Identification of the Single-stranded DNA Binding Surface of the
Transcriptional Coactivator PC4 by NMR*
Sebastiaan
Werten
§,
Rainer
Wechselberger§,
Rolf
Boelens§,
Peter C.
van der Vliet
, and
Robert
Kaptein§¶
From the
Laboratorium voor Fysiologische Chemie,
Universiteit Utrecht, Stratenum, Universiteitsweg 100, 3584 CG Utrecht,
The Netherlands and § Bijvoet Centrum voor Biomoleculair
Onderzoek, Universiteit Utrecht, Padualaan 8, 3584 CH Utrecht, The
Netherlands
 |
ABSTRACT |
The C-terminal domain of the eukaryotic
transcriptional cofactor PC4 (PC4CTD) is known to bind with
nanomolar affinity to single-stranded (ss)DNA. Here, NMR is used to
study DNA binding by this domain in more detail. Amide resonance shifts
that were observed in a
1H15N-HSQC-monitored titration of
15N-labeled protein with the oligonucleotide
dT18 indicate that binding of the nucleic acid occurs by
means of two anti-parallel channels that were previously identified in
the PC4CTD crystal structure. The
-sheets and loops that
make up these channels exhibit above average flexibility in the absence
of ssDNA, which is reflected in higher values of T1
,
reduced heteronuclear nuclear Overhauser effects and faster deuterium
exchange rates for the amides in this region. Upon ssDNA binding, this
excess flexibility is significantly reduced. The binding of ssDNA by symmetry-related channels reported here provides a structural rationale
for the preference of PC4CTD for juxtaposed single-stranded
regions (e.g. in heteroduplexes) observed in earlier work.
 |
INTRODUCTION |
The eukaryotic general transcriptional cofactor PC4 is known to
enhance activated in vitro and in vivo
transcription from various RNA polymerase II promoters, in concert with
members of all major classes of transcriptional activators (1-3). The
activator-dependent stimulation of transcription by PC4 has
been shown to originate mostly from increased recruitment of basal
transcription factors during the early stages of preinitiation complex
(PIC)1 formation (4). It is
likely that this effect is caused by the interactions that have been
reported between PC4 and the activation domains of promoter-binding
activator proteins on the one hand, and basal factors (including TFIIA)
on the other (1, 3). Thus, PC4 appears to act as a bridging factor that
stabilizes the preinitiation complex.
Recently it has become clear that PC4 has several additional functions.
In the first place, the protein was found to be able to repress
transcription under specific conditions (5-7). Repression at moderate
PC4 concentrations is observed in minimal in vitro transcription systems, that normally do not require the basal factor
TFIIH (5, 6), and with respect to aspecific transcription initiation by
RNA polymerase II from DNA ends and unwound regions (6). Repression is
not observed in full-factor in vitro transcription, because
TFIIH is able to overcome repression by PC4 (5, 6). Thus, PC4 appears
to act as what could be called a fidelity-enhancing factor for RNA
polymerase II transcription initiation by repressing adventitious
transcription from incomplete PICs on the one hand, and on the other
hand enhancing transcription from fully assembled PICs, i.e.
those that include TFIIH, generally believed to be the last factor to
enter the PIC during assembly (8). Surprisingly, PC4 has recently also
been found to associate in vivo with the RNA polymerase III
transcription factor TFIIIC and to stimulate transcription by RNA
polymerase III by increasing the rate of reinitiation (9).
Interestingly, copurification of PC4 with the human replication protein
A (RPA) and effects of PC4 observed in a reconstituted SV40 replication
system suggested that the protein may play a further role in DNA
replication (10). Thus, it is emerging that PC4 is an extremely
versatile factor, involved in the processing of genetic information at
multiple levels.
Although stimulation of RNA polymerase II transcription by PC4 is
probably largely effected by protein-protein interactions depending on
the N-terminal half of the protein, the C-terminal half of the protein
in addition comprises a powerful ssDNA-binding domain (3, 4, 11, 12).
The physiological role of this domain has long remained enigmatic.
Recently, binding of single-stranded DNA was found to be entirely
dispensable for transcription activation by PC4 but strictly required
for repression of transcription in the absence of TFIIH (6). Earlier
analysis of PC4CTD (12) has shown that it binds with nanomolar
affinity to juxtaposed stretches of ssDNA running in opposite
directions, such as present in partially melted double-stranded DNA or
heteroduplexes. Optimal binding to such structures requires
approximately 8 unpaired nucleotides in each of the two strands.
