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Molecular and Cellular Biology, June 2000, p. 3860-3869, Vol. 20, No. 11
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
DDP1, a Heterochromatin-Associated Multi-KH-Domain
Protein of Drosophila melanogaster, Interacts Specifically
with Centromeric Satellite DNA Sequences
Alfred
Cortés and
Fernando
Azorín*
Departament de Biologia Molecular i Cellular,
Institut de Biologia Molecular de Barcelona, CSIC, 08034 Barcelona,
Spain
Received 8 December 1999/Returned for modification 21 January
2000/Accepted 2 March 2000
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ABSTRACT |
DDP1 is a single-stranded nucleic acid binding protein of
Drosophila melanogaster that associates with pericentric
heterochromatin. DDP1 contains 15 consecutive KH domains and is
homologous to the highly conserved vigilin proteins that, in
Saccharomyces cerevisiae, are involved in the control of
cell ploidy. DDP1 was identified and purified on the basis of its
binding to the pyrimidine-rich C strand of the centromeric
Drosophila dodeca-satellite. Here, the interaction of DDP1
with the dodeca-satellite C strand was analyzed in detail. This
interaction is sequence specific. In particular, a guanine residue
which is highly conserved in natural dodeca-satellite sequences was
found to be essential for the efficient binding of DDP1. DDP1 binding
was also found to be strongly influenced by the length and extent of
secondary structure of the DNA substrate. Efficient DDP1 binding
required a minimal length of about 75 to 100 nucleotides and was
facilitated by the lack of secondary structure of the substrate. DDP1
also showed a significant affinity for the unstructured pyrimidine-rich
strand of the most abundant centromeric Drosophila AAGAG
satellite. The stoichiometry of the complexes formed with the
dodeca-satellite C strand suggests that, in DDP1, the 15 consecutive KH
domains are organized such that they define two nucleic acid binding
surfaces. These results are discussed in the context of the possible
contribution of DDP1 to heterochromatin organization and function.
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INTRODUCTION |
DDP1 is a multi-KH-domain protein of
Drosophila melanogaster that is found associated with
pericentric heterochromatin (8). DDP1 contains 15 tandemly
organized KH domains and is homologous to the highly conserved vigilin
proteins that have been found in all eukaryotic organisms analyzed to
date, from yeasts to humans (25, 29, 30, 38). Little is
known about the functions of vigilins. They are up-regulated in rapidly
dividing cells (29); in yeast, disruption of the
corresponding gene (SCP160) results in cells with increased
ploidy, suggesting a role in chromosome segregation (38).
The expression of DDP1 complements a 
scp160 deletion in
yeast (8). The association of DDP1 with pericentric heterochromatin also suggests a possible contribution to chromosome segregation. Vigilins could also play a role in RNA metabolism (12, 17, 19-21). They were found to bind in vitro the 3'
untranslated regions (UTRs) of the dystrophin and vitellogenin mRNAs
and were proposed to be responsible for the increased stability of the latter induced by estrogens (12, 17).
Like the vigilins, DDP1 binds single-stranded nucleic acids with high
affinity and specificity (8). Actually, DDP1 was identified
and isolated on the basis of its interaction with the C strand of the
Drosophila dodeca-satellite, a highly repeated DNA sequence
which is localized to the pericentric heterochromatin on chromosome 3 in D. melanogaster (1, 5, 24). In polytene chromosomes, the distribution of DDP1 is not constrained to the regions
containing dodeca-satellite sequences, being associated as well with
other pericentric heterochromatin regions containing no detectable
dodeca-satellite sequences (8). DDP1 is also found at some
discrete sites on the euchromatic chromosome arms, colocalizing with
heterochromatin protein 1 (8). The dodeca-satellite has a
marked Pu-Py strand asymmetry which results in one strand, the C
strand, being enriched in pyrimidines and the complementary strand, the
G strand, being enriched in purines. In vitro, the dodeca-satellite can
form altered DNA structures in which the G strand forms very stable
intramolecular hairpins while the complementary C strand remains
unstructured (13). Other centromeric satellites, such as the
abundant Drosophila AAGAG satellite, show similar structural
properties (6, 7, 14, 27). Formation of these altered DNA
structures could therefore provide an adequate substrate for the
efficient binding of DDP1 to heterochromatin. In vitro, DDP1 was found
to bind the unstructured dodeca-satellite C strand but not the G strand
(8, 13).
