From the Department of Molecular Medicine and Institute of Biotechnology, The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78245
Received for publication, January 2, 2003, and in revised form, January 21, 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Signaling pathways in response to DNA double
strand breaks involve molecular cascades consisting of sensors,
transducers, and effector proteins that activate cell cycle checkpoints
and recruit repair machinery proteins. NFBD1 (a nuclear
factor with BRCT domains protein 1)
contains FHA
(forkhead-associated), BRCT (breast cancer susceptibility gene 1 carboxyl
terminus) domains, and internal repeats and is an early
participant in nuclear foci in response to IR. To elucidate its role in
the response pathways, small interfering RNA (siRNA) directed against
NFDB1 in human cells demonstrated that its absence is associated with
increased radio-sensitivity and delayed G2/M
transition, but not G1 to S. NFBD1 associates with nuclear
foci within minutes following IR, a property similar to histone H2AX,
53BP1, and Chk2, which are all early participants in the DNA damage
signaling cascade. Temporal studies show that H2AX is required for the
foci positive for NFBD1, but NFBD1 is not needed for 53BP1- and
H2AX-positive foci. NFBD1, together with 53BP1, plays a partially
redundant role in regulating phosphorylation of the downstream effector
protein, Chk2, since abrogation of both diminishes phosphorylated Chk2
in IR-induced foci. These results place NFBD1 parallel to 53BP1 in
regulating Chk2 and downstream of H2AX in the recruitment of repair and
signaling proteins to sites of DNA damage.
Molecules participating in the DNA damage signal pathway can be
classified as DNA damage sensors, proximal kinases, transducer kinases,
and effectors (1-3). DNA damage sensors, which initiate the DNA damage
signal cascade, are minimally characterized. Our initial work on NFBD1 showed that it forms IR-induced foci (IRIF)
within 2 min after exposure. Expression of NFBD1-derived BRCT domains
compromised association of both Thr68-phosphorylated Chk2
(Chk2T68P) and Construction of Plasmids--
The RNAi vector BS/U6 was kindly
provided by Y. Shi (Department of Pathology, Harvard Medical School).
Nucleotides sequences from 959 to 981 (GGGCTCAGCCTTTTGGCTTCAT) of
NFBD1, sequence from 767 to 789 (GGGCCTTCCGGATGGCCGGCGG) of H2AX, or
sequence from 1949 to 1971 (GGGTCTGAGGTGGAAGAAATCC) of 53BP1 were used
for construction of the RNAi vectors, BS/U6:NFBD1, BS/U6:H2AX, and
BS/U6:53BP1, respectively. The RNAi expression cassette of NFBD1 was
also inserted into an pCDNA3 vector that directs expression of GFP
and was named pGFP/U6:NFBD1.
Antibodies, Western Blotting, and Immunostaining--
Mouse and
rabbit anti-NFBD1 were generated as described previously (13). Mouse
anti-53BP1 was generated against COOH-terminal 412 amino acids using
standard procedures. Rabbit Cell Culture and Treatments with Irradiation--
MCF7, a human
breast carcinoma cell line, was cultured in Dulbecco's modified
Eagle's medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 50 units of penicillin, and 50 µg/ml streptomycin at 37 °C with 10% CO2.
Cells grown in log-phase were irradiated in a 137Cs
radiation source (Mark I, model 68A Irradiator, JL Shepherd & Associates, CA). The medium was replaced immediately after
irradiation. All the cells were then cultured at 37 °C and harvested
at the indicated time points.
Colony Formation Assay--
MCF7 cells transfected and selected
with RNAi vectors or empty vector were counted and plated into 10 cm
plates. After cell attachment (14 h), the cultures were treated with
irradiation at the indicated doses or mock-treated (0 Gy) and then
incubated for 14 days. After colony formation, cells were fixed and
stained with 2% methyl blue in 50% ethanol. Colony formation was
determined by counting a colony with >50 cells. Averages and
S.D. values were determined from triplicates. Treated sample
percentages were determined by dividing their plating efficiency by the
appropriate mock-control.
G1/S Checkpoint Assay--
Cells were
plated on cover glasses in 35-mm plates. After 24 h, cultures were
either mock-treated or irradiated with 12 Gy of IR and then returned to
the incubator. After 24-h incubation, fresh medium containing 10 mM BrdUrd was added and cultured an additional
3 h. Cells were then washed once with PBS and fixed for BrdUrd
immunostaining using a cell proliferation kit (Amersham Biosciences). BrdUrd-positive cells were scored by fluorescence microscopy and expressed as a fraction of the total cells.
