| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Regular Articles |
Division of Cancer Biology and Genetics [D. A. G, B. D. A. B., K. B. K., S. D.], and Departments of Pathology [D. A. G., S. D.], Biochemistry [B. D. A. B., S. D.], and Oncology [S. D.], Queen’s University Cancer Research Institute, Kingston, Ontario, K7L 3N6 Canada
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
H2AX) after DNA damage. This localization is independent of the damage repair kinase ataxia telangiectasia-mutated kinase (ATM), because hRad9/H2AX colocalization still occurs in ATM-/- fibroblasts. Secondly, hRad9 interacts with replication and checkpoint protein topoisomerase IIß binding protein 1 (TopBP1) before and after DNA damage, and this interaction is dependent on the COOH-terminal 17 amino acids of hRad9. Overexpression of a COOH-terminally deleted form of hRad9 abolishes the colocalization of TopBP1 to H2AX, ablating TopBP1 but not H2AX foci formation. The loss of TopBP1 containing foci, but not of H2AX containing foci, indicates that hRad9 is required for TopBP1 focus formation after damage, but is not required for H2AX formation at DSBs. These results are consistent with a model in which the hRad9/hHus1/hRad1 complex acts as a checkpoint sensor during S phase by rapidly localizing to sites of DNA damage and transducing checkpoint responses by facilitating proper localization of downstream checkpoint proteins, including TopBP1. |
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
H2AX is rapidly phosphorylated (H2AX) at DNA DSBs and a wide array of DNA damage and recombinational products (10, 11, 12)
. By inducing DSBs in a specific partial nuclear volume, Rogakou et al. (10)
were able to demonstrate that H2AX is phosphorylated in discrete regions surrounding damaged DNA, and that these foci formed within 1 min of exposure to DNA damage. Furthermore, localization of 53BP1, BRCA1, and Nbs1 to damage-induced foci is dependent on H2AX (13)
, suggesting that rapid phosphorylation of H2AX at damage sites facilitates recruitment of protein complexes necessary for checkpoint activation and repair.
Several proteins, such as TopBP1, may be required for transducing the checkpoint signal to the downstream checkpoint pathway and normal cellular events such as replication. TopBP1 is the human homologue of fission yeast protein Rad4/Cut5, and is required for replication and the replication and damage checkpoints (14, 15, 16, 17, 18, 19)
. Genetic experiments in fission yeast suggest that Rad4/Cut5 and Rad9 act upstream in the G2 checkpoint signaling pathway (20)
. In human cells, TopBP1 is required for replication, and interacts with DNA polymerase 
, which participates in damage synthesis (20)
. TopBP1 has also been shown to interact with hRad9 by two-hybrid studies and with overexpressed, epitope-tagged hRad9 (20)
. Thus, the close association of hRad9 and TopBP1 and their homologues has persisted throughout evolution, and is integral to the DNA damage response.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-irradiation, performed with 137Cs in a Victoreen Electrometer (Atomic Energy of Canada), at a dose rate of 0.76 Gy/min. UV irradiations were performed at 254 nm at a dose rate of 2 J/m2s. HU (ICN, Costa Mesa, CA) was used at a final concentration of 2 mM, and BM (Faulding, Montreal, Canada) was used at a final concentration of 1 µg/ml. HeLa cells were transfected as described previously (21)
. The Myc-TopBP1 construct was the generous gift of Dr. Junjie Chen (22)
. Transfections were performed as described previously (2)
. hRad9-EGFP stable cells were generated by transfection of HeLa cells expressing the pTet-Off vector (Clontech, Palo Alto, CA) with pTRE/hRad9-EGFP and pTK-Hyg (Clontech), and selecting clones resistant to hygromycin. Cells were synchronized by single thymidine block as described previously (23)
, with the exception that the cells were washed with PBS, and the cells were released into complete DMEM.
Formation DNA DSBs in Partial Nuclear Volumes.
