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J Biol Chem, Vol. 274, Issue 29, 20521-20528, July 16, 1999
From the Life Sciences Division, Lawrence Berkeley National
Laboratory, University of California, Berkeley, California 94720
Genomic sequences with a cluster of ATC sequence
stretches where one strand consists exclusively of well mixed As, Ts,
and Cs confer high base unpairing propensity under negative
superhelical strain. Such base unpairing regions (BURs) are typically
found in scaffold or matrix attachment regions (SARs/MARs) that are thought to contribute to the formation of the loop domain structure of
chromatin. Several proteins, including cell type-specific proteins, have been identified that bind specifically to double-stranded BURs
either in vitro or in vivo. By using
BUR-affinity chromatography to isolate BUR-binding proteins from breast
cancer SK-BR-3 cells, we almost exclusively obtained a complex of
poly(ADP-ribose) polymerase (PARP) and DNA-dependent
protein kinase (DNA-PK). Both PARP and DNA-PK are activated by DNA
strand breaks and are implicated in DNA repair, recombination, DNA
replication, and transcription. In contrast to the previous notion that
PARP and Ku autoantigen, the DNA-binding subunit of DNA-PK, mainly bind
to free ends of DNA, here we show that both proteins individually bind
BURs with high affinity and specificity in an end-independent manner
using closed circular BUR-containing DNA substrates. We further
demonstrate that PARP and Ku autoantigen form a molecular complex
in vivo and in vitro in the absence of
DNA, and as a functional consequence, their affinity to the
BURs are synergistically enhanced. ADP-ribosylation of the nuclear
extract abrogated the BUR binding activity of this complex. These
results provide a mechanistic link toward understanding the functional
overlap of PARP and DNA-PK and suggest a novel role for these proteins
in the regulation of chromatin structure and function.
Specific regions of genomic DNA that exhibit high affinity to the
nuclear matrix in vitro have been identified from various eukaryotic species and are called scaffold/matrix attachment regions (SARs/MARs)1 (1). In recent
years, the biological significance of MARs is emerging (2). In
particular, studies on MARs flanking the immunoglobulin µ heavy chain
(IgH) enhancer showed that these sequences are essential for the B
lymphocyte-specific transcription of a rearranged µ gene (3). These
MARs have also been shown to be required for extending the domain of
chromatin that is accessible to transcription factors and also confer
factor access to enhancer-distal positions. The IgH MARs together with
the µ enhancer can also induce, in a transcription-independent
manner, extensive demethylation across the chromatin domain harboring
these elements (4). Additionally, MARs flanking the immunoglobulin Previously a protein with an apparent molecular mass of 114 kDa was
isolated from breast cancer cells as a BUR-binding protein. The BUR
binding activity of this protein, called p114, correlated with
progression of breast tumorigenesis (17). In this study, we used BUR
affinity chromatography and obtained, almost exclusively, a mixture of
PARP and DNA-PK from the breast cancer cell line, SK-BR-3. PARP has
been implicated in DNA repair in response to DNA damage (18), whereas
DNA-PK has been proposed to function in a variety of nuclear processes
including V(D)J recombination, double-stranded break (DSB) repair, DNA
replication, and transcription (19). PARP and Ku autoantigen, the
DNA-binding subunit of DNA-PK, are mainly known as proteins that bind
to single- and double-stranded DNA ends, respectively (18, 19).
However, we show that either PARP alone or the p70 and p86 subunits of
Ku autoantigen as a heterodimeric complex specifically bind with high
affinity to IgH MARs recognizing internal sequences. We also show that
PARP and the Ku70/86 directly interact and form a protein complex and, as a consequence, synergistically enhance their highly specific binding
to these MARs.
Cell Culture--
Human breast adenocarcinoma cell line SK-BR-3
(ATCC) was used for the studies described here. The cells were grown in
McCoy's 5A medium (Life Technologies Inc.) supplemented with 10%
fetal bovine serum (Tissue Culture Biologicals) and 1×
antibiotic/antimycotic solution (Life Technologies, Inc.).
BUR Affinity Chromatography--
BUR affinity columns were
prepared by coupling either a 24-bp mutated BUR-containing duplex
oligonucleotide (upper strand, 5'-TCTTTAATTTCTACTGCTTTAGAAttc-3') or a 25-bp wild type
BUR-containing duplex oligonucleotide (upper strand,
5'-TCTTTAATTTCTAATATATTTAGAAttc-3'), ligated to an average
length of 25, to freshly prepared CNBr-activated Sepharose CL-4B
(Amersham Pharmacia Biotech) at 200 µg/ml bed volume as described
(20). The differences between the wild type and mutated BUR-containing
oligonucleotides are underlined. The affinity matrices were
equilibrated with 20 mM Hepes, pH 7.9, 20% glycerol, 100 mM KCl, 0.2 mM EDTA, 0.5 mM
dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, and
0.1% Nonidet P-40. Nuclear extract from 5 × 108
SK-BR-3 cells was prepared as described previously (21) and is referred
to as the "0.4 M NaCl extract." This extract was
diluted with equilibration buffer to reduce the salt concentration to 0.2 M. The diluted extract (final volume 15 ml) was mixed
with 10 µg/ml sheared salmon sperm DNA and directly loaded onto a MUT MAR column (0.5 × 4 cm). The flow-through of the MUT MAR column was loaded onto WT MAR column (0.5 × 4 cm), washed first with equilibration buffer, and then the same buffer containing 0.3 M KCl. Bound proteins were then eluted in a stepwise manner
with buffers containing increasing amounts of KCl. Four 0.5-ml
fractions were collected for each concentration of KCl used. The
fractions containing most of the eluted protein (0.4-0.8
M) were combined, dialyzed against 20 mM Tris,
pH 7.4, 50 mM NaCl, 10% glycerol, 0.5 mM
dithiothreitol, and 0.1 mM phenylmethylsulfonyl fluoride, and loaded onto single-stranded DNA cellulose (Amersham Pharmacia Biotech) column (0.5 ml) equilibrated in same buffer. Bound proteins were eluted with a step gradient of NaCl up to 1 M.
Protein Analysis--
Proteins were estimated using QUANTIGOLD
reagent (Diversified Biotech) as described by the manufacturer.
SDS-polyacrylamide gel electrophoresis was performed using standard
procedures, and the proteins were visualized by staining with Silver
Stain Plus kit (Bio-Rad). The identity of BUR affinity column purified
p70 was obtained by analyzing the high pressure liquid
chromatography-purified peptides derived from the tryptic digest of
SDS-polyacrylamide gel-purified p70 using an Applied Biosystems 470 peptide sequencer. The N-terminal sequence of p70 tryptic peptides was
LYRETNEP and ELVYPPDYN, identical to amino acids 288-295 and 527-535
of human Ku-70, respectively (22).