Single-stranded oligonucleotides can be bound with almost equal
affinity, provided these molecules contain a minimum of 16-20
nucleotides. These observations support the idea that ssDNA has to be
bent 180° to create an arrangement of anti-parallel strands similar
to the topology of a heteroduplex for PC4 to bind.
Earlier, the crystal structure of PC4CTD was solved (13). This
structure revealed a novel homodimeric fold in which each monomer
consists of a curved four-stranded
-sheet and a 45°-kinked
-helix, both contributing to a complex dimerization interface. A
prominent feature of the structure is the presence of two channel-like
depressions running in anti-parallel directions. These channels, formed
by the curved
-sheets, are lined both by positively charged and
aromatic residues, suggesting involvement in ssDNA binding in a manner
similar to the DNA binding mode of other single-stranded DNA-binding
proteins such as RPA (14). To investigate whether the channels that
were identified indeed constitute the DNA binding surface of
PC4CTD, we have studied this protein domain and its interaction
with a single-stranded oligonucleotide by means of heteronuclear NMR.
 |
EXPERIMENTAL PROCEDURES |
NMR Sample Preparation--
PC4CTD was overexpressed in
Escherichia coli strain BL21 (DE3) using pET-11a (Novagen)
constructs. The PC4CTD construct encodes amino acids 63-127 of
the full-length protein, preceded by Met-Ala, originating from the
vector sequence. Bacteria were grown at 37 °C in synthetic medium
(6.0 g/liter Na2HPO4, 2H2O, 3.0 g/liter KH2PO4, 0.5 g/liter NaCl, 1.0 mM MgSO4, 0.2 µg/liter FeCl3, 20 µM CaCl2, 0.5 mg/liter thiamine) containing 0.5 g/liter 15NH4Cl as the sole nitrogen source
and either 4.0 g/liter 12C-glucose, or 1.0 g/liter
13C-glucose as the only carbon source. Induction took place
at an A600 nm of 0.5, by the addition of
isopropyl-
-D-thiogalactopyranoside to a final
concentration of 1 mM. After 4 h the cells were
harvested, resuspended in buffer A (20 mM Tris-HCl, pH 7.3, 10% glycerol, 1 mM EDTA, 5 mM dithiothreitol,
10 mM Na2S2O5, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 20 µg/ml leupeptin) containing 500 mM KCl and lysed by
freeze-thawing (once) and sonication for a total of 5 min (in bursts of
20 s). The lysate was then centrifuged at 20,000 × g for 20 min at 4 °C. The supernatant was diluted to 200 mM of KCl by adding buffer A without KCl and immediately loaded onto a heparin-Sepharose column (Amersham Pharmacia Biotech). The column was washed using buffer A at 200 mM KCl and
eluted with buffer A containing 500 mM KCl. Purest
fractions, selected on the basis of Coomassie-stained
SDS-polyacrylamide gel electrophoresis gels, were pooled and, after
dilution with buffer A without KCl to a final concentration of 75 mM KCl, applied to an SP-Sepharose column (Pharmacia),
followed by washing with buffer B (20 mM potassium phosphate, pH 5.5, 5 mM dithiothreitol) containing 75 mM KCl and elution by means of a linear salt gradient
(75-1,000 mM KCl) in buffer B. Peak fractions were further
purified using a Superdex 75 column (Pharmacia) in the same buffer
containing 400 mM KCl and subsequently concentrated by
means of Centricon spin dialysis tubes (molecular mass cut-off 10 kDa,
Amicon). Protein samples at this stage appeared as single bands on
silver-stained polyacrylamide gels. Protein concentrations were
determined using Bio-Rad protein assays (Bio-Rad) employing bovine
gammaglobulin as a standard. Deuterated glycine was added to the final
samples to an end concentration of 2 M, because this
resulted in better long-term stability of the protein at higher temperatures.
The oligonucleotide dT18 was purchased from Carl Roth GmbH
& Co. The DNA was dissolved in buffer B containing 400 mM
KCl and 2 M deuterated glycine to an oligonucleotide
concentration of 11 mM.