Formation of DNA DSBs was preformed by adapting the methods of Rogakou et al. and Limoli and Ward, as follows (10
, 24)
. Live cells stably expressing hRad9-EGFP were grown in delta-T dishes (0.17 mm; Bioptechs, Butler, PA) in the presence of BrdUrd (Cell Proliferation Labeling Reagent, Amersham Biosciences, Piscataway, NJ) for 24 h. Cells were then stained with 1 µg/ml Hoechst dye 33258 where indicated and were overlayed with paraffin oil and were incubated on a heated stage for the duration of the experiment. Cells were visualized using a confocal inverted microscope (Leica TCS SP2 MP inverted Confocal Microscope) using a x100 objective (oil). Localization of hRad9-EGFP was monitored using an Ar/HeNe laser at 488 nm, and emission was measured at 500–535 nm. Using a two-photon laser set at 780 nm, the Hoechst dye was excited in a partial nuclear volume by changing the dimensions of the laser path to a narrow band in the center of the field of view. Immediately after irradiation, the localization of hRad9-GFP was followed for 10 min in 1-min intervals. As a control, cells were also irradiated in the absence of Hoechst dye, which does not induce DNA DSBs when irradiated by the two-photon laser.
Antibodies.
Antibodies used for indirect immunofluorescence experiments were as follows: affinity purified hRad9 chicken polyclonal antibody (2)
, H2AX (Upstate Biotechnology, Charlottesville, VA), TopBP1 (BD, Franklin Lakes, NJ), and Myc (9E10; Santa Cruz Biotechnology, Santa Cruz, CA). Affinity-purified hRad9 antibodies were diluted to 1:200 for immunofluorescence experiments and 1:500 for Western blotting, and the rest of the antibodies were diluted according to manufacturers’ recommendations. For double-labeling experiments, the following secondary antibodies were used when appropriate at a concentration of 2 µg/ml: goat chicken Alexa Fluor 488 (Molecular Probes, Eugene, OR), goat rabbit Alexa Fluor 488 (Molecular Probes), goat mouse Alexa Fluor 555 (Molecular Probes), and goat antirabbit Alexa Fluor 546 (Molecular Probes).
Indirect Immunofluorescence.
Cells were grown on glass coverslips in six-well tissue culture dishes. At indicated times after damage, cells in Fig. 2
were washed twice with PBS and were treated with extraction buffer [50 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM EDTA, 0.5% NP40 (25)
] supplemented with 0.5 mM AEBSF (Bioshop, Burlington, Canada), 20 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, 1 mM Na3VO4, 50 mM ß-glycerol phosphate, and 10 mM NaF, for 30 min at 4°C before fixation. In Figs. 3
and 4
, cells were washed twice with PBS and fixed in 4% paraformaldehyde for 15 min at room temperature, followed by permeabilization in 0.5% Triton-X100 for 10 min at room temperature. In Fig. 5
, cells were fixed in methanol/acetone (50:50) for 10 min at room temperature. Cells were then incubated in blocking solution (0.1% Triton-X100, 5% normal goat serum in PBS) for at least 30 min at 4°C. Cells were incubated with the indicated antibodies diluted in blocking solution for 1 h at room temperature, followed by washing three times with wash solution (0.1% Triton-X100 in PBS). Secondary antibodies diluted in blocking solution were then added for an additional 1–2 h, and coverslips were washed as above. 7-aminoactinomycin D (7-AAD; Molecular Probes) was added before mounting when indicated. A drop of glycerol was added to each coverslip, and coverslips were mounted on slides using nail polish.
|
|
|
|
-Chicken Alexa Fluor 488 was excited at 488 nm, and the emission was measured at 500–535 nm, -rabbit Alexa Fluor 546 and -mouse Alexa Fluor 555 were excited at 514 nm and the emission was measured at 556–618 nm. Images were captured sequentially, so as to minimize signal overlapping, and were processed and merged using PhotoShop (Adobe, San Jose, CA). In Fig. 5
, merged images were reset using a threshold that read only colocalized foci (yellow), and the number of colocalized foci per cell was counted using Image J (NIH, Bethesda, Maryland).
Immunoprecipitations and Immunoblotting.
