|
|
|
|
|
|
|
||
Article |
Phosphorylation of SDT repeats in the MDC1 N terminus triggers retention of NBS1 at the DNA damage–modified chromatin
Correspondence to Jiri Bartek: jb{at}cancer.dk; or Jiri Lukas: jil{at}cancer.dk
DNA double-strand breaks (DSBs) trigger accumulation of the MRE11–RAD50–Nijmegen breakage syndrome 1 (NBS1 [MRN]) complex, whose retention on the DSB-flanking chromatin facilitates survival. Chromatin retention of MRN requires the MDC1 adaptor protein, but the mechanism behind the MRN–MDC1 interaction is unknown. We show that the NBS1 subunit of MRN interacts with the MDC1 N terminus enriched in Ser-Asp-Thr (SDT) repeats. This interaction was constitutive and mediated by binding between the phosphorylated SDT repeats of MDC1 and the phosphate-binding forkhead-associated domain of NBS1. Phosphorylation of the SDT repeats by casein kinase 2 (CK2) was sufficient to trigger MDC1–NBS1 interaction in vitro, and MDC1 associated with CK2 activity in cells. Inhibition of CK2 reduced SDT phosphorylation in vivo, and disruption of the SDT-associated phosphoacceptor sites prevented the retention of NBS1 at DSBs. Together, these data suggest that phosphorylation of the SDT repeats in the MDC1 N terminus functions to recruit NBS1 and, thereby, increases the local concentration of MRN at the sites of chromosomal breakage.
J. Falck's present address is Novo Nordisk A/S, DK-2880 Bagsverd, Denmark.
Abbreviations used in this paper: ATM, ataxia telangiectasia mutated; BRCT, BRCA1 C terminal; CK2, casein kinase 2; DSB, double-strand break; FHA, forkhead-associated; IR, ionizing radiation; MRN, MRE11–RAD50–NBS1; NBS, Nijmegen breakage syndrome; SDT, Ser-Asp-Thr; shRNA, short hairpin RNA; WT, wild type.
 |
Introduction |
|---|
 TopAbstract Introduction ResultsDiscussionMaterials and methodsReferences |
|---|
Prominent among the cellular factors that accumulate at DSBs is the MRE11–RAD50–Nijmegen breakage syndrome 1 (NBS1 [MRN]) complex, an essential genome caretaker that regulates crucial steps of the DSB response such as DSB detection, activity of the ataxia telangiectasia mutated (ATM) kinase (the key upstream component of DSB signaling), cell cycle checkpoints (D'Amours and Jackson, 2002; Petrini and Stracker, 2003; Kobayashi et al., 2004; Stracker et al., 2004), and, as the most recent advancements suggest, induction of apoptosis (Difilippantonio et al., 2007; Stracker et al., 2007). In addition, the MRN complex participates in the resection of DNA ends, an essential step required for an error-free DSB repair by homologous recombination (Jazayeri et al., 2006). Such diverse involvement in the DSB response is reflected by the nuclear dynamics of MRN. Most notably, recent studies revealed that the distribution of NBS1 (and other MRN components) at the DSB sites is not uniform but splits into distinct subcompartments (Lukas et al., 2004a; Bekker-Jensen et al., 2006). Although a fraction of MRN interacts with single-stranded DNA formed after enzymatic DSB resection (consistent with the essential role of MRN in DSB resection), the bulk of the DSB-associated MRN accumulates within the vast regions of chromatin marked by ATM-phosphorylated histone H2AX (
-H2AX). It is indeed this chromatin-associated fraction of MRN that cytologically manifests as the so-called ionizing radiation (IR)–induced foci.
The function of the chromatin-bound MRN has been subjected to intensive investigation and yielded important insights. Thus, it was found that the forkhead-associated (FHA) domain of NBS1 is necessary for its retention at the DSB-flanking chromatin and that its disruption impairs IR-induced foci formation of the entire MRN complex (Zhao et al., 2002; Cerosaletti and Concannon, 2003). Reconstitution experiments in human cells derived from the NBS patients suggested that the NBS1 N terminus, where the FHA domain resides, is important for survival, optimal ATM activity, and the intra–S-phase checkpoint after IR (Tauchi et al., 2001; Zhao et al., 2002; Cerosaletti and Concannon, 2003, 2004; Lee et al., 2003; Horejsi et al., 2004; Cerosaletti et al., 2006). However, because these assays were performed on the background of low levels of hypomorphic NBS1 alleles, the exact role of the chromatin-bound MRN remained a matter of debate. An important breakthrough in this discussion has been recently provided by Difilippantonio et al. (2005, 2007), who generated a mouse strain in which the endogenous NBS1 gene was replaced by a mutant with the disrupted FHA domain. Remarkably, although these mice were viable, they displayed defects in the DNA damage–induced G2/M- and S-phase checkpoints, an increased incidence of chromosomal aberrations, attenuated ATM activity after low doses of IR, and a decreased radiation survival. Although it is possible that the NBS1 N terminus supports other functions than the chromatin tethering of MRN, these results provide the most compelling evidence so far that the chromatin-bound MRN contributes to reach the threshold of the DNA damage signaling (especially after a low dose of IR), thereby guarding against chromosomal instability.
