From the Department of Chemistry, Massachusetts
Institute of Technology, Cambridge, MA 02139 and the ¶ Howard
Hughes Medical Institute, University of Medicine and Dentistry of New
Jersey, Robert Wood Johnson Medical School, Division of Nucleic Acids
Enzymology, Department of Biochemistry,
Piscataway, New Jersey 08854-5635
Received for publication, February 7, 2001, and in revised form, May 7, 2001
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ABSTRACT |
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The structure-specific recognition protein SSRP1,
initially isolated from expression screening of a human B-cell cDNA
library for proteins that bind to cisplatin
(cis-diamminedichloroplatinum(II))-modified DNA,
contains a single DNA-binding high mobility group (HMG) domain. Human
SSRP1 purifies as a heterodimer of SSRP1 and Spt16 (FACT) that
alleviates the nucleosomal block to transcription elongation by RNAPII
in vitro. The affinity and specificity of FACT, SSRP1, and
the isolated HMG domain of SSRP1 for cisplatin-damaged DNA were
investigated by gel mobility shift assays. FACT exhibits both affinity
and specificity for DNA damaged globally with cisplatin compared with
unmodified DNA or DNA damaged globally with the clinically ineffective
trans-DDP isomer. FACT binds the major 1,2-d(GpG)
intrastrand cisplatin adduct, but its isolated SSRP1 subunit fails to
form discrete, high affinity complexes with cisplatin-modified DNA
under similar conditions. These results suggest that Spt16 primes SSRP1
for cisplatin-damaged DNA recognition by unveiling its HMG domain. As
expected, the isolated HMG domain of SSRP1 is sufficient for specific
binding to cisplatin-damaged DNA and binds the major cisplatin
1,2-d(GpG) intrastrand cross-link. The affinity and specificity of FACT
for cisplatin-modified DNA, as well as its importance for transcription
of chromatin, suggests that the interaction of FACT and
cisplatin-damaged DNA may be crucial to the anticancer mechanism of cisplatin.
Eukaryotic cells package DNA into chromatin, the structure of
which impedes essential cellular processes that require DNA for
function. Such processes include replication, recombination, and
transcription. The regulation of gene expression is therefore intimately tied to chromatin structure.
DNA transcription in vitro can be reconstituted by a minimal
set of general transcription factors and RNA polymerase II (1). These
minimal components are insufficient for transcription from reconstituted nucleosomal templates, however, implying the existence of
cellular mechanisms for chromatin remodeling to facilitate access of
the transcription machinery to DNA (2). Two classes of nuclear factors,
ATP-dependent chromatin remodeling enzymes and
histone acetyltransferases, allow the transcription machinery to
assemble and initiate transcription from chromatin templates (3, 4).
Recently, a novel factor,
FACT1
(Facilitates Chromatin
Transcription), which enables transcription elongation past
nucleosomes, was isolated from HeLa nuclear extracts (5, 6). This
factor is a heterodimer of human Spt16/Cdc68 and human SSRP1
proteins (7). FACT is inactivated by chemical cross-linking of the
histone octamer, suggesting that it may function to unravel H2A/H2B
histone dimers from nucleosome cores (7). The remaining H3/H4
tetramer is itself insufficient to repress the elongating RNA
polymerase (8, 9).
FACT is a very abundant nuclear protein complex with an estimated
100,000 copies/HeLa cell (7). The complex is conserved across a diverse
range of organisms, analogous complexes of SPT16/SSRP1 homologs having
been isolated from Xenopus and Saccharomyces
cerevisiae (10-12). Although distinct roles for the two protein
components of FACT have yet to be elucidated, the DNA-binding
high-mobility group (HMG) domain of SSRP1 (13) may target the complex
to nucleosomes (7). Circumstantial evidence suggests that HMG domain
proteins bind to DNA as it enters and exits the nucleosome (14).
Consistent with the hypothesis that the HMG domain of SSRP1 is
responsible for FACT binding to DNA, FACT activity is abolished by
addition of a 5-fold excess of superhelical plasmid DNA competitor (5). The mechanistic details of derepression of transcription elongation by
FACT remain to be elucidated.