Cells were grown in 10-cm tissue-culture dishes and were washed two times with PBS, were lysed in NETN buffer [150 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl, 0.5% NP40 (pH 8.0)] supplemented with 0.5 mM AEBSF, 20 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, 1 mM Na3VO4, 50 mM ß-glycerol phosphate, and 10 mM NaF, and were removed by scraping. Lysates were incubated on ice for 30 min and then were centrifuged at 16,000 x g for 10 min at 4°C. Soluble supernatants were precleared with either -IgY agarose (Promega, Madison, WI) or protein G Sepharose (Amersham, Piscataway, NJ) for 30–60 min at 4°C on an orbital shaker. Immunoprecipitations were conducted with 20 µl of affinity-purified -hRad9 chicken polyclonal antibody with 20 µl of -IgY agarose or 2 µg of -Myc monoclonal antibody with 15 µl of protein G Sepharose, overnight at 4°C. Immunocomplexes were collected for 2 h at 4°C on an orbital shaker. Immunoprecipitated proteins were washed once with 800 µl of NETN buffer and three times with 800 µl of PBS. Samples were then resuspended in 50 µl of 1.5x SDS-PAGE sample buffer, and heated to 95°C for 5 min.
Samples for immunoblotting were treated as described previously (21)
with the exception that horseradish peroxidase-conjugated -mouse (The Jackson Laboratory, West Grove, PA) secondary antibodies were used for Myc and TopBP1 Western blots.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
H2AX after DNA Damage.
, which is consistent with earlier experiments performed by indirect immunofluorescence (2)
. In the presence of BrdUrd, excitation of Hoechst dye generates DNA DSB (24)
. DNA DSB were generated in a partial nuclear area by irradiating a narrow band of the nucleus with a two-photon laser at 780 nm, exciting the Hoechst dye (indicated by the arrow in Fig. 1
). Within 1–2 min of DNA DSB formation, hRad9-EGFP preferentially localized to the damaged DNA and accumulated in a bright band coincident with the laser path (Fig. 1
, column 3). hRad9-EGFP continued to accumulate to the DNA containing DSBs for 5 min after damage (Fig. 1)
and remained associated for at least 20 min after damage. Progressive photobleaching of all of the images was observed over time. Localization of hRad9-EGFP to the subnuclear area along the laser path was dependent on the Hoechst dye (Fig. 1
, column 1), suggesting that it is dependent on the formation of DNA DSBs. Even in the presence of Hoechst, however, rapid (<2 min) localization of hRad9-EGFP to the damaged DNA occurred only in 30% of cells growing asynchronously (Fig. 1
, compare column 3 and 4). To investigate whether localization of hRad9-EGFP to DNA containing a DSB occurs only in a cell cycle subpopulation of cells, we synchronized cells to S phase by single thymidine block and release. hRad9-EGFP localization to DNA containing a DSB was apparent within 2 min of damage in >70% of S-phase cells (Fig. 1
, column 2), suggesting that the function of hRad9 in sensing DNA damage may be acting preferentially during DNA replication.
|
H2AX by indirect immunofluorescence in fixed cells. DNA damage induces the phosphorylation of H2AX (H2AX) in the region surrounding DSBs (10
, 26)
. Many proteins have been found to colocalize with H2AX after DNA damage, including 53BP1, MDC1, and TopBP1 (27, 28, 29)
. To determine whether hRad9 also colocalizes with H2AX after damage, cells were damaged with BM and IR to induce DSBs. Indirect immunofluorescence analysis demonstrates that hRad9 forms extraction-resistant foci, which are enhanced by DNA damage (Fig. 2
, column 1). When cells were synchronized to late S-G2 phase, formation of DNA DSBs by BM resulted in colocalization of hRad9 with H2AX foci within 30 min, in 
60% of cells (Fig. 2a)
. In asynchronous cells, after 8–10 h of recovery from DNA damage, H2AX foci emerged at sites of DSBs in larger, more diffuse foci known to encompass megabase domains of DNA (10)
. These foci largely colocalized with hRad9 foci, in 80% of cells (Fig. 2b)
. These results confirm the live-cell studies (Fig. 1)
, indicating that hRad9 colocalizes with H2AX at sites of DNA containing DSBs and also that it can remain associated at long-time points after damage.