G2/M Checkpoint Assay--
Cells were
plated on cover glasses in 35-mm plates. After 24 h, cultures were
either mock-treated or irradiated with 2-12 Gy of Elimination of NFBD1 Expression Affects IR Sensitivity and
G2/M Checkpoint--
Our initial work on NFBD1
(nuclear factor that contains BRCT domains 1, KIAA0170) showed that it
forms IRIF within 2 min after exposure. Expression of NFBD1-derived
BRCT domains compromised association of both
Thr68-phosphorylated Chk2 (Chk2T68) and
Inactivation of genes essential for DNA damage signal transduction,
including ATM, H2AX, BRCA1, Chk2 and p53, results in IR sensitivity
(see review in Ref. 20). Because NFBD1 is an early participant in this
pathway (13), we then examined IR sensitivity in MCF7 cells expressing
NFBD1 siRNA. As shown in Fig. 1C, NFBD1 RNAi-transfected
cells, but not empty vector, formed significantly less colonies upon IR
exposure. Similar results were also observed in human HeLa and
osteosarcoma U2OS cells (data not shown). To test for the possibility
that IR sensitivity results from defects in checkpoint control, NFBD1
RNAi-transfected MCF7 cells were tested for G1/S or
G2/M checkpoint control. In both temporal and dose-response
protocols (Fig. 1, D and E, respectively), NFBD1 RNAi-transfected MCF7 cells are partially defective in G2/M
checkpoint, while the G1/S checkpoint remains intact (Fig.
1F).
NFBD1 Association with IR-induced Foci Has Kinetics Similar to
NFBD1 Is Downstream of NFBD1 and 53BP1 Regulate Chk2 Redundantly--
The function of
NFBD1 may serve as an adaptor protein similar to the part played by
yeast Rad9 for Rad53, the human homologue of Chk2. To directly test
whether NFBD1 has a role in regulating Chk2, phosphorylation of
Thr68 in cells transfected with NFBD1 RNAi was assayed by
Western blotting using phosphorylation state-dependent
antibodies. As shown in Fig. 3, A and B, the
immunoreactivity detected with these antibodies was partially reduced
relative to total Chk2 protein. These data are comparable with the
partial reduction of Chk2T28 phosphorylation observed in 53BP1
RNAi-transfected cells (15-17). Immunoprecipitation of 53BP1 could
efficiently bring down Chk2 (15). This observation suggested
that 53BP1 might act as an adaptor that facilitates Chk2
phosphorylation. Similarly, Chk2 can be reciprocally
co-immunoprecipitated with NFBD1 (Fig. 3C). Taken together,
these results suggest that NFBD1 binds to Chk2 and mediates its phosphorylation.
To delineate the signaling pathway leading to Chk2 phosphorylation,
Chk2T68-positive IRIF in cells transfected with H2AX, 53BP1, and NFBD1
RNAi expression plasmids were scored. A consistent observation is that
H2AX is essential for Chk2T68 association IRIF. However, in 53BP1 or
NFBD1 RNAi-treated cells, Chk2T68-positive foci were only partially
reduced (Fig. 4, A and
B). Interestingly, in 53BP1 and NFBD1 RNAi double
transfected cells, CHK2T68-positive IRIF was reduced to a level
comparable with H2AX RNAi-transfected cells (Fig. 4, A and
B). These results suggest that NFBD1 and 53BP1 redundantly
regulate the participation of the phosphorylated Chk2T28 in IR-induced
foci.
In response to DSBs, NFBD1 and 53BP1 both appear to play a central role
in transducing DNA damage signals to downstream effectors by serving
perhaps as adaptor proteins connecting the upstream signal from
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
-H2AX and the
Rad9-Rad1-Hus1 (proliferating cell nuclear antigen-like) clamp
complex were considered to be candidates for DNA damage sensors, given
their accessibility to damaged DNA (4, 5). Although the phosphorylation
and recruitment of these molecules to the DNA damage sites are early
actions, the dependence on
ATR/ATM1 for these events
challenges their roles as primary sensors (4, 6). Proximal kinases
(ATM, possibly ATR and DNA-PK), in cooperation with adaptor proteins,
activate a variety of substrates, such as transducer kinases and
effectors (1, 3). In yeast, Rad9 is an adaptor protein that plays a
central role in transducing and amplifying DNA damage signals by
recruiting transducer kinase Rad53 to DNA sites of double strand breaks
(7, 8). Based on sequence similarity (9, 10) and early participation in DNA damage responses, three BRCT domain-containing proteins, 53BP1, BRCA1, and NFBD1 (11-14), are possible mammalian orthologs of yeast Rad9. Recent functional studies indicating that 53BP1 only partially regulates the G2/M checkpoint and Chk2, the human homologue
of scRad53, suggests that additional players participate in this crucial cellular response to DNA damage (15-17).