In this study, we have analyzed in vitro the interaction of DDP1 with
single-stranded nucleic acids. The interaction of DDP1 with the
dodeca-satellite C strand is sequence specific but is also strongly
influenced by the length and extent of secondary structure of the DNA
substrate. DDP1 also shows a significant affinity for the pyrimidine
strand of the Drosophila AAGAG satellite.
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MATERIALS AND METHODS |
DNAs and RNAs.
Table 1
summarizes the different DNAs used in these experiments. Fragments 42R
and 9R were obtained, respectively, from plasmids pBK6E215 and
pBK6E218, which are pBluescript (Stratagene) derivatives carrying
dodeca-satellite sequences inserted at the unique SpeI site
(1). The dodeca-satellite fragments were released by
digestion with SpeI, and the corresponding C strands were
obtained as described earlier (13). All synthetic
oligonucleotides were purified by denaturing polyacrylamide gel
electrophoresis before use. The DNA concentration was determined by UV
spectroscopy as described previously (15). When needed, DNAs
were radioactively labeled by conventional methods.
RNAs were obtained by in vitro transcription with T7 RNA polymerase
(Promega) of plasmids pbsVIT and pbsDYS, which are pBluescript
derivatives carrying the double-stranded VIT and DYS sequences,
respectively (Table
1), flanked by a
SacI site at the 5' end
and an
EcoRI site at the 3' end. Before in vitro
transcription,
plasmids were linearized with
HindIII.
RNA products were visualized
on 2% nondenaturing agarose gels, and
their concentrations were
determined by UV spectroscopy. Transcripts
obtained in this way
contained, in addition to the VIT and DYS
sequences, 15 and 12
nucleotides (nt) of unrelated sequences at their
5' and 3' ends,
respectively.
Proteins.
DDP1 was either purified from SL2 nuclear extracts
or obtained as a recombinant by expression in Escherichia
coli cells (8). Both proteins behaved indistinctly in
electrophoretic mobility shift assay (EMSA) experiments (8).
1/2DDP1 was obtained as a recombinant by the expression in
E. coli of the first 651 amino acids of DDP1 by use of the
PET29-b expression vector (Novagen). 1/2DDP1 was produced
as a fusion protein carrying a C-terminal His6 tag and
purified as DDP1 (8).
EMSA experiments.
EMSA experiments were performed as
described previously (8) with ~0.2 ng of radioactively
labeled DNA and, when necessary, an excess of competitor. Competition
experiments were always performed at a protein concentration providing
75 to 97% binding in the absence of any added competitor. When binding
to fragment 42R was studied, complexes were resolved on native 4%
instead of 5% polyacrylamide gels. For high-resolution analysis, the
protein-DNA complexes were subjected to electrophoresis through
40-cm-long native 5% polyacrylamide gels for 12 h at 150 V. Autoradiographs were recorded on HyperFilm (Amersham) and analyzed
quantitatively on a Molecular Dynamics laser densitometer. The percent
competition was expressed as the percent binding observed in the
presence of competitor DNA relative to the percent binding obtained in the absence of competitor DNA.
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RESULTS AND DISCUSSION |
Efficient binding of DDP1 to the dodeca-satellite C strand depends
strongly on the length and lack of secondary structure of the DNA
fragments.
The affinity of DDP1 for the dodeca-satellite C strand
depends strongly on the length of the DNA fragments; efficient
interaction requires relatively long binding sites. As shown in Fig.
1, the affinity of DDP1
for the dodeca-satellite C strand decreases sharply for fragments
shorter than about 70 nt. Fragments 42R and 12R correspond to the C
strand of naturally occurring dodeca-satellite DNA fragments containing
42 and 12 repeats, respectively. Oligo 9R and oligo 4R, which were
derived from fragment 12R, carry nine and four repeats, respectively
(Table 1). The efficiency of the interaction of DDP1 with these
C-strand fragments was determined through competition experiments. As
judged from the excess of a nonspecific heat-denatured single-stranded
E. coli DNA competitor required to obtain efficient
competition, the affinities of DDP1 for fragment 42R, fragment 12R, and
oligo 9R are very similar, but the affinity for oligo 4R is much lower
(Fig. 1A). For oligo 4R, the binding of DDP1 is completely abolished in
the presence of a 50-fold excess (wt/wt) of nonspecific competitor
(Fig. 1A, 4R, lane 1), while for the longer fragments, significant
binding is still detected in the presence of a 2,500- to 3,000-fold
excess (Fig. 1A, 42R, 12R, and 9R, lanes 3). The lower affinity
detected for oligo 4R was corroborated when the binding of DDP1 to
oligo 9R was competed by oligo 9R itself or oligo 4R (Fig. 1B). A
similar reduced affinity was observed for DNA fragments also containing four repeats but spanning different regions of fragment 12R (data not
shown), indicating that this reduced affinity is not directly associated with the different nucleotide sequences of the fragments used. Consistent with this interpretation, similar results were obtained when the binding of DDP1 to DNA fragments carrying nine (oligo
9Rc) and six (oligo 6Rc) repeats of the C-strand consensus sequence
(TCGGTCCCGTAC) was analyzed (Fig. 1C). These synthetic oligonucleotides differ in length but not in nucleotide sequence (Table
1). The affinity of DDP1 for oligo 9Rc is higher than that for oligo
6Rc, as judged from competition experiments in which the binding of
DDP1 to oligo 9Rc was competed by oligo 9Rc and oligo 6Rc (Fig. 1C).