Immunoblot Analysis--
SDS-polyacrylamide gel electrophoresis
and protein transfer to PVDF membranes (Millipore) was performed
essentially as described (17). Nonspecific sites on the membrane were
blocked by incubation with 5% bovine serum albumin (fraction V, Sigma)
in TST (20 mM Tris-Cl, pH 7.4, 0.5 M NaCl, and
0.05% Tween 20). The blots were then incubated with either anti-PARP
(1:2500 dilution of rabbit polyclonal antibody H-250 from Santa Cruz
Biotechnology Inc.) or anti-Ku70 and anti-Ku86 (1:750 dilution of
monoclonal antibody clones N3H10 and 111, respectively, from
NeoMarkers). The blots were then incubated with appropriate
peroxidase-conjugated secondary antibodies (1:10,000 dilution in TST)
and detected by using SuperSignal chemiluminescence kit (Pierce). The
quantitation of signals was performed by densitometric analysis of the
x-ray films using Molecular Dynamics Personal Densitometer SI and
ImageQuant (version 1.11) software.
ADP-ribosylation of Nuclear Extract--
In vitro
ADP-ribosylation of the nuclear extract was performed using
modification of a previously published procedure (23). Briefly, 5 ml of
nuclear extract containing 25 mg of protein from SK-BR-3 cells was
mixed with an equal volume of a buffer containing 200 mM
Tris-HCl, pH 8, 20 mM MgCl2, and 3 mM dithiothreitol. ADP-ribosylation was induced by the
addition of 10 µg of DNase I (Worthington)-activated salmon sperm DNA
(Sigma) and 10 µM NAD (Calbiochem) to one-half of the
extract. To the other half of the extract (control), only activated DNA
was added. Both batches of extracts were incubated for 5 min at
37 °C, followed by addition of 1 mM 3-aminobenzamide (Calbiochem) and chilling on ice. BUR affinity chromatography was
performed separately for the ribosylated and the control extract as
described above except that the elution was carried out in a single
step with 1 M KCl in equilibration buffer. Southwestern analysis was performed as described previously (20).
Immunoprecipitation--
Fifty µg of 0.4 M NaCl
nuclear extract from SK-BR-3 cells was diluted with an equal volume of
PBS containing 0.2% Nonidet P-40 and precleared with mouse IgG and
protein A/G plus beads (Pierce). Briefly, 1 µl of 0.1 mg/ml mouse IgG
1 and IgG 2a each (Sigma) were added to the extract and incubated at
4 °C for 1 h, followed by the addition of 10 µl of protein
A/G plus beads and incubation at 4 °C for 1 h on a rotating
shaker. The precleared extract was incubated with 2 µl of either
anti-PARP (clone CII-10, PharMingen) or anti-Ku (clone 162, NeoMarkers)
for 8 h at 4 °C, followed by addition of 10 µl of protein A/G
plus beads and further incubation at 4 °C for 8 h. The beads
were then washed four times with 500 µl of PBS each containing 0.1%
Nonidet P-40. The washed beads were resuspended in 20 µl of 2×
SDS-PAGE sample buffer and incubated at 95 °C for 5 min. Solubilized
immunoprecipitates were obtained by centrifugation and analyzed by
7.5% SDS-PAGE. Immunoprecipitates obtained from an equal amount of
nuclear extract were subjected to immunoblot analysis, and the entire
amount of immunoprecipitated protein was loaded on the gel to compare
the results. For GST pull-down assay, the NaCl concentration in the
reaction mixtures was adjusted to 0.2 M, and the
glutathione-Sepharose beads (Amersham Pharmacia Biotech) were washed
four times with 500 µl of PBS each, and the immunoprecipitates were
subjected to SDS-PAGE analysis as described above.
DNA Binding Analysis by EMSA--
Multimeric wild type and
mutant MAR DNA fragments were prepared as described (14). The various
subfragments of the µ enhancer were generated by digesting the 1-kb
Cµ region-containing plasmid (13) with HinfI and
XbaI. DNA binding was analyzed by electrophoretic mobility
shift assay (EMSA) using 0.5 ng of 32P-labeled end-filled
DNA as described (20). Minicircles were prepared by blunt end ligation
of these DNAs using 20 units/µl T4 DNA ligase (New England Biolabs)
followed by treatment with 5 units/µl exonuclease III (New England
Biolabs) to eliminate DNA molecules with free ends. The exonuclease
III-resistant minicircles were gel-purified and were also confirmed to
be resistant to S1 nuclease. Binding reactions with both linear and
circular DNAs contained 50-100-fold excess of
XmnI-ScaI fragment of pBluescript as competitor
DNA (17). After incubation at 25 °C for 15 min, the reaction
products were loaded on 6% native polyacrylamide gels and
electrophoresed at 150 V in 0.5× Tris borate/EDTA buffer and
visualized by autoradiography of the dried gel. Data were quantified by
densitometric analysis of the autoradiograms as described above.
Isolation of BUR-binding Proteins from SK-BR-3 Cells--
For
purification of BUR-binding proteins, we used a combination of mutated
and wild type BUR columns as described previously (17). For the
preparation of BUR affinity columns, we used a 25-bp sequence derived
from the MAR located 3' of the IgH enhancer containing the core
unwinding element. The mutated BUR column was prepared using a 24-bp
sequence derived from the same region as the 25-bp sequence except that
the core unwinding element was mutated (20). Multimerization of wild
type sequence, wild type (25)7 confers high affinity to the
nuclear matrix and high propensity for unwinding; therefore, it is a
BUR. On the other hand, a similarly multimerized mutated sequence,
mutated (24)8 does not possess either feature (2).
Nuclear extract from SK-BR-3 breast cancer cells was passed
successively through a non-MAR (mutated BUR) and a MAR (wild type BUR)
affinity column to select for BUR-specific binding proteins. A 114-kDa
MAR-binding protein from breast cancer cells specifically bound to a
double-stranded BUR affinity column (17) was identified as
poly(ADP-ribose) polymerase.3
This posed an apparent paradox because PARP has been known to bind in a
sequence-independent manner only to the ends and nicks in DNA (24).
Interestingly, we found that three other polypeptides of apparent
molecular masses of 460, 86, and 70 kDa almost exclusively copurified
with p114 from a BUR affinity column (Fig.
1A). Peptide microsequence
analysis of p70 revealed its identity with the p70 subunit of Ku
autoantigen (22). This result suggested that the other proteins that
copurified may represent individual polypeptide subunits of DNA-PK. We
next performed Western blot analysis of various fractions from either
wild type (WT) MAR or mutated (MUT) MAR affinity columns to compare the
binding of PARP and Ku and to verify the immunological identity of the
purified proteins. Since we used identical amounts (20 µl each) of
the column fractions for the immunoblot analysis, the amount of PARP
and/or Ku70/86 present in any given fraction could be directly
quantitated and compared by the corresponding signal on the
immunoblots. We typically observed that less than 10% of the input
amount of PARP and Ku70/86 was bound to the MUT MAR column as judged by
the amount of protein in the column flow-through (Fig. 1B,
compare lanes 1 and 2 in MUT MAR
panels), whereas virtually all of PARP and Ku70/86 were retained
by the WT MAR column as judged by the weak signals in the column
flow-through (Fig. 1B, lane 2 in WT MAR panels).