Human Cdc68/Spt16 is a 119.9-kDa nuclear protein with 36% identity to
its yeast homolog. Previous genetic studies with yeast Cdc68 suggested
a role in modulating chromatin structure to effect both gene repression
and activation (15-17). The domain features of the 81-kDa human SSRP1
protein are shown in Fig. 1 (18). Initially isolated from expression
screening of a human B-cell cDNA library for proteins that bind DNA
modified by the antitumor agent cisplatin
(cis-diamminedichloroplatinum(II)), SSRP1 is expected to
bind distorted DNA structures through its DNA-binding HMG domain (18).
The ability of SSRP1 to recognize DNA modified by cisplatin and the
deleterious effect of cisplatin on transcription suggest a possible
role for SSRP1 and its physiologically relevant complex FACT in the
cisplatin anticancer mechanism (19-21). Although the success of
cisplatin therapy on a variety of cancers, including testicular,
ovarian, and head and neck, has been remarkable since its introduction
in 1979, there are several drawbacks to cisplatin treatment. These
include toxic side effects, inherent or acquired resistance, and
efficacy in only a handful of tumor types (22). A detailed
understanding of its mechanism of action may allow for the rational
design of new antitumor therapies.
Cisplatin reacts with a number of cellular components, but it is
generally accepted that the biologically relevant target is DNA. After
loss of the chloride ligands due to the relatively low concentration of
Cl Like other members of the HMG-domain protein family, SSRP1 is expected
to recognize cisplatin-damaged DNA through this DNA-binding component.
Here we demonstrate the affinity and specificity of FACT for
cisplatin-damaged DNA and the major 1,2-intrastrand cisplatin-DNA lesion in particular. Electrophoretic mobility shift assays reveal that
both the SSRP1 and Spt16 subunits of FACT are necessary for high
affinity binding to cisplatin-modified DNA. The isolated HMG domain of
SSRP1, however, is sufficient for binding the major 1,2-d(GpG)
intrastrand cisplatin-DNA cross-link. The affinity and specificity of
FACT for cisplatin-modified DNA and its role in modulating chromatin
structure during transcription suggest that the interaction of FACT and
cisplatin-damaged DNA may be important in the cisplatin anticancer mechanism.
Oligonucleotide Probes--
A 127-base pair probe was
constructed by digesting a commercially available 123-base pair ladder
(Life Technologies, Inc.) with AvaI restriction enzyme (New England
Biolabs). The digested 123-base pair fragment containing 4-bp
5'-overhangs was purified on a 10% native polyacrylamide gel,
ethanol-precipitated, and quantitated by UV-vis spectroscopy (Hewlett
Packard 8453). The purified probe was modified with cisplatin or
trans-DDP at various platinum:nucleotide ratios
(rf, added platinum:nucleotide ratio) following
published procedures (26). The extent of platination (rb, bound platinum:nucleotide ratio) was determined by flameless atomic absorption spectroscopy (PerkinElmer Life Sciences
HGA-800 AAnalyst 300) and UV-vis spectroscopy. Platinated and
unplatinated 123-base pair probes having recessed 3'-ends were fill-in
labeled with Klenow fragment of DNA polymerase (New England Biolabs)
and [
Double-stranded 156-bp probes were assembled from six
oligonucleotides of lengths 87 nt (fragment 1), 12 nt (fragment
2), 57 nt (fragment 3), 52 nt (fragment 4), 24 nt (fragment 5), and 80 nt (fragment 6) as described previously (28, 29). The 12-nt fragment
d(TCTCG*G*ACTTCT) contained a single
cis-[Pt(NH3)2{d(GpG)-N7 (1),-N7 (2)}] 1,2-intrastrand d(GpG) cross-link at the
site denoted by asterisks. Fragments 3 and 6, which ultimately
constitute the 3' ends of the 156-bp duplexes, were prepared with three
phosphorothioate linkages each to minimize exonuclease
digestion2.
Single-stranded 12-mer oligonucleotides were 5'-end labeled with
[ Plasmids--
cDNA encoding the HMG (residues
539-614) domain of SSRP1 was amplified by PCR using human SSRP1
cDNA and the primers
5'-GCTCTAGAAAGGAGGTGGAGATGAAGAAGCGCAAAGAC-3' and 5'-CTCGCCTCGGCATATGTTAATATTCTTTCATGGCTTT-3'.