Similarly, single-stranded E. coli DNA competed the binding
of DDP1 to oligo 6Rc more efficiently than to oligo 9Rc (data not
shown).
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FIG. 1.
Efficient binding of DDP1 to the dodeca-satellite
C strand depends on the length of the DNA substrate. (A) The binding of
DDP1 to dodeca-satellite C-strand DNA fragments of different lengths is
shown as a function of increasing amounts of heat-denatured
single-stranded (ss) E. coli DNA. The excess quantities
(weight to weight) of competitor used were as follows: panels 42R and
12R, 0 (lanes 0), 150 (lanes 1), 750 (lanes 2), 3,000 (lanes 3), and
9,000 (lanes 4); and panels 9R and 4R, 0 (lanes 0), 50 (lanes 1), 500 (lanes 2), and 2,500 (lanes 3). Quantitative analysis of the results is
shown on the right for fragment 42R, fragment 12R, oligo 9R, and oligo
4R. (B) The binding of DDP1 to oligo 9R is shown as a function of
increasing amounts of oligo 9R (panel 9R) and oligo 4R (panel 4R). The
excess quantities (weight to weight) of competitor used were as
follows: panel 9R, 5 (lane 1), 50 (lane 2), and 500 (lane 3); and panel
4R, 50 (lane 1), 500 (lane 2), and 2,500 (lane 3). Lane 0 shows the
binding obtained in the absence of any added competitor. Quantitative
analysis of the results is shown on the right for oligo 9R and oligo
4R. (C) The binding of DDP1 to oligo 9Rc is shown as a function of
increasing excess quantities (weight to weight) of oligo 9Rc (panel
9Rc) and oligo 6Rc (panel 6Rc): 0 (lane 0), 5 (lanes 1), 50 (lanes 2),
and 500 (lanes 3). Quantitative analysis of the results is shown on the
right for oligo 9Rc and oligo 6Rc. See Table 1 for a description of the
DNA fragments used in these experiments.
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Altogether, these results indicate that the binding of DDP1 to the
dodeca-satellite C strand requires a minimum length of
between six
repeats (72 nt) and nine repeats (104 to 108 nt).
The affinity of DDP1
for DNA fragments above this length threshold
does not increase
significantly. A similar length requirement
was reported for the
binding of the
Xenopus vigilin to nucleic
acids
(
17). The large size of the vigilin binding sites strongly
suggests that their 15 KH domains are actually involved in nucleic
acid
recognition.
The binding of DDP1 to the dodeca-satellite C strand is highly
sensitive to the extent of secondary structure of the DNA fragments.
The affinity of DDP1 for oligo 9R is significantly higher than
that for
oligo 9Rc, as shown by the larger excess of single-stranded
E. coli DNA required to compete binding to the former (Fig.
2A)
as well as by the greater efficiency
of oligo 9R as a competitor
(Fig.
2B). Both DNA fragments are of
similar lengths, containing
nine repeats of the dodeca-satellite C
strand that, in the case
of oligo 9Rc, are perfect repetitions of the
consensus sequence.
The dodeca-satellite C-strand consensus sequence is
slightly palindromic
and, as a consequence, DNA fragments carrying
perfect repetitions
of this sequence form relatively stable fold-back
structures under
the experimental conditions used for DDP1 binding and
EMSA. On
the other hand, the repeats of oligo 9R are not perfect (Table
1) and, as a consequence, oligo 9R does not show any significant
secondary structure. The faster electrophoretic mobility of oligo
9Rc
than of oligo 9R (Fig.