H2AX is phosphorylated by ATM and ATR after DNA damage, but localization to DSB is not dependent on this phosphorylation (12
, 30
, 31)
. Whereas hRad9 is constitutively phosphorylated at multiple residues throughout the normal cell cycle, it becomes hyperphosphorylated after damage at serine 272 by ATM (21
, 32) . ATM-dependent phosphorylation of hRad9 is required for checkpoint activation but not for chromatin binding (7
, 21
, 32) . Therefore, we wondered whether hRad9 would still colocalize with H2AX in AT cells that do not express functional ATM. After -radiation treatment, H2AX foci colocalized with hRad9 foci in 40% of AT cells, indicating that phosphorylation of hRad9 by ATM is not required for hRad9 localization to DNA containing DSBs (Fig. 2c)
. These results are consistent with experiments performed in Saccharomyces cerevisiae, which showed that the hRad9 budding yeast homologue Ddc1 localizes to damaged DNA independently of the ATM homologue Mec1 (33
, 34)
.
hRad9 Interaction with TopBP1 Is Dependent on the hRad9 COOH Terminus.
We next wanted to look at transducers of the checkpoint downstream of hRad9 that also form damage-dependent foci. TopBP1 colocalizes to H2AX foci after DNA damage and was shown to interact with epitope-tagged hRad9 (20
, 29)
. We, therefore, wanted to know whether TopBP1 associates with endogenous hRad9. As shown in Fig. 3a
, endogenous TopBP1 coimmunoprecipitated with hRad9 antibodies. The interaction persisted after DNA damage and after HU treatment, which stalled replication fork progression in S-phase cells. Indirect immunofluorescence showed that in cells transfected with Myc-TopBP1, hRad9 also colocalized with TopBP1 in 70% of cells (Fig. 3b)
, but this colocalization was most apparent after treatment with HU, suggesting that this interaction may be especially important in S-phase cells.
Because TopBP1 is able to interact with the COOH-terminal 148 amino acids of hRad9 by yeast two-hybrid analysis, we generated hRad9 deletion constructs to determine which domain of hRad9 interacts with TopBP1 (20)
. The deletion constructs eliminated the final 59, 36, 31, and 17 amino acids of hRad9, respectively (Fig. 4a)
. Each of these constructs and full length hRad9 was tagged with the Myc epitope to distinguish endogenous and transfected hRad9. In coimmunoprecipitation experiments of cells transiently transfected with the Myc-hRad9 constructs, only full-length Myc-tagged hRad9 was able to coimmunoprecipitate with TopBP1 (Fig. 4b)
. None of the deletion constructs were able to interact with TopBP1. However, because three of the four deletion constructs fully or partially eliminated the NLS of hRad9, we tested to see whether the interaction was abolished simply because hRad9 was sequestered from the nucleus. Cells transiently transfected with Myc-hRad9 WT exhibited nuclear colocalization with endogenous hRad9 (Fig. 4c)
. Myc-hRad9
31, 36, and 59 constructs failed to localize discretely to the nucleus and were dispersed more extensively throughout the cytoplasm as more of the COOH terminus was removed. Myc-hRad917, which contains an intact NLS, however, did localize to the nucleus. Therefore, Myc-hRad9 17 failed to coimmunoprecipitate with TopBP1 despite its nuclear localization. These results suggest that the final 17 amino acids of hRad9 are important for its interaction with TopBP1. Additionally, Myc-hRad9 P5A, which contains five serine/threonine mutations at hRad9 phosphorylation sites (2)
, bound TopBP1 with a reduced affinity. This suggests that the interaction may be regulated in part by these phosphorylation events.
DNA Damage-dependent Focus Formation of TopBP1 Is Dependent on hRad9.
