1 or A2/B1 as are UP1 or HDP-1, respectively.
(ii) The 25-kDa size of uqTBP25 differs from the 22- and 27-kDa
molecular masses of calf thymus UP1 (58) and mouse myeloma HDP-1 (65),
respectively. (iii) Whereas UP1 or HDP-1 bind single-stranded DNA with
little or no sequence preference (51, 65), uqTBP25 associates
selectively with the telomeric sequence d(TTAGGG)n (Tables V
and VI). (iv) Unlike UP1, which stimulates >5- or >10-fold the
copying by DNA polymerase of poly(dC) or of single-stranded DNA
templates, respectively (51), uqTBP25 slightly inhibits copying
of poly(dC) and increases by less than 2-fold copying of
single-stranded DNA (Fig. 5).
A group of related proteins that bind the pre-mRNA 3
splice site r(UUAG/G) as well as the telomeric sequence
d(TTAGGG)n was identified in HeLa cells (71). The size,
antigenicity, nucleic acid binding preference, and partial amino
sequence of most of these proteins suggested that they are identical or
closely related to hnRNP type A2/B1, D, or E (71). However, a 26-kDa
protein designated A26, which has stretches 18 amino acids long
homologous to hnRNP A1, binds d(TTAGGG)n with a high sequence
specificity. Resemblance between human A26 and rat uqTBP25 extends to
their similar molecular mass, their preferential binding of
single-stranded over blunt-ended double-stranded telomeric sequence,
and their high sequence specificity of d(TTAGGG)n binding (71). Yet, some properties distinguish uqTBP25 from A26. Whereas binding competition results indicate that A26 binds r(UUAGGG)n more
tightly than d(TTAGGG)n (71), the
uqTBP25-d(TTAGGG)4 complex has an 11.6-fold lower
Kd than a uqTBP25-r(UUAGGG)4 complex
(Table VI). Additionally, unlike uqTBP25, which binds TeR-4 and TeR-2
DNA with a similar affinity (Table VI), A26 binds TeR-2 DNA less
tightly than TeR-4 DNA (71). Last, A26 fails to bind the substituted
homologues of r(UUAGGG)4
(r(CUAGGG)4, r(UCAGGG)4, r(UUGGGG)4,
or r(UUAAGG)4), but it does bind
r(UUAGAG)4 or
r(UUAGGA)4 (71). By contrast, relative to
d(TTAGAG)4, uqTBP25 binds most weakly
d(TTGGGG)4 and
d(TTAGAG)4, but it does associate relatively
tightly with d(TAAGGG)4 (Table VI).
The amino acid sequence of uqTBP25 indicates that it is probably a derivative of an hnRNP species that is closely related but not identical to hnRNP A1 or A2/B1 (Table VI). Likewise, uqTBP25 is related to but distinct from the single-stranded DNA-binding proteins UP1 and HDP-1. Based on its molecular size and telomeric DNA binding specificity, uqTBP25 is most closely similar to human protein A26 (71). It was argued that the prime target of protein A26 is the pre-mRNA splice site but that it could also be involved in the binding of telomeric DNA (71). In view of the preferential binding by uqTBP25 of telomeric DNA sequence over its RNA homologue and its lack of clear preference for an intact splice site (Table VI), it might be that this protein interacts primarily with the G-strand of telomeric DNA rather than with pre-mRNA. By binding the telomeric G-strand overhang, uqTBP25 may protect it against nucleolytic attack (Fig. 4). Additionally, uqTBP25 might be instrumental in the stabilization of specific structures of telomeric DNA. Hence, by binding tightly single-stranded or unimolecular tetraplex forms of d(TTAGGG)n while binding weakly its bimolecular or tetramolecular tetraplex forms, uqTBP25 may stabilize the monomolecular forms of the G-strand overhang and prevent the generation of multimolecular tetraplex structures.
We are grateful to G. Sarig and Dr. P. Weisman-Shomer for help. We thank the Technion Protein Research Center (Professor Arie Admon) for ably performing peptide microsequencing.