Although the requirement of the NBS1-FHA domain for the MRN focus formation has been established, the way this domain communicates with the DSB-flanking chromatin and/or the associated proteins is less clear. An earlier study suggested that the NBS1-FHA domain directly binds to -H2AX (Kobayashi et al., 2002). However, more recent results showed that the interaction of NBS1 with the DSBs requires MDC1, a large adaptor protein and an important upstream coordinator of the DSB-induced chromatin response (Goldberg et al., 2003; Stewart et al., 2003; Lukas et al., 2004a; Lou et al., 2006). MDC1 contains tandem BRCA1 C-terminal (BRCT) domains, which bind with high specificity to phosphorylated Ser139 of -H2AX (Stucki et al., 2005). As a result, MDC1 is among the first proteins to accumulate at the DSB sites, and its productive assembly in this compartment is a prerequisite for retention of most of the known chromatin-binding DSB regulators, including the MRN complex (Stucki and Jackson, 2006). The key evidence for the causative role of MDC1 for MRN focus formation was provided by several independent studies, which all showed that the siRNA-mediated knockdown of MDC1 by RNA interference in human cells (Goldberg et al., 2003; Stewart et al., 2003; Lukas et al., 2004a) or a complete MDC1 knockout in mouse cells (Lou et al., 2006) prevented MRN retention at the DSB-flanking chromatin. Importantly, kinetic measurements in living cells extended these results by showing that the MDC1 knockdown abolished MRN retention at DSBs from the earliest stages after DNA damage and that in the absence of MDC1, NBS1 could not concentrate at the DSB-flanking chromatin despite the fact that H2AX was efficiently phosphorylated in these compartments (Lukas et al., 2004a).
At lest three additional pieces of evidence suggested that the retention of MRN at the DSB-flanking (and -H2AX decorated) chromatin is not direct but is mediated by interaction with MDC1. First, peptides derived from the phosphorylated -H2AX C terminus interacted with MRN only in the presence of MDC1 (Lukas et al., 2004a). Second, MDC1 can be efficiently copurified with MRN from cell extracts (Goldberg et al., 2003; Stewart et al., 2003; Lukas et al., 2004a). Importantly, this interaction was dependent on the phosphate-binding FHA domain of NBS1, suggesting that the formation of a productive MDC1–MRN complex is phosphorylation dependent (Lukas et al., 2004a). Third, real-time imaging of protein assembly in live cells revealed that NBS1 accumulates at the DSB-flanking chromatin with a dynamics that is indistinguishable from that of MDC1 (but significantly faster than other chromatin-binding proteins such as 53BP1 or BRCA1), an observation that further supported the emerging model of the intimate relationship between MDC1 and the MRN complex (Lukas et al., 2004a; Bekker-Jensen et al., 2005; Mailand et al., 2007).
Although intriguing, these results left several mechanistic questions unanswered: most notably, it remains unclear whether the phosphosubstrate of the NBS1-FHA domain is indeed MDC1 itself or whether the MDC1–NBS1 interaction requires yet another, hitherto unrecognized (phospho)intermediate. Furthermore, the identity of the protein kinases and the phosphorylation sites that generate the recognition signal for the NBS1-FHA domain (and thus promote retention of MRN on the DSB-flanking chromatin) have not been determined. In an attempt to resolve these issues, we performed a detailed analysis of the MDC1–NBS1 interaction in vitro and in cells. We show that the NBS1-FHA domain binds to the N-terminal part of MDC1 enriched in acidic Ser-Asp-Thr (SDT) repeats. Furthermore, we provide evidence that the SDT repeats are constitutively phosphorylated, at least in part, by casein kinase 2 (CK2). These SDT phosphorylations are functionally significant because they trigger productive interaction between MDC1 and NBS1 and determine the retention of MRN at the DSB-modified chromatin.
|
Results |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
|
C) nor mutation of the three major phosphorylation sites targeted by the ATM kinase (NBS13A) had a measurable effect, and these mutants interacted with the MDC1 fragment as efficiently as the wild-type (WT) NBS1 (Fig. 1 E). Together, these data suggest that the MDC1 N terminus contains the necessary structural signature to allow recognition of MDC1 by the FHA domain of NBS1.