The PCR primers contained restriction sites for XbaI and
NdeI (italics) as well as initiation, termination, and
ribosome binding sites (underlined); the last, 5'-AAGGAG, was
positioned nine base pairs upstream of the initiation codon. PCR was
performed with Taq DNA polymerase (Life Technologies, Inc.)
under the following thermal cycler conditions: 5 min at 94 °C
(denaturation cycle); 1 min at 94 °C, 2 min at 50 °C, and 3 min
at 72 °C (25 cycles); 10 min at 72 °C (extension cycle). The
resulting fragments were digested with XbaI and
NdeI (New England Biolabs), purified by using a PCR
purification kit (Qiagen), and ligated into the XbaI and
NdeI sites of pET3a (Novagen) to give p3SSRP.d1 for
expression of the HMG domain. Correct insertion of the PCR fragment was
confirmed by restriction enzyme mapping and DNA sequencing.
Expression and Purification of Recombinant SSRP1 HMG-domain
Peptides--
BL21(DE3) Codon Plus RIL (Stratagene) cells
harboring p3SSRP.d1 were grown at 37 °C in LB containing 100 µg/ml ampicillin and 20 mM methionine. Protein production
was induced at an A600 of 0.7 by addition
of isopropyl Isolation of FACT from HeLa Cells--
FACT was isolated from
HeLa cells as described (5). The purity of native FACT (hFACT) was
assessed by denaturing and native PAGE.
Production of Recombinant SSRP1, Spt16, and FACT--
Methods
for recombinant SSRP1 (rSSRP1) and Spt16 (rSpt16) production and
purification will be reported
elsewhere.3
Recombinant FACT (rFACT) was obtained by mixing equimolar amounts of
pure rSSRP1 and pure rSpt16, followed by gel filtration chromatography.
Gel Mobility Shift Assays--
Gel mobility shift assays with
SSRP1 HMG domain and protein were performed as follows. To assay for
protein recognition of cisplatin-damaged DNA, cisplatin-modified or
unmodified 127-base pair duplexes (0.2 nM, 40,000 cpm) were
titrated with protein in buffer PP250 (10 mM Tris, pH 7.5, 250 mM NaCl, 10 mM MgCl2, 0.5 mM EDTA, 5% glycerol, 1 mM dithiothreitol)
containing 0.2 mg/ml of chicken erythrocyte genomic DNA. Samples were
incubated on ice for 1 h and made 10% in sucrose immediately
prior to loading onto pre-equilibrated native 4% or 5% polyacrylamide
gels (29:1 acrylamide:bisacrylamide, 3.3% cross-linking, 45 mM Tris borate, 1 mM EDTA, pH 8.3).
Electrophoresis was continued for 2 h at 215 V and 4 °C. To
assay for binding of the major 1,2-d(GpG) intrastrand cisplatin-DNA
cross-link, AG*G*A and AGGA 15-bp oligonucleotide duplexes (0.4 nM, 20,000 cpm) were titrated with protein in binding buffer SD250 (10 mM Hepes, pH 7.5, 10 mM
MgCl2, 50 mM LiCl2, 100 mM NaCl, 1 mM spermidine, 0.2 mg/ml bovine
serum albumin, 0.05% Nonidet P-40). Samples were incubated on ice for
30 min and made 10% in sucrose immediately prior to loading onto
pre-equilibrated native 12% polyacrylamide gels (29:1
acrylamide:bisacrylamide, 3.3% cross-linking, 45 mM Tris
borate, 1 mM EDTA, pH 8.3). Electrophoresis was continued
for 1.5-2 h at 300 V and 4 °C. Following electrophoresis, gels were
dried and exposed to a phosphorimaging plate for 12-24 h. The amount
of bound and free oligonucleotide was assessed with a Bio-Rad GS-525
phosphorimaging device.
To examine the affinity and specificity of FACT for cisplatin-damaged
DNA, 127-bp duplexes (0.02-2 nM, 20,000 cpm) undamaged or
globally damaged with cisplatin or trans-DDP, or 156-bp
site-specific modified or unmodified duplexes (0.1 nM,
20,000 cpm) were combined with hFACT, rFACT, rSpt16, or rSSRP1 in
binding buffer PP50 (10 mM Tris, pH 7.5, 50 mM
NaCl, 10 mM MgCl2, 0.5 mM EDTA, 5%
glycerol, and 1 mM dithiothreitol). Chicken erythrocyte
genomic DNA competitor (0.2-2.0 mg/ml) was included in reactions
containing globally modified probes where noted. Samples were incubated
on ice 1 h prior to loading onto running, pre-equilibrated native
4% polyacrylamide gels (29:1 acrylamide:bisacrylamide, 3.3%
cross-linking, 50 mM Tris, pH 8.0, 2 mM EDTA,
380 mM glycine). Electrophoresis was continued at 100 V for
5 h and 4 °C. Gels were dried and exposed to a phosphorimaging
plate or to Kodak X-OMAT film for 12-24 h. Exposed phosphorimaging
plates were analyzed with a Bio-Rad GS-525 phosphorimaging
device and Multi-Analyst software.
