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J Biol Chem, Vol. 274, Issue 31, 22060-22064, July 30, 1999
From the Department of Radiation Oncology, Kimmel Cancer Center of
Jefferson Medical College, Thomas Jefferson University,
Philadelphia, Pennsylvania 19107
Exposure of mammalian cells to DNA
damage-inducing agents (DDIA) inhibits ongoing DNA replication. The
molecular mechanism of this inhibition remains to be elucidated. We
employed a simian virus 40 (SV40) based in vitro DNA
replication assay to study biochemical aspects of this inhibition. We
report here that the reduced DNA replication activity in extracts of
DDIA-treated cells is partly caused by a reduction in the amount of
replication protein A (RPA). We also report that the dominant
inhibitory effect is caused by the DNA-dependent protein
kinase (DNA-PK) which inactivates SV40 T antigen (TAg) by
phosphorylation. The results demonstrate that RPA and DNA-PK are
involved in the regulation of viral DNA replication after DNA damage
and suggest that analogous processes regulate cellular DNA replication
with the DNA-PK targeting the functional homologues of TAg.
Inhibition of DNA replication in eukaryotic cells is one of the
earliest effects of radiation to be reported. Camptothecin (CPT)1 and other DNA
damage-inducing agents (DDIA) exert similar inhibitory effects on DNA
replication in actively growing cells. It has been documented that a
major component of this inhibition derives from a delay in the
initiation of unreplicated origins (1, 2). Although this response was
first observed several decades ago, the underlying molecular mechanisms
remain largely unknown but ATM is thought to play an
important role. More recently, evidence has been presented suggesting
that the inhibition observed in DNA replication in response to DDIA is
equivalent to the activation of a checkpoint in the S-phase and that it
requires in yeast the products of MEC1 and RAD53
genes, homologues of ATM and Chk2, respectively (3-6). It is important
to establish the mechanisms of regulation of DNA replication after DNA
damage and the biochemical functions of the above genes in the process.
We investigated the regulation of DNA replication after DNA damage
using a simian virus 40 (SV40) based in vitro DNA
replication assay, and have shown that extracts of cells exposed to
DDIA have reduced activity for in vitro DNA replication and
inhibit, in a dominant fashion, the ability of extracts from nontreated
cells to promote in vitro DNA replication (7, 8).
In the SV40-based DNA replication assay, replication of plasmids
containing the SV40 origin of DNA (ori+ DNA) replication is
accomplished in vitro with either crude cytoplasmic extracts
or proteins purified from such extracts with SV40 T antigen (TAg) as
the only noncellular protein (9-13). It is believed that all cellular
proteins required in this assay function in a manner similar to that
in vivo (10-14). We have reported that the degree of
inhibition of DNA replication in cells exposed either to x-rays or CPT
is comparable with that measured in vitro using extracts prepared from cells exposed to these agents (7, 8). We subsequently focused on the molecular characterization of this inhibition. In the
present study, we provide evidence that following DNA damage two
processes contribute to the inhibition of in vitro DNA
replication: reduced availability of replication protein A (RPA) and
activation of DNA-dependent protein kinase (DNA-PK).
Cell Culture, X-ray Radiation, and CPT Treatment--
HeLa cell
culture, x-ray radiation, and CPT treatment are as described (7, 8).
MO59J cells were grown in Dulbecco's modified Eagle's medium-F-12
supplemented with 10% iron-supplemented calf serum (Hyclone).
Cytoplasmic Extract Preparation and Extract
Modification--
Procedures used for cytoplasmic extract preparation
either in large scale (109 cells) or in small scale
(107 cells) have been described previously (7, 8, 15). In some experiments, double-stranded DNA (dsDNA) fragments (Stratagene, sonicated salmon sperm DNA, length range from 0.5 to 2 kilobase pairs)
in TE buffer (10 mM Tris·Cl, pH 7.5, 1 mM
EDTA) were added in the cytoplasmic extracts at 30 °C for 10 min.
Wortmannin (Sigma) was added to the extracts (20 µM) at
30 °C for 10 min before assembling replication reactions. In some
experiments, purified recombined RPA (rRPA) or TAg was added to the
extracts at 30 °C for 10 min before assembling the reactions. RPA,
wild type or mutant forms, were prepared as described (8). Detection of
RPA was by Western blotting using standard procedures.