Both columns were then washed with equilibration buffer containing 0.1 M KCl to remove loosely bound proteins. Up to 30% of input PARP and Ku were removed at this step from both WT and MUT MAR columns
(Fig. 1B, lane 3 in respective panels). The
columns were then eluted in two steps of increasing concentration of
KCl to collect bound proteins. The bound PARP and Ku70/86 were almost quantitatively eluted from the WT MAR column with 0.3 M
KCl- and 1 M KCl-containing buffers (Fig. 1B, lanes
4 and 5, respectively, in WT MAR panels). In contrast,
extremely low amounts of PARP and Ku70/86 were left to be eluted from
the MUT MAR column under identical conditions (Fig. 1B, lanes
4 and 5 in MUT MAR panels). Quantification
of the immunoblot signals corresponding to 0.3 and 1 M KCl
eluates from both these columns revealed that the WT MAR column bound
at least 10-fold more of PARP and Ku as compared with that of the MUT
MAR column. These results suggest that both PARP and Ku70/86 exhibit
selective binding to the WT MAR column over the MUT MAR column. The
immunoblot analysis of the 1 M KCl eluate from the WT MAR
column also confirmed the immunological identity of PARP and both
subunits of the Ku autoantigen (Fig. 1B, lane 5 in WT
MAR panels). In addition, by using an antibody against the
catalytic subunit of DNA-PK, we confirmed the identity of p460 as the
catalytic subunit of DNA-PK (data not shown). All three of the proteins
that copurified were thus subunits of DNA-PK, suggesting that PARP
might be physically associated with DNA-PK by interacting with at least
one of its subunits in vivo. To test this hypothesis, we
attempted to separate the individual components of this putative
complex by employing ion exchange column chromatography. However, we
observed that PARP and Ku70/86 always copurified through a variety of
matrices such as DEAE-cellulose and phosphocellulose (data not shown).
This copurification was observed in the presence of up to 0.6 M KCl and 0.1% Nonidet P-40, the conditions that we
typically employed for elution of these proteins from the BUR affinity
matrix. Separation of these proteins to near-homogeneity was ultimately
achieved by taking advantage of the extremely high affinity of PARP to
single-stranded DNA (ssDNA). Both Ku70/86 and PARP bound the ssDNA
cellulose column. However, as depicted in Fig. 1C, p70 and
p86 subunits of Ku autoantigen co-eluted at 0.4 M NaCl
(Fig. 1C, lane 2), whereas PARP eluted at 0.7 M
NaCl (Fig. 1C, lane 3) from the ssDNA cellulose matrix.
ADP-ribosylation Abolishes the BUR Binding Activity of
PARP--
Upon autoribosylation, PARP has been shown to lose its DNA
end binding activity, and this feedback mechanism has been implicated in base excision repair (18, 24). We therefore tested the effect of
autoribosylation of PARP on its double-stranded BUR binding activity.
ADP-ribosylation was stimulated in nuclear extract of SK-BR-3 cells by
the addition of NAD and nicked DNA, and the ribosylated extract was
loaded successively onto mutated and wild type BUR affinity columns.
After washing the columns with equilibration buffer, bound proteins
were eluted in two steps using 0.3 M KCl- and 1 M KCl-containing buffer. Both PARP and Ku70/86 from the ADP-ribosylated extract bound neither the MUT MAR column nor the WT MAR
column and were quantitatively recovered in the flow-through of both
columns as revealed by immunoblot analysis (Fig.
2A, lanes 2 and 3,
respectively). Washing the columns with the equilibration buffer did
not yield either of the two proteins indicating loss of loose binding
(Fig. 2A, lanes 4 and 5). Elution with
0.3 M KCl- and 1 M KCl-containing buffers did
not elute any of the two proteins (Fig. 2A, lanes 6-9)
confirming the lack of binding of both Ku and PARP to either column.
Based on these data, we conclude that ADP-ribosylation of SK-BR-3
nuclear extract resulted in the loss of the BUR binding activity of
both PARP and Ku.
Next, we examined the effect of varying levels of PARP autoribosylation
upon its BUR binding activity by Southwestern analysis. Autoribosylation of column purified preparation of PARP was induced by
the addition of DNase I-activated salmon sperm DNA and various amounts
of unlabeled NAD. The migration of ADP-ribosylated PARP on denaturing
gels was retarded exponentially as a function of NAD concentration
(Fig. 2B, lanes 2-9). After resolving the ADP-ribosylated PARP by SDS-PAGE, the protein was electroblotted onto PVDF membrane and
renatured in situ. The membrane was then incubated with
end-labeled BUR-specific DNA probe (WT(25)7). As depicted
in Fig. 2C, PARP abruptly lost its BUR binding activity upon
ADP-ribosylation at concentrations of NAD exceeding 10 µM
(Fig. 2C, lanes 7-9). Intriguingly, this threshold value is
approximately 100-fold lower than the intracellular concentration of
NAD (25), and yet both endogenous Ku70/86 and PARP do bind to the BUR
column (Fig. 1A). These results suggest that in
vivo ADP-ribosylation of PARP may be tightly regulated to modulate
its BUR binding activity.
Physical Interaction between PARP and Ku Autoantigen in Vivo and in
Vitro--
The BUR affinity co-purification profile of PARP and Ku and
the loss of their binding capacity to the BUR affinity matrix upon
ADP-ribosylation of the nuclear extract strongly suggested that PARP
and Ku form a protein complex, either directly or mediated by DNA.
Direct physical association between Ku and PARP is unprecedented. To
first examine whether PARP and Ku interact physically in
vivo, we employed a coimmunoprecipitation strategy using SK-BR-3
nuclear extracts. Immunoprecipitates obtained from an equal amount of nuclear extract were subjected to Western blot analysis for the identification of interacting partner(s). Antibody clone CII-10, which
recognizes the N-terminal DNA binding domain of PARP, was able to
immunoprecipitate both Ku and PARP (Fig.
3A, lane 3). For
immunoprecipitating Ku autoantigen, we used an antibody (clone 162)
that recognizes the dimer interface of its p70 and p86 subunits (26).
This antibody not only immunoprecipitated Ku autoantigen, but also
pulled down PARP along with it (Fig. 3A, lane 4) indicating that Ku and PARP are indeed physically associated in vivo.
The coimmunoprecipitation signals for both PARP and Ku obtained with either antibody clone CII-10 or clone 162 were significantly greater than that with mouse IgG (Fig. 3A, lane 2). It could be
argued that the physical interaction between these proteins is merely an artifact due to their binding to the ends of the same DNA molecule. However, it is most likely not the case because proteins were extracted
from the nuclei of SK-BR-3 cells using a mild procedure to avoid
genomic DNA contamination. The extraction procedure involved isolation
of nuclei at low ionic strength followed by selective extraction of
proteins by incubating these nuclei in a buffer containing 0.42 M NaCl (21). The absence of trace amounts of genomic DNA
within this extract was confirmed by the total failure of
ADP-ribosylation by endogenous PARP which strictly requires DNA to
activate it. ADP-ribosylation in this extract was induced only after
exogenous DNA was added (data not shown).