TopBP1 contains eight BRCT domains, which are common in checkpoint and DNA repair proteins for mediating protein-protein interactions and DNA binding (35)
. TopBP1 BRCT domains 4 and 5 were shown to directly interact with hRad9 in yeast two-hybrid analysis (20)
. Furthermore, TopBP1 BRCT 5 is required for formation of TopBP1 containing foci after DNA damage (22)
. Taken together, these results suggest that the TopBP1 interaction with hRad9 may be required to localize TopBP1 to sites of DNA damage. To test this hypothesis, we transfected cells with hRad9WT, 59, or 17 constructs, and measured the number of TopBP1 foci that colocalized with H2AX after DNA damage. The transfection efficiency was >40% in all cases, based on the number of GFP positives in a concurrent experiment. The number of TopBP1 foci that colocalized with H2AX per cell was counted, and the frequency of the number of foci/cell was plotted and analyzed (Fig. 5)
. In cells transfected with hRad9WT, TopBP1 significantly colocalized with H2AX, with a median of 13 colocalized foci/cell. In contrast, in cells transfected with hRad959, the number of TopBP1/H2AX colocalized foci per cell was markedly less, with a median of 2 colocalized foci/cell. The lack of colocalization between TopBP1 and H2AX was attributable to a significant reduction in the number of TopBP1 foci that formed, rather than a decrease in the number of TopBP1 foci that overlayed with H2AX (Fig. 5a)
. Similar results were demonstrated for hRad917, with a median of 3 colocalized foci/cell. More than one-half of the hRad959 and 17 transfected cells had <3 colocalized foci/cell, whereas none of the hRad9WT transfected cells had <3 colocalized foci/cell (Fig. 5b)
. This indicates that overexpression of hRad959 and 17 abolishes the damage-dependent focus formation of TopBP1.
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
59 mutant, including the NLS, leaving only the PCNA-like domain and the first one-half of the COOH terminus. This construct is, however, sufficient for interaction with hRad1 and hHus1 (data not shown). Similar hRad9 constructs have been shown to act as dominant negatives (36)
, because most of the cellular pool of hRad1 and hHus1 that bind this domain is recruited to the cytoplasm. Thus, hRad959 is likely abolishing the formation of the TopBP1 foci by sequestering hRad1 and hHus1 in the cytoplasm, and prohibiting the hRad9/hRad1/hHus1 complex from localizing to sites of DNA containing DSBs. In the case of hRad917, which resides in the nucleus and binds hRad1 and hHus1 but lacks the TopBP1-binding domain, a similar phenotype is observed. This demonstrates that abolishing the interaction between hRad9 and TopBP1 is sufficient to disrupt TopBP1 containing foci after DNA damage. Taken together, these data suggest that the hRad9 COOH terminus targets TopBP1 to damage sites.
We have also demonstrated that hRad9 is localized to sites of DNA containing DSBs. Whereas other groups have previously shown that hRad9 is retained at chromatin after DNA damage (6)
, we are able to show that hRad9 is localized specifically to DNA containing a DSB by rapid localization of hRad9-EGFP to a specific subnuclear area of DNA damage, and colocalization of hRad9 with the damage-induced phosphorylated form of H2AX. After DNA damage, virtually all of the H2AX containing foci also contain hRad9, although the converse is not true. We have observed similar relationships between hRad9 containing foci and those containing other damage response proteins.4
The additional hRad9 foci likely results from an excess of nuclear hRad9, but we cannot rule out the possibility that they represent the association of hRad9 with some form of endogenous DNA damage.
Localization of hRad9 to DSBs and colocalization with H2AX occurs rapidly in S-phase cells. Studies using cell-free Xenopus extracts have suggested that the binding of XHus1 to chromatin is dependent on DNA polymerase (37)
, suggesting that hRad9/hRad1/hHus1 acts as sensor of DNA damage during replication. Others have shown, however, that hRad9 binds chromatin after damage in G1 (7)
, although we do not observe hRad9-EGFP accumulation at DNA DSBs in the majority of asynchronous cells, which are 60% G1 phase. It may be that, although hRad9/hRad1/hHus1 is binding to chromatin during G1 phase after DNA damage, we do not observe accumulation of hRad9-EGFP after damage because hRad9 is not competent within this very rapid time period (5 min or less) to localize to the damaged DNA. Phosphorylation of specific hRad9 residues during S phase may enable hRad9 to bind to DNA DSBs very rapidly, and, indeed, we have previously reported a distinct hRad9 phosphorylation that occurs exclusively in G2-M phase cells (21)
.