The SDT-rich region in the MDC1 N terminus mediates interaction with NBS1
Next, we subjected the N-terminal fragment of MDC1 to a bioinformatic analysis and searched for conserved motifs that may potentially mediate the interaction with NBS1. Interestingly, the region between amino acids 210 and 460 (that is, the part of MDC1 identified in the previous section as necessary and sufficient to bind NBS1) contained up to six acidic SDT motifs, some of which are conserved among different vertebrate species (Fig. 2 A and Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200708210/DC1).
To test whether the SDT clusters influence the MDC1–NBS1 interaction, we generated another set of MDC1 fragments either spanning different parts of the SDT-containing region or harboring an in-frame deletion of the entire SDT region (Fig. 2 B). The fragment containing all six SDT repeats efficiently bound to endogenous NBS1 when expressed in the U2OS/shRNA cell line (Fig. 2 C, lane 4). Strikingly, even shorter fragments containing either three N-terminal (SDT1) or four C-terminal (SDT2) repeats still interacted with NBS1, albeit with a lower efficiency than the full-length SDT region (Fig. 2 C, lanes 5 and 6). Conversely, deletion of the entire SDT region completely abolished the ability of the MDC1 N terminus to bind NBS1 under these conditions (Fig. 2 C, lane 3). Together, these data indicate that the SDT repeats within the MDC1 N terminus might be involved in binding to NBS1, and we set out to further explore this possibility.
|
|
|
|
To explore the emerging cross talk between CK2 and the N terminus of MDC1, we tested the impact of CK2 inhibition on SDT phosphorylation in cells. Indeed, preincubation of U2OS cells with a CK2 inhibitor significantly reduced the overall incorporation of radioactive phosphate to the ectopically expressed SDT fragment (Fig. 5 A).
More importantly in this context, the ensuing phosphopeptide analysis revealed that this was accompanied by an 
50% reduction of phosphate incorporation into two prominent SDT repeats (Ser299/Thr301 and Ser453/Ser455; Fig. 5 B). Thus, CK2 can at least partially phosphorylate the SDT repeats in vivo. To further support this conclusion, we immunopurified HA-tagged MDC1 N terminus (Fig. 6 A) or endogenous MDC1 (Fig. 6 B) from cells and subjected these immunocomplexes to an in vitro kinase assay (without any additional substrate).
In both cases, MDC1 copurified with an autocatalytic kinase activity that was strongly attenuated by a specific CK2 inhibitor (Fig. 6, A and B). Interestingly, this MDC1-associated CK2 activity was clearly present in undamaged cells (Fig. 6, A and B; lane 1) and did not appreciably increase after exposing the cells to IR (Fig. 6 A, lane 2). Collectively, the data in Figs. 4–6
show that CK2 can phosphorylate the SDT repeats and suggest that this may contribute to trigger a productive interaction between the MDC1 N terminus and the FHA domain of NBS1.
|
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
At first glance, it appears surprising that CK2, a ubiquitous and constitutively active kinase, regulates MDC1, a protein that functions almost exclusively in stochastic events triggered by genotoxic stress in which it operates strictly locally at the sites of chromosomal lesions. To comprehend this seemingly unusual cross talk, it is important to realize that MDC1 and NBS1 interact already in unstressed cells (Goldberg et al., 2003; Stewart et al., 2003), and, as we show in this study, even this predamage interaction is at least partly regulated by CK2. Based on this observation, the first obvious scenario that comes to mind is that as a result of its constitutive phosphorylation by CK2, MDC1 forms a stable holocomplex with MRN. In this model, the MRN focus formation simply reflects the propensity of MDC1 (as a fourth subunit of the MRN complex) to avidly interact with -H2AX. Although simple and attractive, several pieces of evidence suggest a more subtle way of regulation. First, MRN clearly has functions that do not require MDC1 (Petrini and Stracker, 2003; Falck et al., 2005; Jazayeri et al., 2006), and a strong affinity trap on SDT-phosphorylated MDC1 would likely reduce MRN's flexibility and limit its accessibility to nuclear compartments where it operates independently of MDC1. Indeed, kinetic analysis of NBS1 and MDC1 mobility in living cells revealed marked differences between these two proteins: although both NBS1 and MDC1 become retained at the DSB sites (a feature that determines formation of the cytologically discernible nuclear foci), the residence time of MDC1 at DSBs was nearly one order of magnitude longer compared with NBS1. As a result, although MDC1 becomes immobilized at the DSB-flanking chromatin, NBS1 remains highly mobile, a feature compatible with its flexible exchange among distinct DSB-generated subcompartments (Lukas et al., 2003, 2004a). Thus, the interaction between MDC1 and NBS1 is dynamic, and these two factors do not appear to form a rigid holocomplex.