To demonstrate unequivocally the physical association between Ku and
PARP in the absence of DNA and to verify whether these two proteins
interact in vitro, we incubated bacterially overexpressed and purified glutathione S-transferase (GST)-fused PARP
together with ssDNA cellulose column purified Ku70/86 and captured with glutathione-Sepharose beads. Recombinant PARP associated with Ku70/86
regardless whether purified native Ku70/86 (Fig. 3B, lane 3)
or crude 0.4 M nuclear extract (Fig. 3B, lane 2)
was used as a source of Ku autoantigen. We performed several controls
to ensure the validity of this interaction. First, proteins in the 0.4 M extract alone did not display any nonspecific binding to
the beads (Fig. 3B, lane 4). A parallel pull-down assay with
GST did not capture Ku70/86 (Fig. 3B, lane 5). Western blot
with anti-p110 Rb (Fig. 3B, lowermost panel)
served as an additional control indicating that the retinoblastoma
protein was not associated with GST-PARP or with glutathione-Sepharose
beads under identical conditions. Collectively, these experiments
demonstrate that the physical interaction between Ku autoantigen and
PARP occurs independently of DNA.
Preferential Binding of PARP and Ku to the 3' MAR within the IgH
Enhancer--
PARP has been known to bind solely to nicks and ends of
DNA (24), and in most cases Ku70/86 has been known to bind to free DNA
ends (19). Although recently Ku70/86 has been shown to bind to mouse
mammary tumor virus (MMTV)-negative regulatory element in a
sequence-dependent manner (27), PARP has not yet been shown to bind directly to double-stranded DNA in a sequence-specific manner.
To elaborate the apparent specificity of PARP and Ku70/86 for the BUR
DNA in the affinity matrix, we next performed electrophoretic mobility
shift assays (EMSA) with the purified proteins using well characterized
MARs surrounding the IgH enhancer region (3, 4) in which specific BURs
have been identified (13) (Fig. 4A). Interestingly, Ku-DNA and
PARP·DNA complexes were observed with both 5' (Fig. 4B, top
panel) and 3' (Fig. 4B, bottom panel) linear MAR
substrates but not with the enhancer under the concentrations indicated
(Fig. 4B, middle panel). It should be noted here that Ku70/86 did bind the enhancer fragment, only when its concentration was
raised to at least 20-fold, above the Kd value for the IgH 3' MAR, most probably reflecting end-related binding (data not
shown). Similar analysis using PARP at up to 5-fold of its Kd value for the IgH 3' MAR indicated complete lack
of binding to the enhancer fragment (data not shown), suggesting that
end binding by PARP must occur at much higher concentrations. Additionally, Ku70/86 was able to clearly distinguish wild type (25)2 from that of mutated (24)2 (data not
shown). Between the two IgH MARs, the 3' MAR appeared to be a better
substrate for both PARP and Ku70/86 with at least a 2-fold lower
dissociation constant (Kd) as compared with that of
the 5' MAR, consistent with the presence of a core unwinding element in
the 3' MAR (13). Surprisingly, the binding affinities of Ku70/86 to
these MARs were at least 15-fold higher (Kd of 0.05 and 0.2 nM for the 3' and 5' MAR, respectively) than those
of PARP (Kd of 1.8 and 3 nM for the 3'
and 5' MAR, respectively). Intriguingly, this observation is in direct
contrast with the BUR affinity elution profile of the two proteins
which indicated that PARP required up to 1 M KCl for its
complete elution from the matrix, whereas almost all of Ku was eluted
with 0.6 M KCl. We are currently investigating the
mechanisms underlying the binding of Ku 70/86 and PARP to BURs in
vivo.
Direct binding of Ku 70/86 and PARP to the primary BUR sequence was
confirmed by using covalently closed DNA minicircles as substrates. We
chose the 3' MAR for circularization since both Ku and PARP showed
higher affinity for this substrate in linear form. These circular
substrates were predigested with exonuclease III and S1 nucleases
to eliminate the possible contribution of any structural features
or nicks (data not shown). Interestingly, both Ku70/86 and PARP bound
the 300-bp circular IgH 3' MAR (Fig. 4C, upper panel) with
affinities similar to the corresponding linear substrate
(Kd values of 0.05 and 2 nM,
respectively). Up to three complexes could be identified, indicating
that these proteins were able to bind to one or more sites within this
substrate perhaps in a manner similar to that of the T cell
MAR/SAR-binding protein SATB1 (14). However, both Ku and PARP failed to
bind a minicircle comprising the core enhancer (Fig. 4C, lower
panel) as well as a control circle prepared by ligation of a
400-bp HinfI-XbaI fragment of pUC19 (data not
shown). Furthermore, both Ku and PARP also bound a circular
concatemeric BUR containing seven copies of the core unwinding element
of the Cµ 3'MAR (13), (WT (25)7), with high affinity
(Fig. 4C, lower panel). The binding reached saturation in a
protein concentration-dependent manner and occurred in the
presence of a 50-fold excess of linear DNA competitor. Up to seven
different complexes could be identified, indicating increasing
occupancy of the BURs within the minicircle. These data convincingly
demonstrate that both Ku70/86 and PARP recognize the primary sequence
of the BURs and not the ends of DNA.
Physical Association between PARP and Ku Promotes Their Synergistic
Binding to BURs--
We next examined the possible effects of physical
association between Ku and PARP on their BUR binding activity. We
performed EMSA analysis using 300 bp of linear IgH 3' MAR under
conditions wherein Ku70/86 and PARP were added simultaneously at
concentrations at which neither of them individually bound more than
50% of the DNA substrate (Fig. 5A,
lanes 2 and 4), and the possibility of end-mediated
binding is virtually eliminated. Contrary to the previous notion that
Ku and PARP would compete for their DNA substrates (28), we found that
at least with respect to MARs, they exhibit synergistic binding. When
added together, Ku70/86 and PARP bound more than 90% of the labeled
DNA probe (Fig. 5A, lane 5), indicating an increase of at
least 3-fold over the summation of their individual binding. Increasing
the amount of added Ku (Fig. 5A, lane 3) led to further
enhancement of binding activity (Fig. 5A, lane 6). It
appeared that at 1.8 nM, PARP itself did not bind very well to the MAR substrate (Fig. 5A, lane 4); however, its binding
activity was much enhanced by the addition of only as little as
0.05-0.1 nM Ku70/86 (Fig. 5A, lanes 5 and
6). A similar enhancement in binding was observed using the
circular IgH 3' MAR DNA template. However, since in the case of this
substrate it was difficult to score the intermediate protein-DNA
complexes, and it was difficult to measure the synergy of binding (data
not shown).