NMR Spectroscopy--
NMR experiments were carried out at
305 K (32 °C) on Bruker AMX500, AMX600, Varian UnityPlus 500, and
UnityPlus 750 spectrometers. The protein concentration was 1.5 mM in all experiments.
Protein-DNA titrations were carried out by repeated addition of small
aliquots of an 11 mM solution of dT18 to a 1.5 mM 15N-PC4CTD sample. Formation of the
protein-DNA complex was observed through the recording of a 500 MHz
1H15N-HSQC spectrum after each addition.
15N-T1 and 15N-T1
relaxation and heteronuclear NOE experiments were carried out at 500 MHz as described earlier (15-17), using pulsed-field gradients for
coherence selection in combination with the sensitivity enhancement
scheme (18-20). The longitudinal 15N relaxation rates were
determined from a series of 7 spectra with delays of 32, 64, 128, 256, 512, 768, and 1024 ms. The transverse in-phase 15N
relaxation rates (21) were determined from a series of 7 spectra with
delays of 8, 16, 32, 48, 64, 96, and 128 ms, using a
15N-spin lock with a field strength of 1.4 kHz.
Heteronuclear cross relaxation constants were derived from two 500 MHz
spectra recorded with and without 2.5 s of saturation of the amide protons.
To measure the exchange rate of amide protons, the protein was
first lyophilized and then redissolved in D2O, followed by the recording of a series of 1H15N-HSQC
spectra, up to 200 h after redissolving the protein.
All spectra were processed on Silicon Graphics O2
workstations using the software package NMR Pipe (22). Longitudinal and transversal in-phase relaxation rates and the errors in these parameters were obtained by curve-fitting of a mono-exponential function through the peak intensities according to the
Levenberg-Marquardt algorithm using in-house developed software, as
described by Vis et al. (17).
 |
RESULTS AND DISCUSSION |
The 600 MHz 1H15N-HSQC spectrum of
PC4CTD is shown in Fig. 1. A
nearly complete backbone assignment (listed in Table I) was obtained using double and triple
resonance methods, essentially in the manner described by Vis et
al. (23). Thus, amide 15N and 1H
frequencies were obtained from a high-resolution 600 MHz
1H15N-HSQC spectrum, followed by the collection
of corresponding intra- and inter-residual C
and CO
connectivities from 600 MHz HNCA, HN(CO)CA, HNCO, and HN(CA)CO spectra.
This information allowed in most cases for the unique matching of
inter-residual C
and CO connectivities to the
intra-residue connectivities of the preceding amide. Peptide fragments
identified in this way could then be retrieved in the protein sequence
on the basis of characteristic C
-frequencies (24) and,
if detectable, side chain resonances obtained from a 750 MHz total
correlation spectroscopy-HSQC spectrum. Nearly all H
frequencies could also be obtained from this total correlation
spectroscopy-HSQC spectrum. Remaining ambiguities in the
sequential assignment were resolved using short range
NOEs from a 750 MHz NOE spectroscopy-HSQC.

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Fig. 1.
1H15N-HSQC spectrum
(600 MHz) of PC4CTD, in which backbone assignments have been
indicated. Side chain amide groups are indicated by
horizontal lines but have not been assigned.
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Table I
PC4CTD backbone assignments
Backbone assignment for PC4CTD (in ppm). The first Ala residue
is not part of the PC4 sequence but originates from the expression
vector. For several residues (Ile-66, Glu-93, Glu-95, and Met-96),
multiple sets of signals could be observed.
|
|
To investigate which parts of PC4CTD are involved in the
interaction with ssDNA, a titration was performed with the oligonucleotide dT18. The choice of an 18-mer for our
titration was based on earlier experiments that indicated that
PC4CTD interacts with approximately 16-20 residues of ssDNA
(12). The complex that forms when the oligonucleotide is added exhibits slow exchange, resulting in a simultaneous decrease in the intensity of
the original peaks and increase in the intensity of new signals (data
not shown). Nevertheless, assignment of the new signals was in almost
all cases straightforward, because the perturbations in the spectrum
were relatively modest. Fig.
2A contains a selected region
of the superimposed 1H15N-HSQC spectra of the
free protein and the 1:1 complex showing resonances that are either not
affected (Gln-116), slightly affected (Lys-126), or severely affected
(Asn-106) by the addition of the ssDNA.