Given these findings, the new data presented in this study, and similar results reached by Spycher et al. (see p. 227 of this issue), our current model describing the mechanism of MRN chromatin retention is as follows. In undamaged nuclei (Fig. 8, top), CK2 phosphorylates the SDT repeats of MDC1. Combined with the ability of the NBS1-FHA domain to recognize phosphorylated SDTs, this allows constitutive yet highly dynamic MDC1–MRN interaction, which remains amenable to further regulation and prevents sequestration of any of these factors in rigid aggregates.
The latter feature could be crucial to allow the rapid recognition of DNA breaks by the MRN complex, a process that is independent of MDC1 (see Introduction). After DSB generation (Fig. 8, bottom), the most proximal chromatin modification includes phosphorylation of the H2AX C terminus (-H2AX). Although several DSB regulators contain phosphate interaction motifs and domains, the MDC1-BRCT domain appears to interact with -H2AX with the highest affinity (Stucki et al., 2005), resulting in a rapid coating of the -H2AX–primed chromatin by MDC1. At this point, the MDC1 N terminus becomes important for the ensuing events on the DSB-flanking chromatin (Fig. 8, bottom). We propose that constitutive phosphorylation of the SDT repeats (translated to a continuous generation of binding sites for the NBS1-FHA domain) is likely the most efficient means to avoid any delay in recruiting MRN to the modified chromatin rapidly evolving around the incipient DBS lesions. Indeed, integration of a constitutive signal is consistent with the simultaneous recruitment of MDC1 and NBS1 to DSBs (Lukas et al., 2004a; Mailand et al., 2007) and suggests that in vertebrates, the SDT repeats of MDC1 coevolved with MRN to prime the latter for the fastest possible arrival at the sites of harmful chromosomal lesions.
|
50% compared with CK2-proficient cells). Unlike complete genetic disruption of the dominant SDT repeats (Fig. 7 B), this degree of the SDT phosphorylation impairment was not sufficient to prevent accumulation of NBS1 at the DSB sites (our unpublished data). As alluded to in the preceding paragraphs of this section, this model poses an important conceptual question: how is constitutive and ubiquitous phosphorylation of the MDC1 N terminus transformed to a signal that locally and transiently enhances chromatin affinity for NBS1? The answer to this question is not trivial, but several scenarios come to mind (Fig. 8, bottom). First, it is possible that just the sheer increase in the local density of MDC1 (and the corresponding phosphates on the SDT repeats) may be sufficient to delay MRN at the DSBs to an extent that still allows its dynamic exchange between distinct DSB-generated subcompartments yet prevents its dilution in the undamaged parts of the nucleus. Second, although CK2 activity does not increase after DNA damage, one can envisage that DSBs may locally regulate (inhibit) chromatin-associated phosphatases and thereby decrease the rate of SDT-phosphate turnover. In principle, this may also increase local density of phosphorylated SDT repeats and thereby stabilize the interaction between the MDC1 N terminus and the NBS1-FHA domain. Third, an alternative (but not mutually exclusive) model is based on our observation that the MDC1 N terminus harbors a cluster of six SDT sites and that at least some of them contribute to the MDC1–NBS1 interaction in a cooperative fashion (Fig. 7). Thus, it is possible that although only a subset of the SDT repeats generates the basal level of MDC1–MRN interaction in undamaged cells, the DSB-associated MDC1 undergoes additional conformational changes in its N terminus that may allow phosphorylation of the entire SDT region and, thereby, locally increase the interaction platform for NBS1 and indeed the entire MRN complex. Further studies will be required to discriminate among these scenarios.