To test whether this increase in the binding activity was due to actual
physical association between Ku and PARP versus the proteins
independently occupying the binding sites in the substrate, we used a
truncated form of PARP that does not interact with
Ku.4 In a GST pull-down
assay, the recombinant DNA binding domain (DBD) of PARP fused to GST
captured a barely detectable amount of Ku (Fig. 5B, lane 2),
indicating that they do not form a complex. EMSA analysis indicated
that the recombinant DBD of PARP alone could bind to the MAR substrate
in a manner identical to that of native PARP (Fig. 5A,
compare lane 7 with 4). However, addition of the
same amounts of Ku that led to an increase in the binding activity of
native PARP did not significantly affect the binding activity of
truncated PARP (Fig. 5A, lanes 8 and 9),
suggesting that the synergistic effect that we observed using
full-length PARP and Ku reflects a specific effect and not a
nonspecific stabilization of binding. Taken together, these results
indicated that physical association between Ku and PARP is indeed
critical for their synergistic binding to MARs.
PARP is primarily involved in DNA repair (18), and DNA-PK has been
strongly implicated in double-strand break (DSB) repair and in V(D)J
recombination (19). These biological roles of PARP and DNA-PK are
linked to their common property to be activated by DNA breaks (Refs. 19
and 24 and reviewed in Ref. 29). Consistent with this, PARP has been
known to bind solely to ends and nicks of DNA (24). Although the Ku
autoantigen is mostly known to bind DNA ends, using a closed circular
DNA containing a negative regulatory element from the MMTV long
terminal repeat, Giffin et al. (27) have demonstrated
sequence-specific DNA binding by Ku70/86. In the present study, we
utilized BUR affinity chromatography, and we showed that PARP, Ku70/86,
and the catalytic subunit of DNA-PK were almost exclusively copurified.
PARP and DNA-PK together preferentially bound only the wild type BUR
affinity matrix and not the mutated BUR affinity matrix. In contrast to
the previous notion that PARP only binds to ends and nicks of DNA (24),
we show that PARP specifically binds with high affinity to the BURs surrounding the IgH enhancer using circular DNA substrates and does not
bind the non-BUR core enhancer. Similar binding specificity was also
observed for Ku70/86 using circular DNA substrate. Apparently, under
the experimental conditions employed in this study, the affinity of
either PARP or Ku70/86 to BURs is much higher than their affinity to
the free ends of the linear DNA.
Several lines of evidence implying DNA-PK and PARP interaction have
been reported previously. Studies utilizing PARP and DNA-PK-deficient cells have suggested a functional overlap between these two proteins to
mitigate genomic damage caused by DSBs (28). In a recent report,
Ruscetti et al. (31) demonstrated co-purification of DNA-PK,
PARP, and human heterogeneous nuclear ribonucleoprotein-U proteins by
employing antibody affinity chromatography using antibody against the
p86 subunit of Ku autoantigen. However, they argued that the
interaction among these proteins was mediated only by virtue of binding
to free ends of DNA in cis. Furthermore, they also
demonstrated that ADP-ribosylation of DNA-PK by PARP in
vitro stimulated its kinase activity, suggesting their functional
interaction in response to DNA damage. We have shown that Ku70/86 and
PARP directly interact to make a protein complex independently of DNA and synergistically enhance their binding to BURs. This result, to our
best knowledge, is the first demonstration of a direct physical
association between Ku and PARP, and their synergistic binding to DNA
recognizing specific internal DNA sequences.
The DNA binding domains of PARP and Ku differ significantly as follows:
that of PARP consists of two zinc fingers and resides in the N-terminal
portion of the same 114-kDa polypeptide that harbors the catalytic site
(24), whereas the 70- and 86-kDa Ku subunits, which are distinct
polypeptides, together constitute the DNA-binding partner of the
460-kDa catalytic subunit of DNA-PK (19). The DBD of Ku autoantigen
consists of two separate domains each residing at either terminus of
its p70 subunit. The N-terminal DBD of p70 binds DNA only upon
dimerization with p86, whereas the C-terminal DBD is p86-independent
(30). Results of our Southwestern analysis to monitor the BUR binding
activity of individual Ku subunits indicated that Ku70 or Ku86 alone do
not bind BURs (data not shown), whereas PARP (a 114-kDa homodimeric
protein) does exhibit BUR binding activity (this study). The BUR
binding activity of Ku70/86 therefore must be dependent on
heterodimerization of p70 and p86. Despite the obvious differences in
their DNA binding domains, PARP and Ku70/86 remarkably seem to bind to
similar targets. It is noteworthy, however, that in the in
vitro binding experiments, we have observed that the binding
affinity of Ku70/86 for BURs is at least 10-fold higher than that of
PARP. Surprisingly, the BUR affinity profile indicated that PARP is
bound much more strongly than Ku70/86. Since extremely low amounts of
Ku70/86 are sufficient to increase the binding affinity of PARP
in vitro, it is conceivable that Ku70/86 may assist PARP in
binding to BURs; however, once PARP is bound it does not dissociate
easily from BURs. The effects of post-translational modification(s) of
PARP other than ADP-ribosylation on its BUR binding activity need to be examined.
The apparent specificity of Ku70/86 and PARP for the BUR sequences and
their synergistic binding to these regions may have important
biological implications. PARP has been found within the DNA replication
(32, 33), repair (34), transcription (35), and recombination (36)
complexes. Accumulating evidence has shown that the eukaryotic
replication machinery is concentrated at discrete foci within the
nucleus and are attached to a diffuse nucleoskeleton (37). These foci
often colocalize with the foci of active transcription (38). In
addition, a role for PARP in aging has also been suggested. PARP
associates with p53 protein in vivo, and PARP activation
leads to accelerated loss of telomeres, activation of p53, and
premature senescence (39). Ku70/86 has been shown to be involved in
regulation of glucocorticoid-induced MMTV transcription (27) and has
also been demonstrated to be essential for telomere maintenance in
yeast (40-42). Additionally, Ku70/86 has been shown to be one of the
core factors that bind to a highly conserved AT-rich motif within the
BCL2 major breakpoint region (43), which has also been shown
to be recognized by SATB1.5 Our finding
that PARP and Ku70/86 proteins form a complex suggest that
PARP·DNA-PK complex may be commonly found to be involved in some or
all of the functions mentioned above. At least some of the seminal
roles of PARP and Ku70/86 may be mediated by their association with
BURs, which are the key structural elements of MARs. We speculate that
the PARP·DNA-PK complex may bridge MARs with these multiprotein machineries.
It is likely that the PARP·DNA-PK complex has a role in chromatin
structure. ADP-ribosylation of histones by PARP at the site of DNA
damage has been shown to cause relaxation of the local chromatin
superstructure (44). Also, a recent study has indicated that Ku is
involved in chromosome condensation during G2 and M phases
of the cell cycle (45). Recent studies on factors that are involved in
chromatin assembly and remodeling have suggested their role in the
regulation of cell proliferation (46). Thus, studies on the association
between Ku and PARP and their high affinity binding to BURs may provide
novel insights into some aspects of the mechanistic link between
chromatin structure and various other functions involving DNA.
We thank Drs. Judith Campisi for critical
reading of this manuscript, Mark Smulson for kindly providing human
PARP cDNA construct, and Guy Poirier for the kind gift of
polyclonal anti-PARP antibody that was used in the initial stages of
this work.