In Vitro Assay for DNA Replication--
The plasmid, pSV01 Single-stranded DNA (ssDNA) Binding--
A 25-mer DNA
oligonucleotide (5'-GATCCTCCTCACTACTTCTGGAATG-3' was end-labeled by T4
polynucleotide kinase using 5 pmol of [ DNA-PK Activity--
DNA-PK activity was assayed by using a
commercially available kit following the manufacture's protocols
(SignaTECT DNA-PK assay system, Promega).
TAg Phosphorylation by DNA-PK--
The reaction mixture (25 µl) containing 1 µg of purified TAg, 20 units of DNA-PK (Promega),
25 mM HEPES-KOH, pH 7.5, 12.5 mM
MgCl2, 20% glycerol, 0.1% Nonidet P-40, 1 mM
dithiothreitol, and 50 mM KCl, [ RPA, a ssDNA-binding protein, is a heterotrimeric complex with
subunits of 70, 34, and 14 kDa required in the initiation stages of
in vitro DNA replication and for multiple other processes of DNA metabolism (17-20). To test whether RPA plays a role in the inhibition of DNA replication in the in vitro DNA
replication assay, we measured, by immunoblotting, its abundance in the
extracts of cells treated with radiation or CPT. The amount of RPA p70, the subunit responsible for ssDNA binding, was reduced by approximately 50% (Fig. 1a,
top). In line with a reduction in RPA p70, the binding activity of RPA to ssDNA was also reduced by about 50% in the same
extracts (Fig. 1a, bottom, lanes 4 and
5). Post-translational modifications of RPA p34 have been
implicated in cell cycle regulation and S-phase checkpoint activation
(21, 22). We observed phosphorylation of RPA p34 (8), but could not
establish alterations in the level of this subunit after DNA damage,
probably due to the presence of a form not associated with the RPA
holoenzyme (23). A monoclonal anti-RPA p70 antibody slowed the mobility
of the DNA-protein complex in reactions assembled using cell extracts,
as well as in reactions assembled with rRPA (Fig. 1b,
lanes 2 and 4), confirming that the ssDNA binding
protein is RPA. The amount of RPA p70 in the whole cell lysates of
treated cells was similar to that of nontreated cells (data not shown).
Therefore, we hypothesized that RPA may bind to DNA damage sites after
exposure of cells to DDIA and be retained in the nucleus during extract
preparation, reducing thus its level in the cytoplasmic component. In
support of this hypothesis, addition of dsDNA fragments reduced RPA
binding to ssDNA (Fig. 1c). It is possible that competition
for RPA between damage sites and replication sites contributes to the
inhibition of DNA replication in DDIA-treated cells.
Although the amount of RPA was reduced in extracts from DDIA-treated
HeLa cells, addition of rRPA could not recover DNA replication activity
(Fig. 1d, compare lanes 7 and 8).
Also, a small amount of such extracts mixed with control extracts
strongly inhibited DNA replication (Fig. 1d, compare
lanes 1 and 6). We discovered that the inhibition
of DNA replication could be reversed when extracts of DDIA-treated HeLa
cells were preincubated with wortmannin, a nonspecific inhibitor of
protein kinases, and were supplemented with RPA (Fig. 1d,
lane 10). A similar reversion of DNA replication inhibition
was also observed when extracts of DDIA-treated cells were preincubated
with wortmannin and mixed with control extracts (Fig. 1d,
lane 11). Thus, the reduction in DNA replication activity observed in cytoplasmic extracts of cells exposed to ionizing radiation
or CPT is the combined result of a reduction in RPA activity and an
activation of a wortmannin-sensitive kinase. Other factors essential
for in vitro SV40 DNA replication remain practically unaffected in such extracts.
As mentioned above, dsDNA fragments could reduce RPA binding to ssDNA.
We examined whether addition of dsDNA fragments to extracts of
nontreated cells could simulate some of the characteristics of
inhibition observed in extracts of treated cells. The presence of dsDNA
fragments in the extracts of nontreated cells inhibited DNA replication
in a dose-dependent manner (Fig.
2a, HeLa), and the
inhibition reached a plateau at 50 ng (Fig. 2a,
HeLa). Addition of rRPA relieved somewhat the observed
inhibition, but the effect was relatively small (Fig. 2a,
HeLa, and Fig. 2b, lane 7).