Expression and Purification of SSRP1 HMG Domain--
Full-length
SSRP1 contains a single DNA-binding HMG domain flanked by two short
basic regions (Fig. 1). The minimal HMG
domain was recombinantly expressed and purified. As shown in Fig.
2, the SSRP1 HMG domain was obtained with
>95% purity. Western blots with polyclonal antibodies raised against
the full-length hSSRP1 protein confirmed the identity of the
recombinant protein. Electrospray ionization mass spectrometry of the
protein confirmed the absence of oxidized contaminants. N-terminal
sequencing of each protein yielded the predicted amino acid sequence
with retention of the initial methionine.
Affinity and Specificity of the HMG Domain of SSRP1 for
Cisplatin-damaged DNA--
The HMG domain of SSRP1 is expected to
confer recognition of cisplatin-modified DNA to the full-length SSRP1
protein. To examine the affinity and specificity of the hSSRP1 HMG
domain for cisplatin-modified DNA, a series of 127-bp probes,
unmodified or containing various levels of global cisplatin- or
trans DDP damage were used in bandshift assays with
recombinant hSSRP1 HMG domain. All bandshifts were performed in the
presence of a large excess of nonspecific DNA competitor. The
recombinant hSSRP1 HMG domain retains affinity for cisplatin-modified
DNA, as evidenced by gel-mobility shifts of the domain with a 127-bp
probe damaged globally with cisplatin (Fig.
3A). The interaction between
the domain and cisplatin-modified DNA is not a result of general DNA
affinity for the probe because the domain fails to bind the unmodified
127-mer under identical conditions (Fig. 3A). The domain
does exhibit very low affinity for probes damaged with the clinically
ineffective trans-DDP isomer (data not shown); however,
protein affinity for the cisplatin-damaged probe is significantly
higher than that for the corresponding trans-DDP-damaged
DNA.
The mobility of protein-DNA complexes formed upon incubation of hSSRP1
HMG domain with the cisplatin-modified 127-bp probe decreases with
increasing protein concentration (Fig. 3A). A similar pattern of mobility shifts occurs when probes with increasing damage
levels (0.003-0.044 platinum atoms/nucleotide, 1-12 platinum atoms/duplex DNA) are used (data not shown). Both of these results are
consistent with multiple proteins binding to these long probes at high
platination or protein levels.
Recognition of the Major 1,2-d(GpG) Intrastrand Cisplatin-DNA
Adduct by hSSRP1 HMG Domain--
To confirm that the minimal HMG
domain of hSSRP1 can bind the major 1,2-d(GpG) intrastrand
cisplatin-DNA cross-link, the affinity of the domain for the
oligonucleotide duplex AG*G*A, 5'-CCTCTCAG*G*ATCTTC-3', where asterisks
denote the sites of platinum coordination to the N-7 positions of
adjacent guanines, was investigated. A representative gel mobility
shift with AG*G*A and hSSRP1 HMG domain is shown in Fig. 3B.
The domain fails to shift the corresponding unplatinated duplex under
identical conditions (data not shown).
Isolation of hFACT from HeLa Cells--
Human FACT, the
heterodimer of hSpt16 and hSSRP1, was isolated from HeLa cells as
described (5). The complex contains hSpt16 and hSSRP1, as well as a
minor contaminant at ~40 kDa as judged by SDS-PAGE followed by silver
staining. This contaminant is not necessary for FACT activity (7).
Native PAGE demonstrates that hFACT isolated in this manner contains no
free hSSRP1 or hSpt16.
Gel Mobility Shift Assays of hFACT with DNA Damaged with
Cisplatin--
To assess the ability of hFACT to bind specifically to
cisplatin-modified DNA, gel mobility shift assays were performed with a
series of platinated or unplatinated 127-bp probes (Fig.