*
This work was supported by Grant BCRP 1RB-0381A (to
T. K.-S.) from the Breast Cancer Research Program, University of
California, Sankyo Co. Ltd., Japan, and the United States Department of
Energy Contract DE-A C03-76SF00098 (to T. K.-S.).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: Life Sciences Division,
Lawrence Berkeley National Laboratory, University of California, 1, Cyclotron Rd., Mail Stop 70A-1118, Berkeley, CA 94720.
2
J. D. Alvarez, D. H. Yasui, H. Niida,
T. Joh, D. Y. Loh, and T. Kohwi-Shigematsu, submitted for publication.
3
S. Galande, J. Yanagisawa, C. Lee, Y. Kohwi, and
T. Kohwi-Shigematsu, manuscript in preparation.
4
S. Galande, T. Nishimura, and T. Kohwi-Shigematsu, unpublished observations.
5
M. Ramakrishman, P. A. DiCroce, A. Posner, J. Zehng., T. Kohwi-Shigematsu, and T. Krontiris, submitted for publication.
The abbreviations used are:
SARs/MARs, scaffold/matrix attachment sequences;
IgH, immunoglobulin heavy chain;
BURs, base unpairing regions;
PARP, poly(ADP-ribose) polymerase;
DNA-PK, DNA-dependent protein kinase;
DSBs, double-strand
breaks;
PBS, phosphate-buffered saline;
PAGE, polyacrylamide gel
electrophoresis;
bp, base pairs;
EMSA, electrophoretic mobility shift
assays;
ssDNA, single-stranded DNA;
WT, wild type;
MUT, mutated;
GST, glutathione S-transferase;
DBD, DNA binding domain;
MMTV, murine mammary tumor virus;
PVDF, polyvinylidene difluoride.
Poly(ADP-ribose) Polymerase and Ku Autoantigen Form a Complex and
Synergistically Bind to Matrix Attachment Sequences*
and
ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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enhancer have been shown to be necessary for B cell-specific
demethylation of the locus (5). Several lines of evidence have
demonstrated that various genomic segments containing MARs are often
found at the boundaries of transcription units and near regulatory
elements such as enhancers (see Refs. 6-8, reviewed in Ref. 9),
suggesting that the loop domains of chromatin formed by the attachment
of MARs to nuclear matrix may define units of genetic function
(reviewed in Ref. 1). Additionally, recent studies have linked MARs to
replication as certain MARs colocalize with origins of replication
(10), and the choice of the replication origin within the amplified dihydrofolate reductase domain may be dictated by the alteration in the
local chromatin structure mediated by attachment of DNA to the nuclear
matrix (11). The yeast H4 autonomous replication sequence contains a
DNA sequence element with high unwinding potential and is located
adjacent to the core replication sequence. This unwinding element is
essential for initiation of replication at the H4 origin (12). In
various MARs, there exist base unpairing regions (BURs) of less than
150 bp that readily unwind by continuous base unpairing under negative
torsional stress (2, 13). Mutating the core unwinding element so as to
abolish its unwinding ability greatly reduces binding to the nuclear
matrix (2). BURs are binding targets of cell type-specific proteins
such as the T cell factor SATB1 (14) and the B cell factor Bright (15).
The T cell factor SATB1 binds in vivo to specialized genomic
sequences located at the bases of chromatin loop domains which are
tightly anchored onto the nuclear matrix (16) and is essential for
proper T cell development.2
These results demonstrate the significance of BURs and BUR-binding proteins in gene regulation.
EXPERIMENTAL PROCEDURES
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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View larger version (52K):
[in a new window]
Fig. 1.
Isolation of BUR-binding proteins from
SK-BR-3 nuclear extract. A, elution profile of PARP and
DNA-PK from the BUR affinity column. Nuclear extract from SK-BR-3 cells
was passed successively onto mutated and wild type BUR affinity
columns, and the bound proteins were eluted as described under
"Experimental Procedures." Twenty µl of individual fractions from
the column were resolved by 7.5% SDS-PAGE, and the proteins were
visualized by silver staining of the gel. MUT, mutated BUR;
WT, wild type BUR; FT, flow-through. Molecular
mass markers in kDa are indicated on the left, and the
concentration of KCl in the elution buffer is indicated on the
top. B, immunoblot analysis of MAR affinity
column fractions. Twenty µl of each of the indicated column fractions
were resolved by 7.5% SDS-PAGE analysis and subjected to immunoblot
analysis as described under "Experimental Procedures." Lane
1, column input; lane 2, column flow-through;
lane 3, 0.1 M KCl eluate; lane 4, 0.3 M KCl eluate; lane 5, 1 M KCl
eluate. The column from which fractions were obtained and the antibody
used for immunostaining are indicated on the left and
right side of each panel, respectively. C,
silver-stained 4-15% gradient SDS-polyacrylamide gel (Bio-Rad) of
ssDNA cellulose column fractions. Proteins eluted from the WT BUR
affinity matrix were combined and loaded onto ssDNA cellulose column
and further purified as described under "Experimental Procedures."
Lane 1, molecular mass markers in kDa as indicated on the
left; lane 2, 0.4 M NaCl eluate
containing 120 ng of Ku70/86; lane 3, 0.7 M NaCl
eluate containing 100 ng of PARP.
View larger version (32K):
[in a new window]
Fig. 2.
ADP-ribosylation abolishes the BUR binding
activity of PARP. A, immunoblot analysis of MAR
affinity column fractions with ADP-ribosylation. ADP-ribosylation
reaction was performed in the presence or absence of 10 µM NAD in the nuclear extract which was then subjected to
BUR affinity chromatography as described under "Experimental
Procedures." Usage of antibodies is indicated on the right
side of each panel. Lane 1, MUT MAR affinity column
input; lane 2, MUT MAR affinity column flow-through;
lane 3, WT MAR affinity column flow-through; lane
4, MUT MAR affinity column 0.1 M KCl eluate;
lane 5, WT MAR affinity column 0.1 M KCl eluate;
lane 6, MUT MAR affinity column 0.3 M KCl
eluate; lane 7, WT MAR affinity column 0.3 M KCl
eluate; lane 8, MUT MAR affinity column 1 M KCl
eluate; lane 9, WT MAR affinity column 1 M KCl
eluate. B, autoradiogram displaying in vitro
ADP-ribosylation of PARP. Purified PARP was incubated without
(lane 1) or with indicated amounts of NAD (lanes
2-9) in the presence of 10 µg per ml activated salmon sperm
DNA. Ten nM [32P]NAD was added to each
reaction for autoradiographic detection of ADP-ribosylated PARP. The
reaction mixtures were electrophoresed on 10% SDS-polyacrylamide gels,
and the dried gel was exposed to x-ray film at 
80 °C. Molecular
mass markers are indicated in kDa to the left. C,
Southwestern analysis of in vitro ADP-ribosylated PARP.
Reactions were carried out as described in B except that the
addition of [32P]NAD is omitted.
32P-End-labeled wild type synthetic MAR sequence
(WT(25)7) was used as probe.
View larger version (28K):
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Fig. 3.
Physical association of Ku autoantigen and
PARP in vivo and in vitro.