Pretreatment of extracts with wortmannin did not affect the inhibition
caused by 50 ng of dsDNA (Fig. 2b, lane 6).
However, addition of rRPA to wortmannin-pretreated extracts completely
reversed DNA replication inhibition (Fig. 2b, lane
8). Thus, dsDNA fragments inhibit in vitro DNA
replication via a mechanism similar to that identified for extracts of
cells exposed to radiation or CPT, i.e. by sequestering RPA
and by activating a wortmannin-sensitive kinase. In subsequent experiments, dsDNA fragments were added to the extracts of untreated cells to mimic the DNA damage effect.
Roles of Replication Protein A and DNA-dependent
Protein Kinase in the Regulation of DNA Replication following DNA
Damage*
,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EP,
carrying the minimal origin of SV40 DNA replication (ori+)
was used as a template in the replication reactions. The reaction mixture was incubated at 37 °C for 1 h. Activity present in
acid-insoluble material was determined (6, 7). Replication products
were analyzed by agarose gel electrophoresis as described (7, 8).
-32P]ATP (Life
Technologies, Inc.). Extracts were prepared as described above. Binding
reactions were performed at 37 °C for 15 min with 30 fmol of
radiolabeled probe (2000 cpm/fmol) and 10 µg of total cell protein as
described (16). Bound and free probes were separated on 10% native
polyacrylamide gels in 0.25 × TBE buffer (45 mM Tris
borate, 1 mM EDTA). Gels were dried and exposed to
autoradiography film. A monoclonal antibody against RPA 70 subunit was
used during DNA binding reactions when indicated.
-32P]ATP
(3000 Ci/mmol, 10 µCi/µl, 0.05 µl/sample), 0.25 mM
cold ATP, and (when indicated) 0.6 µg of ori+ plasmid DNA
were incubated at 30 °C for 10 min. Then the reactions were either
mixed with the 2× electrophoresis sample loading buffer and
electrophoresed in 10% SDS-polyacrylamide gel electrophoresis for
autoradiography or supplemented with 12.5 µl of termination buffer
(7.5 M guanidine hydrochloride) for 32P
phosphorylation counting. The gels for the former samples were subsequently dried and exposed to autoradiography film. The latter samples were collected onto Whatman GF-C glass fiber filters, washed
with 10% trichloroacetic acid followed by repeated washing with
deionized water. 32P phosphorylation was determined by
liquid scintillation counting. The phosphorylation activity of TAg is
calculated in picomoles of ATP/minute/20 units of DNA-PK.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
View larger version (60K):
[in a new window]
Fig. 1.
RPA DNA binding activities and in
vitro DNA replication in cytoplasmic extracts of DDIA
treated cells. a: top, Western blot for RPA p70 level in
cytoplasmic extracts of HeLa cells (C, control;
R, irradiated, CPT, CPT-treated). Extracts
prepared as described under "Experimental Procedures."
a: bottom, RPA binding to ssDNA measured by a
gel-shift assay with the same extracts. rRPA was used as a positive
control. b, a monoclonal anti-RPA antibody against p70
supershifts the DNA-protein complex in HeLa cell extracts and in a
reaction assembled with rRPA. c, as in
a, but for reactions incubated in the presence of
different amounts of dsDNA fragments (0, 1, 5, 10, 20, 50, and 100 ng
in lanes 1, 2, 3, 4,
5, 6, and 7, respectively).
d, SV40 DNA replication reactions (25 µl) were assembled
using the indicated amount of extracts (S-100) from untreated,
irradiated (upper gel) or CPT-treated (lower gel)
cells. Shown are the effects of added rRPA (2 µg) and wortmannin (20 µM) on DNA replication activity. Results of reactions
assembled by mixing extracts of treated (R or
CPT) and nontreated cells are also shown. Reactions
assembled without TAg were included in all experiments and consistently
showed only background levels of DNA synthesis (about 1 pmol
incorporation; results not shown).
View larger version (31K):
[in a new window]
Fig. 2.