4A). Cisplatin- and
trans-DDP-damaged probes used in these experiments were
modified at similar levels. FACT retains some nonspecific affinity for
all probes in the absence of competitor; however, the band of lowest
mobility observed in lanes 2, 5, and 8 corresponds to the position of free protein (data not shown) and
disappears in the presence of excess competitor (Fig. 4A). A
specific protein-DNA complex is formed upon incubation of FACT with the
127-bp probe modified globally with cisplatin. This complex persists
even in the presence of a 3.6 × 105-fold excess of
nonspecific competitor DNA (Fig. 4A, lanes 5 and 6) and remains robust in NaCl concentrations of at least 250 mM (data not shown). The mobility of the putative
FACT-cis127-bp complex is unchanged by increasing platinum damage
levels within the range of 1-8 platinum atoms/127-bp duplex (data not
shown).
Binding of FACT to the Major 1,2-d(GpG) Intrastrand Cisplatin-DNA
Cross-link--
Native hFACT forms a specific complex with a 156-bp
probe containing a single, centered 1,2-d(GpG) intrastrand
cisplatin-DNA cross-link but not with the corresponding unmodified
probe (Fig. 4B).
Binding of Individual FACT Subunits to Cisplatin-modified
DNA--
Recombinantly produced rSSRP1, as well as rSpt16 and rFACT,
were used to confirm that the SSRP1 subunit confers cisplatin-modified DNA binding activity on the complex. Proteins obtained as described under "Experimental Procedures" are pure as judged by SDS-PAGE followed by Coomassie staining (Fig.
5A). Silver-stained native PAGE confirms that both rSpt16 and rSSRP1 exist as monomers (data not
shown) and demonstrates that the rFACT preparation contains no free
rSSRP1 or rSpt16 (Fig. 5B). Furthermore, rFACT produced in
this manner is competent for in vitro RNA polymerase
transcript elongation on chromatin templates.3
The recombinant complex yields a bandshift pattern with the
cis127-bp probe identical to that of native FACT (compare Figs. 6A and 4A). As with
native FACT, rFACT fails to bind the trans-DDP-modified probe under these conditions (data not shown). In contrast, gel mobility assays with each of the recombinant FACT subunits indicate that the Spt16 subunit fails to bind the cis127-bp probe (Fig. 6B) and that the SSRP1 subunit gives rise to only a very
weak shift that disappears in the presence of excess competitor (Fig. 6C). Although native and recombinant FACT form specific
protein-cis127-bp complexes at protein concentrations as low as 3 nM (data not shown), rSSRP1 fails to yield a bandshift
pattern similar to that of rFACT or native FACT even at protein
concentrations of 900 nM (Fig. 6C).
Interaction of the Isolated HMG Domain of SSRP1 with
Cisplatin-damaged DNA--
Fragments of SSRP1 interact with DNA
damaged globally with cisplatin in filter binding assays (18). This
interaction is expected to be mediated by the DNA-binding HMG domain of
SSRP1 (Fig. 1). Several proteins containing one or more HMG domains bind to cisplatin-damaged DNA, including HMG1 (32, 33), HMG2 (33, 34),
SSRP1 (18), Ixr1 (35, 36), hUBF (37), LEF1 (38), and SRY (39). In
particular, full-length HMG1 binds DNA damaged globally with cisplatin
but not DNA damaged with its clinically inactive isomer
trans-DDP (32). The HMG domains of HMG1, HMG2, SSRP1, and
other HMG-domain proteins that bind cisplatin-damaged DNA are likely to
be the key elements in protein recognition of cisplatin-DNA lesions.
Evidence supporting this conclusion has been described for both HMG
domains of HMG1 (27, 40). To determine whether the HMG domain of SSRP1
is sufficient for cisplatin-modified DNA binding, a fragment
corresponding to residues 539-614 was expressed and purified from
Escherichia coli. This recombinant HMG domain was used in
gel mobility shift experiments with globally cisplatin-modified and
unmodified probes. The isolated domain is sufficient for specific
binding to DNA damaged globally with cisplatin. The domain is selective
for cisplatin-modified DNA with respect to both unmodified DNA and DNA
containing trans-DDP adducts. In addition, like other
isolated HMG domains (27, 40), the HMG domain of SSRP1 binds
oligonucleotide probes containing the major 1,2-d(GpG) intrastrand
cisplatin-DNA cross-link. The affinity of this interaction depends on
the DNA sequence flanking the drug-DNA lesion as seen for HMG domains A
and B of HMG1 (27).