2A) reflects the formation of fold-back
structures on the former (
27). Similar results were obtained
when the affinity of DDP1 for oligo 4R, carrying four imperfect
repeats, was compared to that for oligo 4Rc, carrying four perfect
repeats of the C-strand consensus sequence (data not shown). These
results indicate that the affinity of DDP1 is strongly influenced
by
the extent of secondary DNA structure of the substrate. Binding
of
Xenopus vigilin to the vitellogenin and dystrophin mRNAs was
also found to be favored by mutations decreasing the degree of
secondary structure of the substrate (
17). Interestingly,
natural
dodeca-satellite sequences are rarely built by perfect
repetitions
of the consensus sequence (
1,
24). As a
consequence, the
C strand of naturally occurring dodeca-satellite
fragments shows
in general a very low degree of secondary structure
(
13,
27)
and is therefore a good substrate for DDP1 binding.
Actually,
DDP1 showed similar high affinities for several different
naturally
occurring C-strand fragments that, under these experimental
conditions,
were unstructured (data not shown).
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FIG. 2.
Efficient binding of DDP1 to the dodeca-satellite C
strand depends on the extent of secondary structure of the DNA
substrate. (A) The binding of DDP1 to oligo 9R (panel 9R) and oligo 9Rc
(panel 9Rc) is shown as a function of increasing excess quantities
(weight to weight) of single-stranded (ss) E. coli DNA: 0 (lanes 0), 25 (lanes 1), 250 (lanes 2), and 1,000 (lanes 3). (B) The
binding of DDP1 to oligo 9R is shown as a function of increasing
amounts of oligo 9R (panel 9R) and oligo 9Rc (panel 9Rc). Excess
quantities (weight to weight) of competitor used were as follows: panel
9R, 5 (lane 1), 50 (lane 2), and 500 (lane 3); and panel 9Rc, 50 (lane
1), 500 (lane 2), and 2,500 (lane 3). Lane 0 shows the binding obtained
in the absence of any added competitor. Quantitative analysis of the
results are shown to the right of each panel for oligo 9R and oligo
9Rc. See Table 1 for a description of the DNA fragments used in these
experiments.
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The interaction of DDP1 with the dodeca-satellite C strand is of
high affinity and specificity.
The results reported above indicate
that, upon melting, the dodeca-satellite could provide sufficiently
long unstructured DNA fragments for efficient DDP1 binding. Others have
shown that vigilins can also specifically recognize various
single-stranded DNA and RNA sequences (10, 12, 17, 37). In
particular, the Xenopus vigilin was shown to specifically
interact with sequences of the 3' UTRs of the Xenopus
vitellogenin and human dystrophin mRNAs (12, 17). The
question then arises as to what extent the interaction of DDP1 with the
dodeca-satellite is specific. Here, we have analyzed the relative
affinity of DDP1 for the dodeca-satellite C strand and the vigilin
binding sites of the 3' UTRs of the vitellogenin and dystrophin mRNAs.
Figure 3 shows the interaction of DDP1
with synthetic oligonucleotides spanning the vigilin binding sites of
the vitellogenin (oligo VIT) and dystrophin (oligo DYS) mRNAs (17). To avoid any length dependence effects, these
oligonucleotides were exactly the same length (104 nt) as oligo 9R
(Table 1). As shown in Fig. 3A, single-stranded E. coli DNA
competes the binding of DDP1 to oligo VIT and oligo DYS much more
efficiently than to oligo 9R, indicating a significantly higher
affinity for the latter. In good agreement with these results, binding
to oligo 9R occurs at a protein concentration (2.5 µl) lower than
that required for oligo DYS (6 µl) or oligo VIT (7 µl).
Corroborating the lower affinity of DDP1 for the vigilin binding sites
of the vitellogenin and dystrophin mRNAs, oligo VIT and oligo DYS
compete the binding of DDP1 to oligo 9R very poorly, since no
significant competition is observed even in the presence of a 500-fold
excess of these competitors (Fig. 3B). Similar results were obtained when the RNA versions of oligo VIT and oligo DYS were used as competitors (Fig. 3B, rVIT and rDYS). Fragments rVIT and rDYS appear to
compete DDP1 binding better than the corresponding DNA versions.