A, Western blot analysis of immunoprecipitates from nuclear
extracts. Immunoprecipitates obtained after incubating the precleared
nuclear extract from SK-BR-3 cells with appropriate antibody and
protein A/G plus beads were dissolved in SDS-PAGE sample buffer and
electrophoresed on 7.5% SDS-polyacrylamide gels. The resolved proteins
were subsequently electroblotted onto PVDF membrane and stained with
appropriate antibody as described (14). Lane 1, 0.4 M nuclear extract from SK-BR-3 cells; lane 2,
mouse IgG control immunoprecipitate; lane 3, anti-PARP
(clone CII-10) immunoprecipitate; lane 4, anti-Ku70/86
(clone 162) immunoprecipitate. The upper panel is stained
with anti-PARP (H-250) and the lower panel is stained with
anti Ku-86 (clone 111) plus anti-Ku 70 (clone N3H10). B,
Western blot analysis following GST pull-down assay. GST-PARP was used
as a bait to form complex with Ku70/86 supplied in either purified form
or as in nuclear extract. The complexes were recovered by low speed
centrifugation and processed as described under "Experimental
Procedures." Lane 1, GST-PARP incubated with
glutathione-Sepharose beads (beads); lane 2, GST-PARP
incubated with 0.4 M nuclear extract and beads; lane
3, GST-PARP incubated with purified Ku and beads; lane
4, 0.4 M extract incubated with beads; lane
5, GST incubated with 0.4 M nuclear extract and beads;
lane 6, 0.4 M extract alone as an internal
marker to indicate the position of each protein on the immunoblot. The
membrane was first stained with anti-PARP (top panel),
successively stripped, and reprobed with anti-Ku (middle
panel) and anti-p110 Rb (bottom panel).
View larger version (45K):
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Fig. 4.
Primary sequence recognition by Ku
autoantigen and PARP. A, schematic representation of
the region flanking Cµ intronic IgH enhancer. Shaded area
represents the region that is stably unpaired under superhelical stress
and at Na+ concentrations of 50 mM or higher
(13). BURs are indicated by a line on top of the
horizontal bar (13). Solid boxes within BURs
represent SATB1-binding sites (14). Double-headed arrows
demarcate positions of the MAR and enhancer elements. C, the
binding of native Ku70/86 and PARP to the three sequence elements
within the Cµ intronic enhancer region was independently analyzed by
EMSA as described under "Experimental Procedures." The
panels to the left depict EMSA using increasing
amounts of Ku70/86, and the panels to the right
depict EMSA using increasing amounts of PARP. The linear probes used
were as follows: top panel, 350-bp 5' MAR; middle
panel, 200-bp enhancer; bottom panel, 300-bp 3' MAR.
C, EMSA of Ku70/86 and PARP binding to minicircles.
32P-End-labeled DNA fragments were circularized by blunt
end ligation, and the binding of column purified native Ku70/86 and
PARP was analyzed by EMSA as described under "Experimental
Procedures." Top panel, circular IgH 3' MAR; middle
panel, circular IgH enhancer; bottom panel, WT
(25)7. Concentrations of Ku70/86 and PARP used (in
nM) are indicated on top.
View larger version (40K):
[in a new window]
Fig. 5.
Synergistic binding of Ku autoantigen and
native PARP to linear Cµ 3' MAR.
A, EMSA analysis. Column purified native Ku70/86 and PARP
were either individually or together incubated with
32P-labeled IgH 3' MAR fragment, and the complexes were
resolved by 6% native polyacrylamide gel electrophoresis. Lane
1, control; lanes 2 and 3, Ku70/86 alone;
lane 4, PARP alone; lanes 4 and 5,
Ku70/86 plus PARP; lane 6, recombinant DBD of PARP
(GST-PARP-DBD) alone; lanes 8 and 9;
Ku70/86 plus GST-PARP-DBD. Concentrations of proteins added to each
reaction are indicated on top. B, GST pull-down
assay. GST-PARP-DBD was incubated with glutathione-Sepharose beads
(Beads) alone (lane 1) or with Ku and the beads
(lane 2). As a control, Ku70/86 was incubated alone with the
beads (lane 3). The complexes were recovered by low speed
centrifugation and processed as described under "Experimental
Procedures." The Western blot was stained with polyclonal
anti-GST-PARP (top panel) and then stripped and reprobed
with antibodies against both p70 and p86 subunits of Ku (bottom
panel). Lane 4 (bottom panel only) depicts
the position of Ku subunits.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
Recipient of BCRP Postdoctoral Fellowship 3FB-0053.
ABBREVIATIONS
REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
Gasser, S. M.,
and Laemmli, U. K.
(1987)
Trends Genet.
3,
16-22
2.
Bode, J.,
Kohwi, Y.,
Dickinson, L.,
Joh, T.,
Klehr, D.,
Mielke, C.,
and Kohwi-Shigematsu, T.
(1992)
Science
255,
195-197 3.
Forrester, W. C.,
van Genderen, C.,
Jenuwein, T.,
and Grosschedl, R.
(1994)
Science
265,
1221-1225 4.
Jenuwein, T.,
Forrester, W. C.,
Fernández-Herrero, L. A.,
Laible, G.,
Dull, M.,
and Grosschedl, R.
(1997)
Nature
380,
269-272[CrossRef]
5.
Kirillov, A.,
Kistler, B.,
Mostoslasky, R.,
Cedar, H.,
Wirth, T.,
and Bergman, Y.
(1997)
Nat. Genet.
13,
435-441
6.
Cockerill, P. N.,
and Garrard, W. T.
(1986)
Cell
44,
273-282[CrossRef][Medline]
[Order article via Infotrieve]
7.
Cockerill, P. N.,
Yuen, M.-H.,
and Garrard, W. T.
(1987)
J. Biol. Chem.
262,
5394-5397 8.
Mirkovitch, J.,
Mirault, M.-E.,
and Laemmli, U. K.
(1984)
Cell
9,
223-232
9.
Nelson, W. G.,
Pienta, K. J.,
Barrack, E. R.,
and Coffey, D. S.
(1986)
Annu. Rev. Biophys. Chem.
15,
457-475[CrossRef][Medline]
[Order article via Infotrieve]
10.
Largarkova, M. A.,
Svetlova, E.,
Giacca, M.,
Falaschi, A.,
and Razin, S. V.
(1998)
J. Cell. Biochem.
69,
13-18[CrossRef][Medline]
[Order article via Infotrieve]
11.
Pemov, A.,
Bavykin, S.,
and Hamlin, J. L.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
14757-14762 12.
Umek, R. M.,
and Kowalski, D.
(1988)
Cell
52,
559-567[CrossRef][Medline]
[Order article via Infotrieve]
13.
Kohwi-Shigematsu, T.,
and Kohwi, Y.
(1990)
Biochemistry
29,
9551-9560[CrossRef][Medline]
[Order article via Infotrieve]
14.
Dickinson, L. A.,
Joh, T.,
Kohwi, Y.,
and Kohwi-Shigematsu, T.