Interaction of FACT and SSRP1 with Cisplatin-damaged DNA--
Both
SSRP1 and its physiologically relevant complex with Spt16, FACT, are
expected to bind distorted DNA structures including cisplatin-DNA
cross-links by means of the HMG domain of SSRP1. Here we show that FACT
has both affinity and specificity for DNA damaged globally with
cisplatin with respect to undamaged DNA. A tightly bound complex
results from incubation of FACT with cisplatin-modified DNA as
evidenced by its resilience in the presence of 3.6 × 105-fold excess of nonspecific DNA or >50-fold excess of
unplatinated competitor DNA. Like other HMG-domain proteins, FACT binds
the major 1,2-intrastrand cross-links of cisplatin-modified DNA. The 1,2-intrastrand cisplatin-DNA cross-links are the most abundant DNA
adducts formed following cisplatin treatment (41). These adducts are
thought to be crucial to cisplatin toxicity because geometric
constraints prohibit the clinically inactive trans-DDP from
forming 1,2-intrastrand adducts, although trans-DDP can form DNA cross-links similar to the less abundant cisplatin-DNA lesions (21). Notably, FACT fails to bind DNA damaged with the clinically ineffective trans-DDP isomer.
We then sought to confirm that the SSRP1 subunit confers the ability to
recognize cisplatin-modified DNA on the FACT complex. As anticipated,
Spt16, which contains no putative DNA-binding domains, has no affinity
for either cisplatin-damaged or undamaged probes. Unexpectedly, SSRP1
fails to form a high-affinity complex with the same cisplatin-modified
probe in gel mobility shift assays under conditions sufficient for
hFACT binding. The complex of rSSRP1 and rSpt16, however, is capable of
binding cisplatin-damaged DNA with affinity and specificity comparable
with hFACT in identical bandshift assays. A >300-fold excess of rSSRP1
fails to afford a protein-DNA complex of affinity comparable with that
of hFACT or rFACT, suggesting that protein concentration discrepancies are unlikely to explain the lack of a bandshift with rSSRP1. The observed differences in affinity between the protein-DNA complex formed
by hFACT or rFACT and that formed by SSRP1 suggest that Spt16 primes
SSRP1 for cisplatin-modified DNA recognition. We suggest that, in the
absence of Spt16, SSRP1 adopts a fold that renders its HMG domain
inaccessible to cisplatin-modified DNA. When Spt16 is present, a
conformational change occurs unveiling the HMG domain of SSRP1 and
leading to the observed binding interaction.
Functional Implications--
Because modulation of chromatin
structure is essential to many cellular processes that use DNA as a
substrate, including replication and transcription, FACT may effect
such processes. The specific interaction of SSRP1 and FACT with DNA
damaged with the anticancer drug cisplatin as judged by electrophoretic
mobility shift assays implicates both SSRP1 and FACT in the mechanism
of cisplatin cytotoxicity. The binding of SSRP1 or FACT to
cisplatin-damaged DNA could mediate cellular sensitivity to the drug by
shielding its DNA cross-links from repair, allowing the drug-DNA
lesions to persist and eventually leading to cell death (28, 29, 35,
39, 42-44). Moreover, presence of cisplatin-DNA adducts could titrate
SSRP1 or FACT from its normal binding sites, thereby disrupting
SSRP1/FACT function(s) (45, 46). Finally, the formation of stable
FACT/SSRP1 complexes at cisplatin-DNA cross-links could lead to
stalling of the RNAPII transcription machinery, ubiquitination of
RNAPII, and proteolysis resulting ultimately in cell death (30, 31).
Any or all of these activities may contribute to the mechanism of
cisplatin-mediated cytotoxicity.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ions in the cell, cisplatin coordinates preferentially
to the N-7 atoms of two adjacent purine nucleotides by forming mainly intrastrand d(GpG) and d(ApG) DNA cross-links (23, 24). Such adducts
bend the duplex toward the major groove and unwind the DNA helix. The
minor groove is concomitantly flattened and widened. Delineation of the
cellular processing of cisplatin-DNA adducts is of great importance to
unraveling its mechanism of cytotoxicity. Therefore, much work has
focused on identifying and characterizing proteins that bind
1,2-intrastrand cross-links with high affinity and specificity (25).