Finally, we note that the role of CK2 in the DNA damage response is not unprecedented. The most striking parallel with our findings has been provided by Loizou et al. (2004), who reported that CK2 phosphorylates XRCC1, an adaptor protein involved in single-strand DNA break repair. This and the subsequent studies (Clements et al., 2004; Bekker-Jensen et al., 2007; Iles et al., 2007; Kanno et al., 2007) showed that CK2-mediated phosphorylation was required for the interaction of XRCC1 with other repair proteins such as polynucleotide kinase, aprataxin, and XIP1 (the latter protein is also known as APLF and PALF, respectively). It is noteworthy that all of these proteins interacted with XRCC1 by their FHA domains, which is in striking analogy to the MDC1–NBS1 interaction described in this study. In addition, an exciting conceptual parallel between the CK2-XRCC1 and CK2-MDC1 interplay is provided by the fact that in both cases, CK2 targets large scaffold proteins that by themselves do not posses enzymatic activity but rather appear to integrate upstream signaling to organize other proteins at the vicinity of DNA lesions. The fact that CK2 targets different adaptor proteins engaged in distinct DNA repair processes (single-strand breaks and DSBs) suggests that phosphorylation of these molecular adaptors coevolved to increase the efficiency of the genome surveillance machinery. Although the recent genetic data strongly support the role of MRN chromatin retention for survival and genomic stability after DSB-generating insults (see Introduction), the CK2-mediated phosphorylation of XRCC1 facilitates the single-stranded break repair by promoting the recruitment of polynucleotide kinase and aprataxin and by protecting XIP1 from proteolytic degradation (Clements et al., 2004; Loizou et al., 2004; Bekker-Jensen et al., 2007; Iles et al., 2007; Kanno et al., 2007). When combined together, the previous findings and our new data support the emerging model that one important function of CK2 is to increase the efficiency of genome surveillance by targeting interaction matchmakers and thereby promoting the local concentration and/or stability of DNA damage regulators at sites of genetic lesions.
|
Materials and methods |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
Plasmids
HA-tagged human MDC1 (pcDNA3-neo) was a gift from D. Stern (Yale University, New Haven, CT). HA-MDC1 fragments were generated by PCR and subcloned into a pcDNA4/TO (Invitrogen) plasmid. To the shorter N-terminal MDC1 fragments, a triple nuclear localization signal was added by cloning the fragments into pCMV-myc/nuc (Invitrogen) vector and thereafter subcloning back into pcDNA4/TO by PCR. Myc-NBS1-pCMV plasmids were described previously (Horejsi et al., 2004). The QuikChange Site-Directed Mutagenesis kit (Stratagene) was used to generate point mutations; deletions were made using the Phusion Site-Directed Mutagenesis kit (New England Biolabs, Inc.) and performed according to the manufacturer's instructions. Plasmid constructs were verified by sequence analysis.
Antibodies, chemicals, and immunochemical techniques
HA antibodies F7 and Y11 were purchased from Santa Cruz Biotechnology, Inc. The rabbit Nbs1 and Myc antibodies originated from Abcam. Rabbit and sheep polyclonal MDC1 antibodies were provided by S. Jackson (Gurdon Institute, Cambridge, UK). The specific CK2 inhibitor InSolution DMAT was obtained from EMD and was used in concentrations specified in the figure legends. For immunoprecipitation, cells were lysed, and proteins were extracted in a high salt buffer (20 mM Tris, pH 7.5, 400 mM NaCl, 0.5% NP-40, 1 mM EDTA, and protease and phosphatase inhibitor mixture). After centrifugation of the lysates, an equal volume of the same buffer without NaCl was added to produce a final NaCl concentration of 200 mM. Kinase assays and immunoblotting were performed as previously described (Falck et al., 2005; Mailand et al., 2006) except for the MDC1-associated kinase assay, in which no exogenous substrate was added.
In vivo labeling and phosphopeptide mapping
U2OS cells transfected with the indicated constructs were labeled for 4 h in phosphate-free DME with 20 mM Hepes, pH 7.2, and 5% dialyzed phosphate-free DME with 1 mCi/ml 32P (PBS43; GE Healthcare). Two-dimensional phosphopeptide and phospho–amino acid mapping were performed as described previously (Blume-Jensen et al., 1995; Hansen et al., 1996). Tryptic phosphopeptides were separated on an electrophoresis system (HTLE-7000; CBS Scientific Company, Inc.) at 2,000 V for 30 min at 14°C. The second dimension chromatography was performed in isobutyric acid buffer for 14–16 h. Separated peptides were subjected to phosphorimager analysis (FLA-3000; Fuji).