Effect of wortmannin and RPA on dsDNA-induced
inhibition of in vitro DNA replication in reactions
assembled with extracts of untreated cells. a, extracts of
HeLa ( 
, 
) or MO59J (
, 
) cells were preincubated with
different amounts of dsDNA fragments for 10 min at 30 °C and were
then added to the reaction mixture without (, ) or with 2 µg of
rRPA (, ) for in vitro DNA replication. The results
have been normalized to the replication activity measured in reactions
assembled in the absence of dsDNA fragments. b, the products
of in vitro DNA replication in reactions assembled with HeLa
cell extracts in the presence or absence of 50 ng of dsDNA fragments.
The effect of rRPA and wortmannin on DNA replication activity is
indicated.
The inhibition of DNA replication after DNA damage is thought of as an
active process that favors DNA repair. To identify the target of
wortmannin in the extracts from DDIA-treated cells, we tested DNA
replication activity in extracts of repair-deficient cell lines. MO59J
cells are deficient in dsDNA break rejoining and sensitive to ionizing
radiation-induced cell killing (24). We found that when MO59J cell
extracts were supplemented with DNA fragments, both RPA binding to
ssDNA (Fig. 1c, MO59J) and in vitro
DNA replication (Fig. 2a, MO59J) were reduced to
a degree similar to that observed in HeLa cell extracts. However,
contrary to the results with the extracts of HeLa cells, RPA alone
completely reversed this inhibition (Fig. 2a,
MO59J, and Fig.
3a). These results indicated
that the wortmannin-sensitive protein kinase was absent from MO59J cell
extracts.
|
It has been reported that MO59J cells lack any detectable DNA-PK activity (25) and have a low expression of ATM (26). It is well known that AT cells show reduced inhibition of DNA replication after DNA damage (27). At the concentration used (20 µM), wortmannin inhibits both DNA-PK and ATM kinase. To examine whether wortmannin acts in MO59J cell extracts by inhibiting ATM or by inhibiting DNA-PK, we prepared extracts from AT cells and performed experiments similar to those outlined with M059J cell extracts. Unexpectedly, restoration of DNA replication in AT cell extracts after addition of DNA fragments required both RPA and wortmannin, indicating that the inhibitory mechanism of in vitro DNA replication does not utilize ATM (data not shown). We measured, therefore, the DNA-PK activity in the extracts of DDIA-treated HeLa cells. The DNA-PK activity was higher in the extracts of treated HeLa cells than in control extracts when no activating dsDNA was added to the reactions (Fig. 3b, in control buffer). Importantly, addition of purified DNA-PK to M059J cell extracts supplemented with DNA fragments re-established the wortmannin requirement for the restoration of DNA replication activity (Fig. 3c). These results identify the wortmannin-sensitive kinase that inhibits DNA in vitro replication as DNA-PK.
DNA-PK (28, 29) is a serine/threonine kinase consisting of a 465-kDa
catalytic subunit (DNA-PKcs) and a heterodimeric regulatory complex
termed Ku. As targets of DNA-PK we considered two essential factors for
in vitro SV40 DNA replication: RPA and TAg. Both proteins
are known to be phosphorylated by DNA-PK and have been implicated in
regulatory steps of DNA replication (29, 30). To investigate whether
RPA or TAg contribute to the inhibition of DNA replication observed in
extracts of DDIA-treated HeLa cells, we separately preincubated these
proteins with a small amount of extract from treated HeLa cells to
allow for possible post-translational modifications. The mixture was
then used to assemble reactions with extracts of nontreated cells.
Wortmannin was added to these reactions after the end of the
preincubation period to neutralize the inhibitory effect of the treated
cell extract that accompanied RPA or TAg. The results indicated that
this preincubation did not alter RPA activity (data not shown), but
dramatically reduced TAg activity for DNA replication (Fig.
4a). To further confirm that
the modulation of RPA by DNA-PK has no effect on the DNA replication
activity, we used a mutant RPA with a deletion in the N-terminal domain
of RPA p34. The deletion removes DNA-PK phosphorylation sites without
affecting the activity of the enzyme for in vitro DNA
replication (31, 32). The effect of this mutant protein (data not
shown) on DNA replication was practically identical to the effect of
the wild type protein. This suggests that phosphorylation of RPA by
DNA-PK does not modify its activity in supporting DNA replication in
extracts of either treated or nontreated cells. Despite the fact that
in vitro observations suggest no role for phosphorylated RPA
in the regulation of DNA replication after DNA damage, the possibility
that this form of RPA plays a role in vivo should be left
open.
|
To confirm that the modification of TAg in extracts of treated HeLa
cells was mediated by DNA-PK, we preincubated TAg with purified DNA-PK.