In asynchronous cells, hRad9 colocalized to H2AX after long periods of recovery from DNA damage. Approximately 8–12 h after DNA damage, many damage response and repair proteins, including H2AX, are known to localize to IR-induced foci (IRIF). However, because repair is expected to be completed by this time frame, it has been suggested that these foci represent irreparable sections of DNA in which damage repair proteins have amassed (38)
.
We have also shown here that hRad9 localizes to DNA containing DSBs independently of ATM. This result is consistent with experiments performed in S. cerevisiae, illustrating that hRad9/hRad1/hHus1 homologues Ddc1/Rad17/Mec3 and ATR/ATRIP homologues Mec1/Ddc2 localize independently to damage sites (33 , 34) . Additionally, the damage-induced phosphorylation of hRad17 by ATR is dependent on the hHus1 binding to chromatin, presumably at sites of damage (8) . Thus, our finding that hRad9 localizes to DSB independently of ATM supports a model in which the hRad9/hRad1/hHus1 complex is localized independently to DNA damage sites.
Modeling of hRad9/hRad1/hHus1 to a PCNA-like complex has led to the suggestion that this complex acts as a landing platform for other repair and checkpoint proteins to sites of damaged DNA (4) . Localization of hRad9 to sites of DNA damage implicates the hRad9/hRad1/hHus1 complex as an immediate sensor of structural changes in DNA during replication. In this model, TopBP1 may be an example of hRad9/hRad1/hHus1-dependent recruitment of checkpoint and repair proteins to damage sites. TopBP1 and other checkpoint and repair proteins may then be phosphorylated by ATR/ATM, which independently localize to DNA damage. Indeed, localization to DNA damage may be required for the ATM-dependent phosphorylation of hRad9 after DNA damage, because localization to DNA damage sites is independent of ATM. TopBP1 recruitment to the hRad9/hRad1/hHus1 complex by hRad9 at sites of DNA damage may be an important function of the DNA damage checkpoint, because TopBP1 contains multiple BRCT domains that are capable of interacting with other BRCT domains containing proteins (39) . Thus, localization of the hRad9/hRad1/hHus1 complex to DNA damage sites may nucleate the many protein-protein interactions necessary for checkpoint activation and DNA repair.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 Supported by Canadian Institutes of Health Research (CIHR) Grants MOP-14352 and MOP-36526 (to S. D.) The confocal microscope was purchased with funding from the Canadian Foundation for Innovation (CFI) and the Ontario Innovation Trust (OIT) for the Protein Function Discovery Facility. Confocal microscopy was funded in part by CIHR Mult-User Equipment and Maintenance Grant MT-7827. S. D. is a Cancer Care Ontario Scientist. D. A. G. is a recipient of a Natural Sciences and Engineering Research Council of Canada studentship. B. D. A. B. is a recipient of a United States Army Breast Cancer Research Studentship DAMD1798-1-8080. 
2 To whom requests for reprints should be addressed, at Division of Cancer Biology and Genetics, Queen’s University Cancer Research Institute, Botterell Hall Room 364, Queen’s University, Kingston, Ontario, K7L 3N6 Canada. Phone: 613-533-6923; Fax: 613-533-6830; E-mail: sd13{at}post.queensu.ca
3 The abbreviations used are: NLS, nuclear localization signal; AT, ataxia telangiectasia; ATM, AT-mutated kinase; ATR, ATM- and Rad3-related; BM, bleomycin; BRCT, BRCA1 COOH-terminal; BrdUrd, bromodeoxyuridine; DSB, double-strand break; GFP, green fluorescent protein; EGFP, enhanced GFP; HU, hydroxyurea; IR, ionizing radiation; TopBP1, topoisomerase IIß binding protein 1; PCNA, proliferating cell nuclear antigen; WT, wild type; H2AX, phosphorylated H2AX; AEBSF, 4-(2-aminoethyl)-benzenesulfonylflurode.HCl; 53BP1, p53 binding protein 1.