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Fig. 2.
HSQC-monitored titrations of PC4CTD
with the single-stranded oligonucleotide dT18.
A, selected HSQC region showing peaks from residues Asn-106,
Gln-116, and Lys-126 in the absence (dotted contour lines)
and presence (solid contour lines) of equimolar amounts of
the oligonucleotide dT18. Arrows indicate the
perturbations caused by the addition of ssDNA. B, combined
chemical shift perturbations for 15N and 1H
( NH) along the PC4CTD sequence. Perturbations
were calculated as Euclidean distances between peak maxima,
i.e.  NH = (( 15N)2 + ( 1H)2)1/2, where
 15N and  1H denote the
15N and 1H chemical shift changes (in Hz),
respectively. In case of degeneracy, the perturbation of the peak
having the highest intensity is shown. In the case of residues Lys-78
and Ile-83, no unambiguous assignments could be made for the complexed
state; for these residues, minimal perturbation values are shown. The
secondary structure of PC4CTD has been indicated schematically
below the graph. Residues Lys-78 and Ile-83 could not be assigned
unambiguously in the complex; perturbations for these residues in the
graph are lower limits.
|
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Fig. 2B lists all perturbations observed. The perturbations
are given as Euclidean distances (in Hz) between the peak maxima in the
HSQC spectra with and without an equimolar amount of dT18. Although small shifts (around 10 Hz) in peak positions are observed for
residues throughout the protein, all perturbations exceeding 20 Hz map
to the
-sheet region. Especially large shifts of 30 Hz or more are
seen for the outermost parts of
-sheets,
2 and
3, and the loop
that connects them (residues Phe-77, Lys-78, Gly-79, Lys-80, and
Val-81), the "
-ridge" that separates the two anti-parallel
channels (residues Trp-89, Met-90, Asp-91, and Lys-101), and the region
that connects sheet
4 to the
-helix (residues Leu-105 and
Asn-106). In addition, a surprisingly large perturbation was found for
the N-terminal Ala residue, which originates from the expression vector
sequence but is located in close proximity to the ends of the
anti-parallel channels in the crystal structure. In Fig.
3, the perturbations of Fig.
2B are shown in the PC4CTD crystal structure by
means of color coding, with colors ranging from green (indicating no
perturbation) through white to red (corresponding to a perturbation of
30 Hz or more). As this figure shows, the data support a model in which
the ssDNA is held in between the
2
3-arms and the
-ridge of the
protein.

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Fig. 3.
Crystal structure of PC4CTD in which
the results from Fig. 2B have been indicated by color
coding. The color spectrum used to represent the perturbations
ranges from green ( NH = 0 Hz) through white
( NH = 15 Hz) to red ( NH = 30 Hz or more).
Unassigned residues and residues that were inaccessible because of
overlap are shown in dark gray.
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Consistent with current views concerning protein-ssDNA interactions
(25), we find a large number of positively charged and aromatic
residues at the binding surface with their side chains exposed to
solution (Lys-68, Arg-70, Tyr-71, Arg-75, Phe-77, Lys-78, Lys-80,
Arg-86, Tyr-88, Trp-89, Lys-97, Arg-100, and Lys-101). Interestingly,
superposition of one of the PC4 anti-parallel channels onto the ssDNA
binding channel found in the RPA-ssDNA cocrystal structure revealed
that several of these residues (Arg-75, Phe-77, Arg-86, Tyr-88, Trp-89,
and Arg-100) occupy positions with respect to the channel that
correspond well to the positions of similar residues in the two RPA
subunits (13, 14). This indicates that PC4 and RPA may have similar
ways of contacting ssDNA even though the
-strand topology is
markedly different. For residues Tyr-71, Arg-75, Phe-77, Lys-78,
Lys-80, Arg-86, Trp-89, and Lys-101, large ssDNA-induced amide
resonance perturbations were observed in our titration. We expect these
residues to make side chain contacts with the DNA. For Trp-89,
involvement in binding has recently been confirmed by mutagenesis (6).
For residues Lys-68, Arg-70, Tyr-88, Lys-97, and Arg-100, no
assignments could be obtained, but the position of these residues with
respect to the binding channels strongly suggests that their side
chains are also involved in binding.