However, significant binding is still observed in the presence of a
2,500-fold excess of the RNA competitors (Fig. 3B, rDYS and rVIT, lanes
4). On the other hand, the affinity of DDP1 for the RNA version of the
dodeca-satellite C strand was shown not to be significantly different
from that for its DNA form (8). These results show that DDP1
binds the dodeca-satellite C strand with a higher affinity than the
vigilin binding sites of the Xenopus vitellogenin and human
dystrophin mRNAs.
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FIG. 3.
Comparison of the affinity of DDP1 for the vigilin
binding sites of the Xenopus vitellogenin and human
dystrophin mRNAs to its affinity for the dodeca-satellite C strand. (A)
The binding of DDP1 to oligo DYS (panel DYS) and oligo VIT (panel VIT)
is shown as a function of increasing excess quantities (weight to
weight) of single-stranded (ss) E. coli DNA: 0 (lanes 0), 25 (lanes 1), 250 (lanes 2), and 1,000 (lanes 3). Quantitative analysis of
the results is shown below for oligo DYS ( ) and oligo VIT ( ). The
results obtained with oligo 9R ( ) (Fig. 2) are included for
comparison. (B) The binding of DDP1 to oligo 9R is shown as a function
of increasing excess quantities (weight to weight) of oligo DYS (panel
DYS), oligo VIT (panel VIT), and the RNA transcripts corresponding to
the DYS (panel rDYS) and VIT (panel rVIT) sequences: 5 (lanes 1), 50 (lanes 2), 500 (lanes 3), and 2,500 (lanes 4). Lane 0 shows the binding
obtained in the absence of any added competitor. Quantitative analysis
of the results is shown below for oligo DYS, oligo VIT, rDYS, and rVIT.
The results obtained with oligo 9R (Fig. 2) are included for
comparison. See Table 1 for a description of the DNA fragments used in
these experiments.
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As shown earlier, in
Drosophila polytene chromosomes, DDP1
is found associated with the chromocenter spanning most of the
pericentric heterochromatin (
8), colocalizing with the
dodeca-satellite-rich
regions on chromosome 3, but also is present at
the pericentric
region of chromosome 2, where no dodeca-satellite
sequences are
detected; these findings suggest a possible association
of DDP1
with other heterochromatin sequences. Consistent with this
hypothesis,
DDP1 also binds in vitro other centric satellite DNAs with
significant
affinity. Figure
4 shows the
interaction of DDP1 with oligo CTCTT
(Table
1), which corresponds to
the pyrimidine-rich strand of
the AAGAG satellite, the most abundant
repetitive DNA of
Drosophila,
which is present at the
pericentric region of chromosome 2 (
23).
As judged from the
amount of nonspecific single-stranded
E. coli DNA required
to efficiently compete DDP1 binding (Fig.
4A), the
affinity of DDP1 for
oligo CTCTT is significantly higher than
those for oligo 9Rc, oligo
VIT, and oligo DYS, which all have
lengths similar to oligo CTCTT.
Similar results were obtained
when the efficiency of oligo CTCTT to
compete the binding of DDP1
to oligo 9R was analyzed (Fig.
4B).
However, oligo CTCTT competes
DDP1 binding to oligo 9R less efficiently
than oligo 9R itself
(Fig.
4B); a 5-fold excess of oligo 9R is
sufficient to mostly
abolish DDP1 binding, but significant binding is
still detected
in the presence of a 500-fold excess of oligo CTCTT.
These results
indicate that DDP1 shows a significant affinity for the
unstructured
pyrimidine strand of the
Drosophila AAGAG
satellite, albeit lower
than that for the dodeca-satellite C strand.
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FIG. 4.
DDP1 binds the pyrimidine-rich strand of the
Drosophila AAGAG satellite. (A) The binding of DDP1 to oligo
CTCTT is shown as a function of increasing excess quantities (weight to
weight) of single-stranded (ss) E. coli DNA: 0 (lane 0), 25 (lane 1), 250 (lane 2), and 1,000 (lane 3). (B) The binding of DDP1 to
oligo 9R is shown as a function of increasing excess quantities (weight
to weight) of oligo CTCTT: 0 (lane 0), 5 (lane 1), 50 (lane 2), and 500 (lane 3). Quantitative analysis of the results is shown to the right of
each panel for oligo CTCTT. The results obtained with oligo 9R (),
oligo 9Rc ( ), oligo DYS (), and oligo VIT (), taken from Fig.
2 and 3, are included for comparison. See Table 1 for a description of
the DNA fragments used in these experiments.