4 D. A. G. and S. D., unpublished observations.
Received 12/18/02. Revised 4/16/03. Accepted 6/ 6/03.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
in establishing the ATR. Hus1 DNA replication checkpoint. J. Biol. Chem., 277: 27088-27093, 2002.This article has been cited by other articles:
|
|
 |
|
  E. Sjottem, C. Rekdal, G. Svineng, S. S. Johnsen, H. Klenow, R. D. Uglehus, and T. Johansen The ePHD protein SPBP interacts with TopBP1 and together they co-operate to stimulate Ets1-mediated transcription Nucleic Acids Res., October 8, 2007; 35(19): 6648 - 6662. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Lee, A. Kumagai, and W. G. Dunphy The Rad9-Hus1-Rad1 Checkpoint Clamp Regulates Interaction of TopBP1 with ATR J. Biol. Chem., September 21, 2007; 282(38): 28036 - 28044. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Jeon, K. Y. Lee, M. J. Ko, Y. S. Lee, S. Kang, and D. S. Hwang Human TopBP1 Participates in Cyclin E/CDK2 Activation and Preinitiation Complex Assembly during G1/S Transition J. Biol. Chem., May 18, 2007; 282(20): 14882 - 14890. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. L. Ball, M. R. Ehrhardt, D. A. Mordes, G. G. Glick, W. J. Chazin, and D. Cortez Function of a Conserved Checkpoint Recruitment Domain in ATRIP Proteins Mol. Cell. Biol., May 1, 2007; 27(9): 3367 - 3377. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Ogiwara, A. Ui, F. Onoda, S. Tada, T. Enomoto, and M. Seki Dpb11, the budding yeast homolog of TopBP1, functions with the checkpoint clamp in recombination repair Nucleic Acids Res., July 13, 2006; 34(11): 3389 - 3398. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. J. Lupardus and K. A. Cimprich Phosphorylation of Xenopus Rad1 and Hus1 Defines a Readout for ATR Activation That Is Independent of Claspin and the Rad9 Carboxy Terminus Mol. Biol. Cell, April 1, 2006; 17(4): 1559 - 1569. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. K. Pandita, G. G. Sharma, A. Laszlo, K. M. Hopkins, S. Davey, M. Chakhparonian, A. Gupta, R. J. Wellinger, J. Zhang, S. N. Powell, et al. Mammalian rad9 plays a role in telomere stability, s- and g2-phase-specific cell survival, and homologous recombinational repair. Mol. Cell. Biol., March 1, 2006; 26(5): 1850 - 1864. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. B. Smilenov, H. B. Lieberman, S. A. Mitchell, R. A. Baker, K. M. Hopkins, and E. J. Hall Combined Haploinsufficiency for ATM and RAD9 as a Factor in Cell Transformation, Apoptosis, and DNA Lesion Repair Dynamics Cancer Res., February 1, 2005; 65(3): 933 - 938. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Jurvansuu, K. Raj, A. Stasiak, and P. Beard Viral Transport of DNA Damage That Mimics a Stalled Replication Fork J. Virol., January 1, 2005; 79(1): 569 - 580. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Yin, A. Zhu, Y. J. Jin, Y.-X. Liu, X. Zhang, K. M. Hopkins, and H. B. Lieberman Human RAD9 checkpoint control/proapoptotic protein can activate transcription of p21 PNAS, June 15, 2004; 101(24): 8864 - 8869. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Wang, C.-L. Hsu, J. Ni, P.-H. Wang, S. Yeh, P. Keng, and C. Chang Human Checkpoint Protein hRad9 Functions as a Negative Coregulator To Repress Androgen Receptor Transactivation in Prostate Cancer Cells Mol. Cell. Biol., March 1, 2004; 24(5): 2202 - 2213. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