-H2AX within IRIF (13). These data suggest the
involvement of NFBD1 in early cellular responses to DNA damage. In the
present study, we investigate the precise role of NFBD1 in DNA damage
response signaling pathway by using RNAi to suppress NFBD1 protein
levels and found that its absence is associated with increased
radio-sensitivity and delayed G2/M transition, but not
G1 to S. NFBD1, together with 53BP1, plays a partially
redundant role in regulating phosphorylation of the downstream effector
protein, Chk2. These studies place NFBD1 parallel to 53BP1 in
regulating Chk2 and downstream of H2AX in the recruitment of repair and
signaling proteins to sites of DNA damage.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
-
-H2AX was purchased from
Upstate Biotechnology (Lake Placid, NY) and rabbit
-Chk2T68P was
from Cell Signaling Technology (Beverly, MA). Rabbit anti-NBS1 and
anti-Chk2 antisera were purchased from GeneTex (San Antonio, TX).
Immunoprecipitation, Western blotting, and immunostaining were
performed as described previously (13, 18). Immunofluorescence images
were captured using a Zeiss fluorescence microscope (Zeiss, Axiplan2).
-radiation and
then returned to the incubator. After 1-4 h, cells were gently washed
with PBS, fixed with 4% paraformaldehyde in PBS, and then stained with
DAPI. Mitotic cells in prometaphase, metaphase, anaphase, and telophase
were identified by fluorescence microscopy, scored, and expressed as a
fraction of the total cells.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
-H2AX within
IRIF (13). NFBD1 phosphorylation in response to ionizing IR is mediated
by ATM (14). Together, these data suggest the involvement of NFBD1 in
early cellular responses to DNA damage. To investigate the precise role
of NFBD1 in DNA damage response signaling pathway, we generated a
vector-based RNAi construct (19) for the in vivo expression
of a 22-base pair RNA duplex that targets NFBD1. The human breast
cancer cell line, MCF7, transfected with the NFBD1 siRNA construct
demonstrated significantly reduced expression of NFBD1 protein about
50-60 h after transfection (Fig.
1B, compare lane 1 with lanes 3 and 4). In a population of
G418-selected cells expressing siRNA, NFBD1 was nearly undetectable
(Fig. 1B, lanes 5 and 6), indicating
effective repression by this approach. Using a GFP/NFBD1 siRNA dual
expression plasmid, it is apparent that NFBD1 immunofluorescence is
inversely correlated with GFP fluorescence, which is not observed using
control vectors (Fig. 1B, compare rows labeled
RNAi, RNAi/NeoR, and
RNAi/NeoR/RNAi with
vector).
View larger version (34K):
[in a new window]
Fig. 1.
Elimination of NFBD1 expression affects IR
sensitivity and G2/M checkpoint. A,
Western blot assay of the expression of NFBD1 in MCF7 cells transfected
with plasmid (GFP/U6:NFBD1), which directs synthesis NFBD1 siRNA.