Structural distortions imposed on DNA by cisplatin 1,2-intrastrand
cross-links are recognized by members of the HMG domain protein family
including SSRP1 with notable specificity over unmodified
double-stranded DNA.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]dCTP (PerkinElmer Life
Sciences), cold dATP, dTTP, and dGTP to give 127-base pair
oligonucleotide duplexes. Radiolabeled probes were purified with G25
Quick Spin columns (Roche Molecular Biochemicals), ethanol-precipitated, and quantitated by scintillation counting (Beckman LS 6500). The radiolabeled 15-bp oligonucleotide
duplexes AG*G*A (d(CCTCTCAG*G*ATCTTC)/d(GAAGATCCTGAGAGG), where
asterisks denote the sites of cis-diammineplatinum(II)
cross-linking via the N-7 positions of adjacent guanines and AGGA, the
unplatinated analog) were prepared according to published procedures
(27).
-32P]ATP and T4 polynucleotide kinase. Fragments 3, 4, and 5 were 5'-end phosphorylated with cold ATP and T4 polynucleotide
kinase. Three complementary duplexes, each with 6-nt overhangs, were
created by annealing each fragments 1 and 6, fragments 2 and 5, and
fragments 3 and 4 (200 pmol each) and subsequently were treated with
DNA ligase (New England Biolabs) to afford radiolabeled 156-bp
duplexes. Ligation mixtures were ethanol-precipitated and purified on
6% preparative denaturing polyacrylamide gels. Subsequent
autoradiography allowed single-stranded 156-mers to be excised and
isolated from the gel by electroelution and ethanol precipitation. DNA
duplexes were obtained by reannealing single-stranded 156-nt
oligonucleotides, followed by purification on 10% native
polyacrylamide gels, electroelution, and ethanol precipitation. The
resulting 156-bp probes were quantitated by scintillation counting.
-D-thiogalactopyranoside to a final
concentration of 1 mM. After an induction period of 5 h, cells were collected by centrifugation, resuspended in cold lysis buffer (50 mM Tris, pH 7.0, 20 mM NaCl, 10 mM EDTA, 10 mM EGTA, 10 mM
MgCl2, 0.035%
-mercaptoethanol, 1 mM
Pefabloc, 1 mM DNase I, 2 mM dithiothreitol, 20 mM methionine, 2 mM
NaS2O4), and lysed by sonication. Debris was
removed by centrifugation, and cellular proteins were precipitated with
55% saturated ammonium sulfate. Recombinant protein was precipitated
with 98% saturated ammonium sulfate, collected by centrifugation,
resuspended in buffer A (20 mM Tricine, pH 8.3, 5 mM dithiothreitol, 1 mM EDTA, 20 mM methionine, 2 mM NaS2O4), and
dialyzed against the same buffer. The desalted protein solution was
loaded onto a cation-exchange SP-Sepharose Fast Flow (Amersham
Pharmacia Biotech) column washed with buffer A. Proteins were
eluted with a linear gradient of 0-1 M NaCl in buffer A. Eluted proteins were detected by SDS-PAGE followed by Coomassie
staining. Fractions containing SSRP1 HMG domain were pooled, dialyzed,
concentrated, and applied to a Superdex 75 column (Amersham Pharmacia
Biotech) washed with buffer B (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4,
1.4 mM KH2PO4, 20 mM
methionine, 2 mM NaS2O4, pH 7.3).
Proteins were eluted with column buffer, and fractions containing pure
SSRP1 HMG domain were pooled, concentrated, frozen in liquid nitrogen,
and stored at
80 °C. SSRP1 HMG domain concentrations were
determined from A280 values by using an
extinction coefficient (
280 = 18,600 M
1 cm
1)
calculated by quantitative amino acid analysis.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Human SSRP1 domain structure. Schematic
representation of the domain structure of human SSRP1. The amino acid
sequence and predicted secondary structure of the DNA-binding portion
are depicted. The positions of the three -helices of the highly
homologous HMG1 domain B are shown above the sequence.
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Fig. 2.
Characterization of SSRP1 HMG domain.
Purity of the HMG domain of SSRP1 as judged by 16.5% Tricine SDS-PAGE
followed by Coomassie staining.
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Fig. 3.