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The high affinity of DDP1 for the dodeca-satellite C strand suggests
that this interaction is sequence specific. Consistent
with this
interpretation, the substitution of part of the dodeca-satellite
sequences of oligo 9R with unrelated DNA sequences results in
a
significant decrease in DDP1 binding (data not shown). To better
understand the sequence determinants of the interaction of DDP1
with
the dodeca-satellite C strand, the binding of DDP1 to oligo
6Rc, which
carries six repeats of the C-strand consensus sequence,
and to variants
carrying specific base substitutions was analyzed
(Table
1). The
affinity of DDP1 for oligo 6RcT, in which the
three central cytosine
residues of each repeat were substituted
with thymines (Table
1), is
higher than that for oligo 6Rc (Fig.
5A).
This increased affinity likely reflects the lower degree
of secondary
structure of oligo 6RcT. As mentioned above, oligo
6Rc forms relatively
stable fold-back structures to which the
central cytosines importantly
contribute through the formation
of very stable C · G pairs.
Changing the three central cytosine
residues to thymines prevents these
base-pairing interactions
and, therefore, the formation of the
fold-back structures. The
lower electrophoretic mobility of oligo 6RcT
than of oligo 6Rc
reflects the lack of secondary structure of the
former (Fig.
5A).
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FIG. 5.
Specific residues contribute importantly to the binding
of DDP1 to the dodeca-satellite C strand. (A) The binding of DDP1 to
oligo 6Rc (panel 6Rc) and oligo 6RcT (panel 6RcT) is shown as a
function of increasing excess quantities (weight to weight) of
single-stranded (ss) E. coli DNA: 0 (lanes 0), 10 (lanes 1),
50 (lanes 2), and 200 (lanes 3). Quantitative analysis of the results
is shown on the right for oligo 6Rc and oligo 6RcT. (B) The binding of
DDP1 to oligo 6RcT (panel 6RcT), oligo 6RcTG1 (panel 6RcTG1), oligo
6RcTG2 (panel 6RcTG2), and oligo 6RcTG3 (panel 6RcTG3) is shown as a
function of increasing excess quantities (weight to weight) of
single-stranded E. coli DNA: 0 (lanes 0), 20 (lanes 1), 40 (lanes 2), 100 (lanes 3), 200 (lanes 4), and 400 (lanes 5).
Quantitative analysis of the results is shown below for oligo 6RcT,
oligo 6RcTG1, oligo 6RcTG2, and oligo 6RcTG3. See Table 1 for a
description of the DNA fragments used in these experiments.
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Significant effects are observed when specific base substitutions are
incorporated into the sequence of oligo 6RcT (Fig.
5B).
A slight
decrease in DDP1 affinity is observed when either the
first or the
third guanine residue of each repeat is changed to
a cytosine, oligo
6RcTG1 or oligo 6RcTG3, respectively (Fig.
5B).
However, when the
second guanine is substituted by a cytosine,
oligo 6RcTG2, the affinity
of DDP1 decreases strongly (Fig.
5B).
It must be noted that, according
to their electrophoretic behavior,
none of these three base
substitutions has a significant effect
on the extent of secondary
structure of the corresponding DNA
fragments. Interestingly, this
second guanine residue is highly
conserved in natural dodeca-satellite
sequences (
1,
24),
being present in about 96% of all the
repeats analyzed. In comparison,
the first and third guanine residues
mentioned above are less
well conserved, being present in about 80 to
85% of the repeats.
These results indicate that specific residues
which are highly
conserved in naturally occurring sequences contribute
importantly
to the interaction of DDP1 with the dodeca-satellite C
strand.
Altogether, these results show that, although DDP1 binds in vitro a
variety of different substrates, its interaction with
the
dodeca-satellite C strand is of the highest affinity. Sequence-specific
interactions importantly contribute to the efficient binding of
DDP1 to
the dodeca-satellite C strand. It is known that nucleic
acid
recognition by the KH fold is, to some extent, sequence specific
and
that homologous KH domains of slightly different amino acid
sequences
show different nucleic acid binding preferences in vitro
(
3,
4,
10,
17,
19,
31,
32,
37). It is therefore
likely that the lower
affinity of DDP1 for the rest of the single-stranded
nucleic acids
tested here reflects less favorable sequence-specific
interactions.