Lane 1, MCF7; lane 2, MCF7 transfected with
control vector, pCDNA3/GFP. Lanes 3 and 4,
MCF7 transfected with GFP/U6:NFBD1 for 24 h (lane 3)
and 48 h (lane 4). Lane 5,
GFP/U6:NFBD1-transfected MCF7 cells were selected with 800 µg/ml G418
for 2 weeks and labeled as RNAi-selected in B. Lane
6, G418-resistant MCF7 cells were transfected with pBS/U6/NFBD1
and labeled as
RNAi/NeoR/RNAi in
B. Cell extracts were separated by SDS-PAGE and then
immunoassayed with antibodies to NFBD1 (upper panel) and p84
(lower panel). B, immunostaining analysis of
NFBD1 expression in MCF7 cells transfected with control vector or
GFP/U6:NFBD1. Cells were fixed and stained with purified rabbit
anti-NFBD1. Panels identified as NFBD1 represent
immunofluorescence staining with anti-NFBD1, and GFP
represents GFP-expressing cells. DAPI indicates cells
stained with 4',6-diamidino-2-phenylindole. C, colony
formation assay for IR sensitivity. NFBD1 RNAi-transfected or empty
vector-transfected cells were irradiated with the indicated doses of
IR, and the surviving cells were scored as colonies. D and
E, IR-induced G2/M checkpoint was examined by
counting mitotic phase cells either at the indicated hours after
irradiation with 12 Gy (D) or at 2 h after the cells
were irradiated with indicated dose (E). F,
G1/S checkpoint control was done by a BrdUrd incorporation
assay to monitor S phase entry using NFBD1 RNAi transfected or empty
vector controls.
-H2AX and 53BP1--
Previous studies showed that the DNA damage
response factors,
-H2AX, RAD51, 53BP1, and CHK2 form IRIF at sites
of DSB (Review in 20). The kinetics of recruitment to DSB sites is
useful in exploring their functional relationship. As high dose IR
treatment may mask the kinetics of early IRIF formation as reported
previously (13), cells were irradiated with 1 Gy, and IR-induced
nuclear foci were scored. Similar to
-H2AX and 53BP1, the percentage of cells with NFBD1-positive IRIF increased immediately after IR
treatment and diminished quickly after 60 min (Fig.
2A), implying that these
proteins have nearly identical kinetics of foci association and
participate at the early stages of DSB response. In contrast, CHK2T68-positive IRIF peaked within 30 min after IR, remained at high
levels for 6 h, after which they slowly diminished (Fig. 2A), suggesting that phosphorylation of Chk2T68
and/or its association with IRIF is a later event compared with that of
NFBD1, 53BP1, or
-H2AX. RAD51- and BRCA1-positive foci were detected
at a much later stage under conditions of low dose IR exposure (Fig.
2A).
View larger version (27K):
[in a new window]
Fig. 2.
Factors recruitment in early DNA damage
responses to DSB. A, kinetics of IR-induced foci
formation positive for factors involved in DNA damage responses. MCF7
cells were exposed to 1 Gy IR and kept in culture for the indicated
times. Treated cells were immunostained with antibodies specific for
each the proteins indicated, and the percentage of the cell nuclei
containing more than five foci was calculated. B-E,
upper panels are mock-treated cells, and lower
panels are cells treated with NFBD1 RNAi (B and
D) or H2AX RNAi (C) or 53BP1 RNAi (E).
The percentage of cells either RNAi-transfected or empty vector
controls with more than five nuclear foci in each cell was counted and
the percentage indicated in the corresponding representative
photomicrograph.
-H2AX and Parallel to 53BP1 in Early
Signal Transduction Hierarchy--
To determine the relationship of
NFBD1, 53BP1, and
-H2AX in early responses to DSB, cells transfected
with plasmids directing expression H2AX, NFBD1, or 53BP1 siRNA were
scored for IRIFs positive for each protein. In the H2AX
RNAi-transfected cells, NFBD1, similar to 53BP1 (16), was not detected
at IRIF (Fig. 2C). In the 53BP1 RNAi-transfected cells, the
presence of NFBD1 in IRIF was unchanged (Fig. 2E).
Conversely, in NFBD1 RNAi-treated cells, IRIFs positive for 53BP1 and
-H2AX was not altered (Fig. 2,
B and D). These results suggest that H2AX is
upstream and is required for the recruitment of NFBD1 and 53BP1 into
the DSB signal transduction cascade. However, association of NFBD1 and
53BP1 with DSB may represent two independent events downstream of
H2AX.
View larger version (36K):
[in a new window]
Fig. 3.