Comparison with other single-stranded DNA-binding proteins suggests a
particularly important role in ssDNA binding for the ends of
-strands,
2 and
3, and the loop
2
3 that connects them
(residues 77-80). This part of the protein is reminiscent of typical
ssDNA-binding loops found in many other single-stranded DNA-binding
proteins, like the L45-loop of the OB-fold proteins (26).
It contains an aromatic residue (Phe-77) and several positively charged
residues (Lys-78 and Lys-80, with Arg-75 located nearby in
-strand
2). In the two RPA subunits, the corresponding strands and the
connecting loop (
4'
5') are seen to bind ssDNA through stacking of
the aromatic residue Phe-269 (in subunit A) or Phe-386 (subunit B) onto
a DNA base, as well as through interactions of the positively charged
residue Lys-263 (A) or Arg-382 (B) with the phosphate backbone. The NMR
data are in agreement with a similar role of the
2
3-loop of PC4
in ssDNA binding, because the ssDNA-induced amide resonance
perturbations of all residues in this region are among the largest
observed. The importance of the
4'
5'-loop region for ssDNA
binding is further supported by a recent mutagenesis study (6).
Temperature factors from the crystallographic structure determination
of PC4CTD (13) suggested that the
2
3-loop and the
-ridge exhibit above average mobility. To assess the flexibility of
these regions in solution, we carried out relaxation measurements
(15N-T1, 15N-T1
, and
1H15N-NOE) for the backbone amides. Results are
shown in Fig. 4. Although 15N-T1 did not show significant variation along
the protein sequence, 15N-T1
values are
indeed higher in this region than in the rest of the protein.
Considerably lower heteronuclear 1H15N-NOE
values were observed in the
2
3-loop and the
-ridge compared with the rest of the molecule. Consistent with these relaxation data,
fast hydrogen exchange was observed in both the
2
3-loop and the
-ridge (Fig. 4, lower panel), whereas most of the amides in the core of the protein exhibit either medium or slow exchange. Thus, the regions of PC4 that mediate ssDNA contacts are relatively flexible in solution. Upon formation of the complex with
dT18, T1
values for amide nitrogens
throughout the protein are reduced, most likely because of the larger
size of the complex (Fig. 5).
However, the reduction of signals from residues in the
2
3-loop
region is significantly larger than this uniform decrease in signal
intensity. This effect may be caused by attenuation of the higher
mobility in this region, which can be understood in terms of
stabilization by ssDNA contacts.

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Fig. 4.
Values of 15N-T1,
15N-T1 , heteronuclear
-1H15N-NOE and hydrogen
exchange rate for PC4CTD backbone amides. The exchange
rates were classified as follows. Fast (F), no peak was
observed in the first HSQC spectrum after redissolving the lyophilized
protein in D2O); medium (M), peaks were still
observable in the first spectrum, but the intensity significantly
decreased in 200 h; slow (S), no significant decrease
in peak intensity could be detected in 200 h. The secondary
structure of PC4CTD has been indicated schematically below the
graph.
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Fig. 5.
15N-T1 values
for the amides in free (gray) and bound PC4CTD (black).
The values obtained from curve fitting of peak intensities are shown
with error bars indicating single standard deviations. The secondary
structure of PC4CTD has been indicated schematically below the
graph.
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|
DNA binding by the two channels as demonstrated here is consistent with
and provides support for existing biochemical data in several ways. In
the first place, high affinity for heteroduplex structures and the
apparent requirement for two juxtaposed single-stranded regions (12)
are readily explained by the present model: each binding channel can
contact one of the strands running in opposite directions, as
illustrated in Fig. 6. Moreover, the
binding site size (approximately 8 nucleotides in each of the two
single-stranded regions) as determined from the affinities for both
heteroduplexes and single-stranded oligonucleotides of increasing size
(12), corresponds closely to what would be expected on the basis of the
channel length in the PC4 structure: if it is assumed that ssDNA bound
by PC4 is similar in conformation to ssDNA bound by RPA, 8 nucleotides
should span a distance of nearly 40 Å, which is also approximately the
length of the channels (Fig. 6).

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Fig. 6.