(1992)
Cell
70,
631-645[CrossRef][Medline]
[Order article via Infotrieve]
15.
Herrscher, R. F.,
Kaplan, M. H.,
Lelsz, D. L.,
Das, C.,
Sceurmann, R.,
and Tucker, P. W.
(1995)
Genes Dev.
9,
3067-3082 16.
de Belle, I.,
Cai, S.,
and Kohwi-Shigematsu, T.
(1998)
J. Cell Biol.
141,
335-348 17.
Yanagisawa, J.,
Ando, J.,
Nakayama, J.,
Kohwi, Y.,
and Kohwi-Shigematsu, T.
(1996)
Cancer Res.
56,
457-462 18.
Satoh, M. S.,
and Lindahl, T.
(1992)
Nature
356,
356-358[CrossRef][Medline]
[Order article via Infotrieve]
19.
Jackson, S. P.,
and Jeggo, P. A.
(1995)
Trends Biochem. Sci.
20,
412-415[CrossRef][Medline]
[Order article via Infotrieve]
20.
Kohwi-Shigematsu, T.,
deBelle, I.,
Dickinson, L. A.,
Galande, S.,
and Kohwi, Y.
(1998)
Methods Cell Biol.
53,
323-354[Medline]
[Order article via Infotrieve]
21.
Dignam, J. D.,
Lebovitz, R. M.,
and Roeder, R. G.
(1983)
Nucleic Acids Res.
11,
1475-1489 22.
Reeves, W. H.,
and Sthoeger, Z. M.
(1989)
J. Biol. Chem.
264,
5047-5052 23.
Shah, G. M.,
Poirier, D.,
Duchaine, C.,
Brochu, G.,
Desnoyers, S.,
Lagueux, J.,
Verreault, A.,
Hoflack, J. C.,
Kirkland, J. B.,
and Poirier, G. G.
(1995)
Anal. Biochem.
227,
1-13[CrossRef][Medline]
[Order article via Infotrieve]
24.
de Murcia, G.,
and Menessier-de Murcia, J.
(1994)
Trends Biochem. Sci
19,
172-176[CrossRef][Medline]
[Order article via Infotrieve]
25.
Wright, S. C.,
Wei, Q, S.,
Kinder, D. H.,
and Larrick, J. W.
(1996)
J. Exp. Med.
183,
463-471 26.
Wang, J.,
Satoh, M.,
Pierini, A.,
Schmitt, J.,
Chou, C. H.,
Stunnenberg, H. G.,
Roeder, R. G.,
and Reeves, W. H.
(1994)
J. Cell Sci.
107,
3223-3333[Abstract]
27.
Giffin, W.,
Torrance, H.,
Rodda, D. J.,
Préfontaine, G. G.,
Pope, L.,
and Haché, R. J. G.
(1996)
Nature
380,
265-268[CrossRef][Medline]
[Order article via Infotrieve]
28.
Morrison, C.,
Smith, G. C. M.,
Stingl, L.,
Jackson, S. P.,
Wagner, E. F.,
and Wang, Z.-Q.
(1997)
Nat. Genet.
17,
479-482[CrossRef][Medline]
[Order article via Infotrieve]
29.
Jackson, S. P.
(1996)
Curr. Opin. Genet. & Dev.
6,
19-25[CrossRef][Medline]
[Order article via Infotrieve]
30.
Wang, J.,
Dong, X.,
and Reeves, W. H.
(1998)
J. Biol. Chem.
273,
31068-31074 31.
Ruscetti, T.,
Lehnert, B. E.,
Halbrook, J.,
Trong, H. L.,
Hoekstra, M. F.,
Chen, D. J.,
and Peterson, S. R.
(1998)
J. Biol. Chem.
273,
14461-14467 32.
Simbulan-Rosenthal, C. M.,
Rosenthal, D. S.,
Hilz, H.,
Hickey, R.,
Malkas, L.,
Applegren, N.,
Wu, Y.,
Bers, G.,
and Smulson, M. E.
(1996)
Biochemistry
35,
11622-11633[CrossRef][Medline]
[Order article via Infotrieve]
33.
Dantzer, F.,
Nasheuer, H.-P.,
Vonesch, J.-L.,
de Murcia, G.,
and Ménissier-de Murcia, J.
(1998)
Nucleic Acids Res.
26,
1891-1898 34.
Masson, M.,
Niedergang, C.,
Schreiber, V.,
Muller, S.,
Ménissier-de Murcia, J.,
and de Murcia, G.
(1998)
Mol. Cell. Biol.
18,
3563-3571 35.
Meisterernst, M.,
Stelzer, G.,
and Roeder, R. G.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
2261-2265 36.
Borgreffe, T.,
Wabl, M.,
Akhmedov, A.,
and Jessberger, R.
(1998)
J. Biol. Chem.
273,
17025-17035 37.
Hozák, P.,
Jackson, D. A.,
and Cook, P. R.
(1994)
J. Cell Sci.
107,
2191-2202[Abstract]
38.
Hassan, A. B.,
Errington, R. J.,
White, N. S.,
Jackson, D. A.,
and Cook, P. R.
(1994)
J. Cell Sci.
107,
425-434[Abstract]
39.
Vaziri, H.,
West, M. D.,
Allsopp, R. C.,
Davison, T. S.,
Wu, Y.-S.,
Arrowsmith, C. H.,
Poirier, G. G.,
and Benchimol, S.
(1997)
EMBO J.
16,
6018-6033[CrossRef][Medline]
[Order article via Infotrieve]
40.
Gravel, S.,
Larrivée, M.,
Labrecque, P.,
and Wellinger, R. J.
(1998)
Science
280,
741-744 41.
Laroche, T.,
Martin, S. G.,
Gotta, M.,
Gorham, H. C.,
Pryde, F. E.,
Louis, E. J.,
and Gasser, S. M.
(1988)
Curr. Biol.
8,
653-656
42.
Nugent, C. I.,
Bosco, G.,
Ross, L. O.,
Evans, S. R.,
Salinger, A. P.,
Moore, J. K.,
Haber, J. E.,
and Lundblad, V.
(1998)
Curr. Biol.
8,
657-660[CrossRef][Medline]
[Order article via Infotrieve]
43.
DiCroce, P. A.,
and Krontiris, T. G.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
10137-10141 44.
de Murcia, G.,
Huletsky, A.,
Lamarre, D.,
Gaudreau, A.,
Pouyet, J.,
Daune, M.,
and Poirier, G. G.
(1986)
J. Biol. Chem.
261,
7011-7017 45.
Muñoz, P.,
Zdzienicka, M. Z.,
Blanchard, J.-M.,
and Piette, J.
(1998)
Mol. Cell. Biol.
18,
5797-5808 46.
Roth, S. Y.,
and Allis, C. D.
(1996)
Cell
87,
5-8[CrossRef][Medline]
[Order article via Infotrieve]
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|
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|
|
|
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|
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|
 |
|
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|
|
|
|
|
|
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|
|
|
|
 |
|
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|
|
|
|
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|
|
|
|
|
|
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|
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|
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|
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|
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