Elimination of NFBD1 expression associates
with reduced phosphorylation Chk2 at Thr68 in response to
IR. A, NFBD1 RNAi or empty vector control MCF7 cells
were irradiated with 16 Gy. Cell lysates were processed at the
indicated times, separated by SDS-PAGE, and then immunoassayed with
antibodies to NFBD1 (top panel), Chk2T68 (second
panel), Chk2 (third panel), and p84 (bottom
panel). B, Chk2 phosphorylation at Thr68 in
response to different dose of IR. NFBD1 RNAi-transfected or empty
vector control MCF7 cells were irradiated with the indicated doses of
IR. Cell lysates were prepared 1 h post-irradiation and processed
as described in the legend to A. C, Chk2 was reciprocally
co-immunoprecipitated with NFBD1. MCF7 were mock-treated ( ) or
treated with IR (10 Gy) (+) and harvested after 2 h. Cell extracts
were immunoprecipitated with preimmune sera (Pre, lane
2), anti-NFBD1 (lanes 3 and 4), or anti-Chk2
(lanes 5 and 6) antibodies and analyzed by
Western blot probed with anti-NFBD1 (top panel) and
anti-Chk2 (second panel) antibodies. One-tenth of the cell
extracts was directly used for Western blot (lane 1),
serving as references.
View larger version (21K):
[in a new window]
Fig. 4.
NFBD1, like 53BP1, redundantly regulates
CHK2. A, empty vector control, H2AX RNAi, NFBD1
RNAi, 53BP1 RNAi, and NFBD1-53BP1 double RNAi-transfected cells were
irradiated with 8 Gy. At 2 h post-irradiation, cells were
immunostained with anti-Chk2T68p antibody. The percentage of the cells
with more than five nuclear foci was calculated. B,
representative photomicrographs of IR-induced Chk2T68-positive foci are
shown. Panels 1 and 13, double stained with
-NFBD1 and
-53BP1; panel 4,
-H2AX; panel
7,
-53BP1; panel 10,
-NFBD1; panels 2,
5, 8, 11, and 14,
-CHK2T68p; panels 3, 6, 9,
12, and 15, DAPI. Panels 1-3 were
transfected with empty vector. Panels 4-6 were transfected
for H2AX RNAi. Panels 7-9 were transfected for 53BP1 RNAi.
Panels 10-12 were transfected for NFBD1 RNAi. Panels
13-15 were transfected for both 53BP1 and NFBD1 RNAi.
Arrows indicate RNAi-targeted cells. C, a model
for the NFBD1 DNA damage checkpoint signaling pathway. In response to
DSBs, NFBD1 and 53BP1 both play a central role in transducing DNA
damage signals to Chk2.
-H2AX to the downstream effector Chk2 (Fig. 4C). This
model is consistent with the role of Rad9 function in budding yeast. In
yeast, Rad9 is an adaptor protein that plays a central role in
transducing and amplifying DNA damage signals by recruiting transducer
kinase Rad53 to DNA sites of double strand breaks (7, 8). Chk2 is a
major target of ATM, which phosphorylates threonine 68 to activate its
kinase activity in response to DNA damage. Activated Chk2, in turn,
phosphorylates p53 at Ser20, CDC25A at Ser123,
and CDC25C at Ser216, contributing to the G1/S,
S, and G2/M checkpoint (see review in Ref. 21). The
critical question of how adaptor proteins help mediate activation of
Chk2 at DSB sites remains to be solved. Our results showing redundancy
of NFBD1 and 53BP1 in regulating the recruitment of Chk2T68 to DNA DSB
sites probably underscores the importance of this step in mammalian
cells. Because of the dissimilarity of their structures (9, 10), it is
anticipated that NFBD1 and 53BP1 may also function in different
branching pathway.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Stanley Fields for providing partial 53BP1 cDNA and Shi Yang for providing pBS/U6 construct. We thank Paula Garza and Diane Jones for antibody preparation and Dave Sharp and Wen-Hwa Lee for critically reading the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant CA 85605 (to P.-L. C.).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: Dept. of Molecular
Medicine and Inst. of Biotechnology, The University of Texas Health
Science Center at San Antonio, 15355 Lambda Dr., San Antonio, TX 78245. Tel.: 210-567-7353; Fax: 210-567-7377; E-mail:
chenp0@uthscsa.edu.