The PC4 ssDNA binding surface. The two
models illustrate how PC4CTD can interact in a very similar
fashion with 16-20 nucleotides of ssDNA (A) and an eight
mismatch heteroduplex (B), the two optimal binding sites
that were found in an earlier study (12). Protein monomers are shown in
light blue and dark blue. DNA is shown in green with ribbons
representing the phosphate backbone and base pairs are shown as
horizontal bars. An arrow (40 Å) is drawn to
indicate the scale of both models. Positively charged and aromatic side
chains, on the basis of our NMR study, that we expect to mediate
contacts have been indicated in red. Involvement in DNA binding of the
side chains shown in yellow is suggested by both our NMR data and a
recent mutagenesis study (6).
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The backbone resonances of the residues Met-69, Arg-70, Tyr-88, and
Lys-97-Arg-100 of PC4CTD could not be assigned because the
corresponding signals were missing in all spectra. Interestingly,
several other residues (Glu-93-Met-96) that are in close proximity to
the ones that were unassigned give rise to multiple sets of signals,
suggesting conformational heterogeneity. Because all of these residues
are located in the region of the PC4CTD structure that connects
the tips of the
-ridge (which themselves produce well defined
nondegenerate resonances) to the core of the protein dimer, it is
tempting to speculate that the orientation of the tips with respect to
the protein core is ill-defined in solution. Interestingly, the tips
contain several negatively charged residues (Asp-91, Glu-93, and
Glu-95) that seem to interfere with the interaction of PC4CTD
with an 8-nucleotide heteroduplex in the hypothetical model published earlier (see Ref. 13 and Fig. 6B), because these charges
would be in close proximity to the phosphate backbone of the DNA.
However, such DNA molecules are bound with very high affinity (12),
suggesting that steric hindrance does not occur. A possible explanation
for this is that the tips may be able to adopt alternative
conformations in which they have moved "inward" compared with the
crystal structure (i.e. toward each other). Such alternative
conformations may allow the
-ridge to be inserted into the
heteroduplex opening more easily. Conformational heterogeneity would be
consistent with the NMR degeneracy that is found for the amino acids in
the hinge region and the fact that crystallographic temperature factors for this region are significantly higher than the average for PC4CTD (13).
So far only a single protein (other than the direct homologues of PC4
in various species) has been characterized that contains a region
homologous to the PC4 C terminus. This protein, the yeast factor
Sub1/Tsp1 (27, 28), is also an RNA polymerase II transcriptional coactivator but differs from PC4 in several important respects. In the
first place, Sub1/Tsp1 is much larger than PC4 (33 kDa) and bears no
homology to PC4 outside the region corresponding to the PC4
ssDNA-binding domain. Furthermore, the reported mechanisms for
coactivation reported for Sub1/Tsp1 (28) and PC4 (4) differ
considerably. Nevertheless, the homology between PC4CTD and
Sub1/Tsp1(40-105) is very high (73% conserved, 49% identical), suggesting a similar fold and function. Indeed, Sub1/Tsp1 has been
reported to bind to ssDNA (27). The fact that two otherwise quite
different transcriptional cofactors both contain this type of ssDNA
binding domain suggests that interaction with ssDNA or heteroduplexes
is part of a general mechanism that helps regulate transcription in
eukaryotes. Indeed, direct evidence for a role of ssDNA binding by PC4
in regulation of transcription was recently provided (6). The
identification of DNA-contacting residues described here and the
construction of point mutants that do not interact with ssDNA (6) will
be helpful in the further elucidation of the precise role of the PC4
C-terminal domain and the homologous domain in Sub1/Tsp1, in
transcription and other processes.
 |
ACKNOWLEDGEMENTS |
Marco Tessari is acknowledged for
help with computer programs. We thank Petra Düx for critical
reading of the manuscript and discussions.
 |
FOOTNOTES |
*
This work was supported by the Netherlands Foundation for
Chemical Research (SON) with financial support from the Netherlands Organization for Scientific Research (NWO).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Bijvoet Centrum
voor Biomoleculair Onderzoek, Padualaan 8, 3584 CH Utrecht, The Netherlands. Tel.: 31-30-2533787; Fax: 31-30-2537623; E-mail: kaptein{at}nmr.chem.uu.nl.
The abbreviations used are:
PIC, preinitiation
complex; RPA, replication protein A; ss, single-stranded; NOE, nuclear
Overhauser effect.
 |
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