However, sequence-specific interactions are not
the only factor
governing the efficient binding of DDP1 or vigilins
in general to
single-stranded nucleic acids. As shown here and
elsewhere
(
17), both the length and the degree of secondary
structure
of the substrate also have a strong influence on the
binding of
vigilins. Actually, the affinity of DDP1 for oligo
9Rc, which contains
nine repeats of the C-strand consensus sequence
and is structured, is
not significantly different from that for
oligo VIT or oligo DYS. This
finding suggests that DNA sequences
other than the dodeca-satellite C
strand could also be recognized
by vigilins, provided that they are
sufficiently long and unstructured.
The localization of DDP1 at the
pericentric heterochromatin on
chromosome 2 of
Drosophila,
together with its significant affinity
for the pyrimidine strand of the
AAGAG satellite, suggests that
this DNA could also be a substrate for
DDP1 binding in vivo. Our
results do not exclude at all the possibility
that vigilins could
also bind in vivo specific RNAs. Increasing
evidence suggests
that RNA binding proteins, such as hnRNP K, also play
a role in
DNA metabolism (
26,
28,
33-36). The mechanisms
regulating in
vivo the binding of these proteins to their different
possible
target nucleic acids are still largely
unknown.
A model for the interaction of DDP1 with the dodeca-satellite C
strand.
Determination of the stoichiometry of the interaction of
DDP1 with the dodeca-satellite C strand suggests that DDP1 contains two
independent nucleic acid binding surfaces. When DDP1 is bound to DNA
fragments of the dodeca-satellite C strand in the presence of
increasing protein concentrations, the formation of complexes accommodating more than one protein molecule is observed. Figure 6A shows the determination of the
stoichiometry of the complexes formed with various C-strand fragments.
For these experiments, the binding of 1/2DDP1, a shorter
protein construct that carries only the first seven KH domains of DDP1,
was also analyzed. The maximum number of protein molecules that can be
incorporated into the complex depends on the length of the DNA fragment
and the size of the protein construct (Table
2). For instance, the shortest, oligo
6Rc, accommodates only one DDP1 but two 1/2DDP1 molecules, while fragment 12R can accommodate up to two DDP1 or four
1/2DDP1 molecules (Fig. 6A). From these data, the number of
protein KH domains per dodeca-satellite repeat involved in the
interaction can be estimated (Table 2). In all cases, the stoichiometry
of the complexes closely corresponds to two KH domains per
dodeca-satellite repeat. This estimate is less precise for complexes
formed with oligo 9R and oligo 9Rc. These DNA fragments can accommodate
up to two DDP1 molecules; however, even at very high protein
concentrations, only a small percentage of complexes contains two DDP1
molecules (Fig. 6A), indicating that the second DDP1 molecule enters
the complex with great difficulty. Therefore, in these cases, the actual stoichiometry of the complexes will be somewhere between those
of the complexes containing one and two DDP1 molecules, 1.7 and 3.3 KH
domains per DNA repeat, respectively. Similarly, fragment 42R
accommodates at least eight 1/2DDP1 molecules. However, in
this case, complexes of higher stoichiometry are also formed, but they
could not be resolved electrophoretically (Fig. 6A). These results
indicate that the interaction of DDP1 with the dodeca-satellite C
strand occurs at a defined stoichiometry of approximately two KH
domains per DNA repeat.
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|
FIG. 6.
Determination of the stoichiometry of the DDP1-C-strand
complexes. (A) The binding of DDP1 and 1/2DDP1 to oligo 6Rc
(panels 6Rc), oligo 9Rc (panels 9Rc), oligo 9R (panels 9R), fragment
12R (panels 12R), and fragment 42R (panels 42R) is presented as a
function of increasing protein concentrations: a threefold-higher
protein concentration was used in lanes 2 than in lanes 1. The
arrowheads indicate protein-DNA complexes of increasing stoichiometry.
The position of the free DNA probe is indicated by zero. (B) Possible
models for the interaction of DDP1 with the dodeca-satellite C strand
(see the text for details). (C) High-resolution EMSA of the complexes
containing one DDP1 molecule formed with fragment 12R (lane 12R), oligo
9Rc (lane 9Rc), and oligo 6Rc (lane 6Rc).
|
|
Two possible models can account for these results. Either each DNA
repeat is recognized by two KH domains (Fig.
6B, top) or,
alternatively, each KH domain binds one DNA repeat, but the
organization
of the KH domains is such that there are two nucleic acid
binding
surfaces in the protein (Fig.