Affinity and specificity of SSRP1 HMG domain
for cisplatin-damaged DNA. A, gel mobility shift assays
of SSRP1 HMG domain with a 127-bp DNA probe undamaged or damaged
globally with cisplatin. 32P-Labeled 127-bp or cis127-bp
(0.012 Pt/nt, 3 Pt/duplex) probe (0.2 nM) was incubated
with 0, 260 nM, or 2.6 µM SSRP1 HMG domain as
indicated and subjected to electrophoresis in the presence of 0.2 mg/ml
chicken erythrocyte genomic DNA. B, gel mobility shift assay
of SSRP1 HMG domain and AG*G*A 32P-labeled probe
demonstrates ability of domain to bind the major 1,2-d(GpG) intrastrand
cisplatin-DNA adduct. Radiolabeled AG*G*A probe (0.4 nM)
was incubated with 0 or 100 nM SSRP1 HMG domain as
indicated and electrophoresed.
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Fig. 4.
Affinity and specificity of hFACT for
cisplatin-damaged DNA. A, native hFACT demonstrates
both high affinity and specificity for cisplatin-damaged DNA with
respect to undamaged DNA or DNA damaged with the clinically ineffective
trans-DDP isomer. Native FACT (130 nM) was
incubated with 32P-labeled 127-bp, cis127-bp (0.012 Pt/nt,
3 Pt/duplex) or trans127-bp (0.015 Pt/nt, 3-4 Pt/duplex) probe (0.2 nM) in the presence or absence of 2 mg/ml nonspecific
chicken erythrocyte genomic DNA. B, native hFACT recognizes
the major 1,2-d(GpG) intrastrand cisplatin-DNA lesion. Native hFACT
(130 nM) was subjected to gel mobility shift assay with
32P-labeled 156-bp probe containing a single, centered
1,2-d(GpG) intrastrand cisplatin adduct (0.1 nM) or the
corresponding undamaged 156-bp duplex (0.1 nM).
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[in a new window]
Fig. 5.
Characterization of recombinant FACT.
A,) 6% SDS-PAGE of purified recombinant rFACT (lane
1), rSSRP1 (lane 2), and rSpt16 (lane 3)
followed by Coomassie staining. B, purified rFACT contains
no free rSSRP1 or rSpt16 as judged by native 4-20% PAGE followed by
silver staining.
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[in a new window]
Fig. 6.
Interaction of rSSRP1, rSpt16, and rFACT with
cisplatin-damaged DNA. Radiolabeled cis127-bp duplexes (0.03 Pt/nt, 7-8 Pt/duplex) were titrated with varying amounts of rFACT
(A), rSpt16 (B), or rSSRP1 (C) in the
presence or absence of 0.2 mg/ml of nonspecific chicken erythrocyte
genomic DNA and subjected to electrophoresis.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Y. Mikata for providing the fragments necessary for assembly of the 156-bp probes, S. Yuditskaya for experimental assistance, A. Loyola and the members of the Reinberg laboratory for technical and experimental expertise, P. Pil for polyclonal antisera to SSRP1 and SSRP1 cDNA, and members of the Lippard laboratory for helpful suggestions. DNA sequencing, N-terminal sequencing, and electrospray mass spectrometry services were provided by the Massachusetts Institute of Technology Biopolymers Laboratory. We acknowledge the Harvard Microsequencing Facility for amino acid analysis.
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FOOTNOTES |
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* This work was supported by NCI National Institutes of Health Grant CA34992 (to S. J. L.) and by grants from the National Institutes of Health and Howard Hughes Medical Institute (to D. R.).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.
§ National Science Foundation Predoctoral Fellow.
To whom correspondence should be addressed: Dept. of
Chemistry, Massachusetts Institute of Technology, Rm. 18-590, Cambridge, MA 02139. Tel.: 617-253-1892; Fax: 617-258-8150; E-mail:
lippard@lippard.mit.edu.
Published, JBC Papers in Press, May 8, 2001, DOI 10.1074/jbc.M101208200
2 S. S. Marla and S. J. Lippard, personal communication.
3 S. Oh and D. Reinberg, personal communication.
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ABBREVIATIONS |
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The abbreviations used are: FACT, Facilitates Chromatin Transcription; HMG, high mobility group; cisplatin and cis-DDP, cis-diamminedichloroplatinum(II); bp, base pair(s); trans-DDP, trans-diamminedichloroplatinum(II); nt, nucleotide(s); PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; cis127-bp, 127-bp probe damaged globally with cisplatin; trans-127-bp, 127-bp probe damaged globally with trans-DDP.
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