Published, JBC Papers in Press, January 24, 2003, DOI 10.1074/jbc.C300001200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
ATR, ataxia telangiectasia and
Rad3-related;
ATM, ataxia
telangiectasia mutated;
NFBD1, a
nuclear factor with BRCT
domains protein 1;
BRCT, breast cancer
susceptibility gene 1 carboxyl terminus;
siRNA, small interfering RNA;
53BP1, tumor suppressor p53 binding protein 1;
DSB, double strand break;
-H2AX, phosphorylated H2AX at serine 139;
Chk2T68P, phosphorylated Chk2 at threonine 68;
PBS, phosphate-buffered
saline;
BrdUrd, bromodeoxyuridine;
DAPI, 4',6-diamidino-2-phenylindole;
IR, irradiation;
IRIF, IR-induced foci;
Gy, gray;
GFP, green fluorescent protein.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Zhou, B. B., and Elledge, S. J. (2002) Nature 408, 433-439[CrossRef] |
2. | Khanna, K. K., and Jackson, S. P. (2001) Nat. Genet. 27, 247-254[CrossRef][Medline] [Order article via Infotrieve] |
3. | Melo, J., and Toczyski, D. (2002) Curr. Opin. Cell Biol. 14, 237-245[CrossRef][Medline] [Order article via Infotrieve] |
4. | Paull, T. T., Rogakou, E. P., Yamazaki, V., Kirchgessner, C. U., Gellert, M., and Bonner, W. M. (2000) Curr. Biol. 10, 886-895[CrossRef][Medline] [Order article via Infotrieve] |
5. |
Kaur, R.,
Kostrub, C. F.,
and Enoch, T.
(2001)
Mol. Biol. Cell
12,
3744-3758 |
6. | Bao, S., Tibbetts, R. S., Brumbaugh, K. M., Fang, Y., Richardson, D. A., Ali, A., Chen, S. M., Abraham, R. T., and Wang, X. F. (2001) Nature 411, 969-974[CrossRef][Medline] [Order article via Infotrieve] |
7. |
Sun, Z.,
Hsiao, J.,
Fay, D. S.,
and Stern, D. F.
(1998)
Science
281,
272-274 |
8. | Gilbert, C. S., Green, C. M., and Lowndes, N. F. (2001) Mol. Cell 8, 129-136[Medline] [Order article via Infotrieve] |
9. | Koonin, E. V., Altschul, S. F., and Bork, P. (1996) Nat. Genet. 13, 266-268[Medline] [Order article via Infotrieve] |
10. | Callebaut, I., and Mornon, J. P. (1997) FEBS Lett. 400, 25-30[CrossRef][Medline] [Order article via Infotrieve] |
11. |
Schultz, L. B.,
Chehab, N. H.,
Malikzay, A.,
and Halazonetis, T. D.
(2000)
J. Cell Biol.
151,
1381-1390 |
12. |
Rappold, I.,
Iwabuchi, K.,
Date, T.,
and Chen, J.
(2001)
J. Cell Biol.
153,
613-620 |
13. |
Shang, Y. L.,
Bodero, A. J.,
and Chen, P.-L.
(2003)
J. Biol. Chem.
278,
6323-6329 |
14. |
Xu, X.,
and Stern, D. F.
(2003)
J. Biol. Chem.
278,
8795-8803 |
15. |
Wang, B.,
Matsuoka, S.,
Carpenter, P. B.,
and Elledge, S. J.
(2002)
Science
298,
1435-1438 |
16. | Fernandez-Capetillo, O., Chen, H. T., Celeste, A., Ward, I., Romanienko, P. J., Morales, J. C., Naka, K., Xia, Z., Camerini-Otero, R. D., Motoyama, N., Carpenter, P. B., Bonner, W. M., Chen, J., and Nussenzweig, A. (2002) Nat. Cell Biol. 4, 993-997[CrossRef][Medline] [Order article via Infotrieve] |
17. | DiTullio, R. A., Mochan, T. A., Venere, M., Bartkova, J., Sehested, M., Bartek, J., and Halazonetis, T. D. (2002) Nat. Cell Biol. 4, 998-1002[CrossRef][Medline] [Order article via Infotrieve] |
18. |
Chen, P. L.,
Chen, C. F.,
Chen, Y.,
Xiao, J., Z. D., S.,
and Lee, W. H.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
5287-5292 |
19. |
Sui, G.,
Soohoo, C.,
Affar el, B.,
Gay, F.,
Shi, Y.,
Forrester, W. C.,
and Shi, Y.
(2002)
Proc. Natl. Acad. Sci. U. S. A.
99,
5515-5520 |
20. |
Rouse, J.,
and Jackson, S. P.
(2002)
Science
297,
537-551 |
21. | Bartek, J., Falck, J., and Lukas, J. (2001) Nat. Rev. Mol. Cell. Biol. 2, 877-886[CrossRef][Medline] [Order article via Infotrieve] |