6B, bottom). This second
possibility
appears more likely. First, although it is not precisely
known
how many bases are directly involved in the interaction with a
single KH domain, it appears unlikely that a dodeca-satellite
C-strand
repeat which is only 11 to 12 nt long would be sufficiently
long to
accommodate two relatively large KH domains. In this respect,
it is
interesting to note that the shortest sequence known to
be bound by a
single KH domain is 15 nt long (
4). Second, the
binding of
two KH domains to a single dodeca-satellite C-strand
repeat would
necessarily imply that each of the two KH domains
recognizes a
different nucleotide sequence; therefore, equivalent
protein-DNA
interactions would take place only at every other
KH domain of DDP1.
However, given the repetitive character of
both substrates, it appears
more likely that each KH domain would
maintain equivalent molecular
interactions with the dodeca-satellite
C strand. Furthermore, if DDP1
contained two nucleic acid binding
surfaces, a second DNA fragment
could be accommodated in the complexes
formed with short DNA fragments
but not in those formed with large
fragments. Consistent with this
hypothesis, the complexes formed
with oligo 6Rc and oligo 9Rc which,
under routine EMSA conditions,
behave as a single molecular species
containing only one DDP1
molecule, are resolved into two closely
migrating species when
subjected to a longer electrophoretic run (Fig.
6C, 9Rc and 6Rc).
On the other hand, under these high-resolution EMSA
conditions,
only a single complex is observed with fragment 12R (Fig.
6C,
12R). All these considerations strongly suggest that DDP1 contains
two nucleic acid binding surfaces. Interestingly, in crystals
of the
KH3 domain of Nova-1 or Nova-2, the lattice is composed
of symmetric
tetramers of independent KH domains that define two
opposite nucleic
acid binding surfaces and two different protein-protein
interfaces
(
22).
The results reported here and elsewhere (
8) indicate that
DDP1 is a single-stranded DNA binding protein whose affinity
for
double-stranded DNA is extremely low. Therefore, the association
of
DDP1 with heterochromatin suggests that this specialized chromosomal
structure contains regions of single-stranded DNA that could be
recognized by DDP1. The formation of single-stranded DNA at
heterochromatin
blocks could originate from their characteristic
enrichment on
highly repetitive satellite DNA sequences, which is
likely to
promote strand slippage events during DNA replication. In
this
respect, the dodeca-satellite, like general satellites showing
Pu-Py strand asymmetry, appear especially suited for the binding
of
DDP1 or vigilins in general, since its two strands show drastically
different structural properties (
6,
7,
13,
14,
27).
The
pyrimidine-rich strand could remain unstructured, providing
long
single-stranded DNA stretches, as required for efficient
DDP1 binding.
On the other hand, the high tendency of the purine-rich
strand to form
intramolecular fold-back structures could help
prevent reannealing of
the two complementary strands. Mechanisms
involving specific
protein-DNA interactions could stabilize, propagate,
or even induce the
formation of single-stranded DNA at heterochromatin.
DDP1 could play
such a role(s). DDP1 could also contribute to
some of the
characteristic properties of heterochromatin. Heterochromatin
appears
to be involved in homologous as well as ectopic chromosome
pairing both
at the centromeric regions and throughout the chromosome
(
2,
9,
11,
16,
18). In
Drosophila, the frequent association
of the
bwD locus with the centric
heterochromatin of chromosome 2 or the
clustering of different
heterochromatin regions to form the chromocenter
likely involves
heterochromatin-mediated pairing events (
9,
11). The
possibility that DDP1 contains two nucleic acid binding
surfaces
suggests a potential contribution to heterochromatin
pairing. A single
DDP1 molecule could bind noncontiguous single-stranded
DNA stretches,
linking together heterochromatin regions of the
same chromosome or of
sister chromosomes and thereby contributing
to chromosome pairing
and/or
cohesion.
| |
ACKNOWLEDGMENTS |
This work was financed by grants from the Spanish DGES (PB96-812)
and the CIRIT of the Generalitat de Catalunya (SGR97-55). A.C. was a
recipient of a doctoral fellowship from the CIRIT. This work was
carried out within the context of the Centre de Referència en
Biotecnologia of the Generalitat de Catalunya.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Departament de
Biologia Molecular i Cellular, Institut de Biologia Molecular de
Barcelona, CID-CSIC, Jordi Girona Salgado 18-26, 08034 Barcelona,
Spain. Phone: 3493-4006137. Fax: 3493-2045904. E-mail:
fambmc{at}cid.csic.es.
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Abstract
Introduction