The in vitro DNA replication activity decreased dramatically
in reactions assembled using TAg preincubated with DNA-PK, and this
inhibition could be completely reversed by nontreated TAg (Fig.
4b). Addition of [-32P]ATP during the
preincubation period confirmed phosphorylation of TAg by DNA-PK (Fig.
4c). Phosphorylation and inactivation of TAg for DNA
replication was inhibited by the presence of ori+ DNA in
the preincubation reaction (Fig. 4c), suggesting that binding of TAg to the replication origin reduced the phosphorylation of
TAg and thus its inactivation.
The results presented here provide evidence that following DNA damage two processes contribute to the inhibition of in vitro DNA replication: reduced availability of RPA and activation of DNA-PK. Similar processes may also be involved and partly regulate cellular or viral DNA replication in vivo after exposure of cells to DDIA. DNA-PK may inhibit DNA replication by phosphorylating functional homologues of TAg. Recruitment of RPA to DNA damage sites may contribute to this down-regulation. The latter pathway may explain the inhibition of DNA replication in irradiated MO59J cells, which is found to be similar to that observed in cell lines with normal DNA-PK activity (24). The results shown in Fig. 2a support this hypothesis.
There is strong evidence that severe combined immunodeficiency mice
have greatly reduced DNA-PK activity (33-35) and are as a result
hypersensitive to radiation and deficient in DNA double strand breaks
rejoining. Recently it has been reported that DNA-PK may play a role in
tumor suppression (36). Here, we report yet another function of DNA-PK:
the regulation DNA replication after DNA damage. Although our results
did not implicate ATM in the inhibition of in vitro DNA
replication following DNA damage, the evidence for an important role of
this kinase in the regulation of DNA replication in vivo is
overwhelming and suggests that multiple processes regulate DNA
replication after DNA damage. The in vitro assay allowed
us to study one specific aspect of this regulation.
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ACKNOWLEDGEMENTS |
|---|
We thank Drs. M. J. Allalunis-Turner for
MO59J cells, J. Hurwitz for pSV01EP plasmid and hybridoma cells
producing antibodies against RPA P34 or RPA P70, and M. Wold for
p11d-tRPA and ptRPA 25 321-33 constructs.
| |
FOOTNOTES |
|---|
* This work was supported by Grants CA76203 (to Y. W.), CA56706 (to G. I.), and P30-CA56036 and T32-CA09137 (to X. Y. Z) from the NCI, DHHS.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 and reprint requests should be addressed.
Tel.: 215-955-2045; Fax: 215-955-2052; E-mail:
ya.wang@mail.tju.edu.
| |
ABBREVIATIONS |
|---|
The abbreviations used are: CPT, camptothecin; DDIA, DNA damage-inducing agents; SV40, simian virus 40; RPA, replication protein A; DNA-PK, DNA-dependent protein kinase; ori+, SV40 origin of DNA; TAg, SV40 T antigen; rRPA, recombined RPA; ssDNA, single stranded DNA; dsDNA, double-stranded DNA; DNA-PKcs, DNA-PK catalytic subunit.
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Y. Shi, G. E. Dodson, S. Shaikh, K. Rundell, and R. S. Tibbetts Ataxia-telangiectasia-mutated (ATM) Is a T-antigen Kinase That Controls SV40 Viral Replication in Vivo J. Biol. Chem., December 2, 2005; 280(48): 40195 - 40200. [Abstract] [Full Text] [PDF]
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 M. Martin, A. Genesca, L. Latre, I. Jaco, G. E. Taccioli, J. Egozcue, M. A. Blasco, G. Iliakis, and L. Tusell Postreplicative Joining of DNA Double-Strand Breaks Causes Genomic Instability in DNA-PKcs-Deficient Mouse Embryonic Fibroblasts Cancer Res., November 15, 2005; 65(22): 10223 - 10232. [Abstract] [Full Text] [PDF]
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J. Park, T. Seo, H. Kim, and J. Choe Sumoylation of the Novel Protein hRIP{beta} Is Involved in Replication Protein A Deposition in PML Nuclear Bodies Mol. Cell. Biol., September 15, 2005; 25(18): 8202 - 8214. [Abstract] [Full Text] [PDF]
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S.-J. Park, S. L. M. Ciccone, B. Freie, A. Kurimasa, D. J. Chen, G. C. Li, D. W. Clapp, and S.-H. Lee A Positive Role for the Ku Complex in DNA Replication Following Strand Break Damage in Mammals J. Biol. Chem., February 13, 2004; 279(7): 6046 - 6055. [Abstract] [Full Text] [PDF]
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 W. D. Block, Y. Yu, and S. P. Lees-Miller Phosphatidyl inositol 3-kinase-like serine/threonine protein kinases (PIKKs) are required for DNA damage-induced phosphorylation of the 32 kDa subunit of replication protein A at threonine 21 Nucleic Acids Res., February 10, 2004; 32(3): 997 - 1005. [Abstract] [Full Text] [PDF]
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 A. Kashishian, H. Douangpanya, D. Clark, S. T. Schlachter, C. T. Eary, J. G. Schiro, H. Huang, L. E. Burgess, E. A. Kesicki, and J. Halbrook DNA-dependent protein kinase inhibitors as drug candidates for the treatment of cancer Mol. Cancer Ther., December 1, 2003; 2(12): 1257 - 1264. [Abstract] [Full Text] [PDF]
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H. Wang, J. Guan, H. Wang, A. R. Perrault, Y. Wang, and G. Iliakis Replication Protein A2 Phosphorylation after DNA Damage by the Coordinated Action of Ataxia Telangiectasia-Mutated and DNA-dependent Protein Kinase Cancer Res., December 1, 2001; 61(23): 8554 - 8563. [Abstract] [Full Text] [PDF]
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R. Ohki, T. Tsurimoto, and F. Ishikawa In Vitro Reconstitution of the End Replication Problem Mol. Cell. Biol., September 1, 2001; 21(17): 5753 - 5766. [Abstract] [Full Text] [PDF]
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K. B. Peters, H. Wang, J. M. Brown, and G. Iliakis Inhibition of DNA Replication by Tirapazamine Cancer Res., July 1, 2001; 61(14): 5425 - 5431. [Abstract] [Full Text] [PDF]
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G. S. Brush and T. J. Kelly Phosphorylation of the replication protein A large subunit in the Saccharomyces cerevisiae checkpoint response Nucleic Acids Res., October 1, 2000; 28(19): 3725 - 3732. [Abstract] [Full Text] [PDF]
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J. Guan, S. DiBiase, and G. Iliakis The catalytic subunit DNA-dependent protein kinase (DNA-PKcs) facilitates recovery from radiation-induced inhibition of DNA replication Nucleic Acids Res., March 1, 2000; 28(5): 1183 - 1192. [Abstract] [Full Text] [PDF]
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S. J. DiBiase, Z.-C. Zeng, R. Chen, T. Hyslop, W. J. Curran Jr., and G. Iliakis DNA-dependent Protein Kinase Stimulates an Independently Active, Nonhomologous, End-Joining Apparatus Cancer Res., March 1, 2000; 60(5): 1245 - 1253. [Abstract] [Full Text]
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J.-S. Liu, S.-R. Kuo, M. M. McHugh, T. A. Beerman, and T. Melendy Adozelesin Triggers DNA Damage Response Pathways and Arrests SV40 DNA Replication through Replication Protein A Inactivation J. Biol. Chem., January 14, 2000; 275(2): 1391 - 1397. [Abstract] [Full Text] [PDF]
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J.-S. Park, S.-J. Park, X. Peng, M. Wang, M.-A. Yu, and S.-H. Lee Involvement of DNA-dependent Protein Kinase in UV-induced Replication Arrest J. Biol. Chem., November 5, 1999; 274(45): 32520 - 32527. [Abstract] [Full Text] [PDF]
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Y. Wang, J. Guan, H. Wang, Y. Wang, D. Leeper, and G. Iliakis Regulation of DNA Replication after Heat Shock by Replication Protein A-Nucleolin Interactions J. Biol. Chem., June 1, 2001; 276(23): 20579 - 20588. [Abstract] [Full Text] [PDF]
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