CK2 kinase assay
1 µg of purified GST or GST-MDC1 fragments was phosphorylated with 100 U of recombinant CK2 kinase in the presence of 10 µCi -[32P]ATP for 15 min at 37°C according to the manufacturer's instructions. The reaction was stopped by the addition of Laemmli sample buffer, and phosphorylated proteins were visualized by resolving the samples on SDS-PAGE followed by autoradiography.
In vitro binding assay
2 µg of purified GST-MDC1(181–480) fragments were left untreated or phosphorylated with recombinant CK2 (New England Biolabs, Inc.) for 30 min at 37°C in the presence of 1 mM ATP. The GST proteins were then bound to glutathione–Sepharose beads (GE Healthcare), and the beads were washed three times in EBC buffer (Mailand and Diffley, 2005). The beads were then incubated with in vitro–translated 35S-labeled Nbs1 for 2 h at 4°C, washed four times with EBC buffer, resuspended in Laemmli sample buffer, and resolved by SDS-PAGE and autoradiography.
Laser microirradiation and microscopy
Induction of localized DSBs by microirradiation was performed as described previously (Lukas et al., 2003, 2004a; Bekker-Jensen et al., 2005, 2006). In brief, U2OS cells and their derivatives were grown on glass coverslips in the presence of 10 µM BrdU for 24 h and subsequently exposed to the pulsed UVA laser (
= 337 nm) along a narrow track spanning the entire nuclear diameter. After an additional hour, the cells were fixed in 4% formaldehyde and subjected to immunostaining. Images were acquired with a confocal microscope (LSM 510; Carl Zeiss, Inc.) through a plan-Neofluar 40x NA 1.3 oil immersion objective (Carl Zeiss, Inc.). For dual color imaging, secondary antibodies coupled to AlexaFluor dyes with excitation wavelengths of 488 and 568 nm were used. Image acquisition and basic image processing were performed with LSM software (Carl Zeiss, Inc.).
Online supplemental material
Fig. S1 shows sequence alignment of the SDT-rich MDC1 regions in several vertebrate species. Fig. S2 provides in vivo evidence for the simultaneous phosphorylation of Ser and Thr residues within the individual SDT repeats. Fig. S3 shows that constitutive down-regulation of MDC1 by shRNA impairs retention of NBS1 at the sites of DNA damage. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200708210/DC1.
|
Acknowledgments |
|---|
This work was supported by grants from the Danish Cancer Society, Danish National Research Foundation, European Union (integrated project DNA Repair and Active p53), European Science Foundation (EuroDYNA), Danish Research Council, Swedish Society for Medical Research Stockholm, and the John and Birthe Meyer Foundation.
Submitted: 31 August 2007
Accepted: 19 March 2008
|
References |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
Bartek, J., and J. Lukas. 2007. DNA damage checkpoints: from initiation to recovery or adaptation. Curr. Opin. Cell Biol. 19:238–245.[CrossRef][Medline]
Beausoleil, S.A., M. Jedrychowski, D. Schwartz, J.E. Elias, J. Villen, J. Li, M.A. Cohn, L.C. Cantley, and S.P. Gygi. 2004. Large-scale characterization of HeLa cell nuclear phosphoproteins. Proc. Natl. Acad. Sci. USA. 101:12130–12135.
Bekker-Jensen, S., C. Lukas, F. Melander, J. Bartek, and J. Lukas. 2005. Dynamic assembly and sustained retention of 53BP1 at the sites of DNA damage are controlled by Mdc1/NFBD1. J. Cell Biol. 170:201–211.
Bekker-Jensen, S., C. Lukas, R. Kitagawa, F. Melander, M.B. Kastan, J. Bartek, and J. Lukas. 2006. Spatial organization of the mammalian genome surveillance machinery in response to DNA strand breaks. J. Cell Biol. 173:195–206.
Bekker-Jensen, S., K. Fugger, J.R. Danielsen, I. Gromova, M. Sehested, J. Celis, J. Bartek, J. Lukas, and N. Mailand. 2007. Human Xip1 (C2orf13) is a novel regulator of cellular responses to DNA strand breaks. J. Biol. Chem. 282:19638–19643.
Blume-Jensen, P., C. Wernstedt, C.H. Heldin, and L. Ronnstrand. 1995. Identification of the major phosphorylation sites for protein kinase C in kit/stem cell factor receptor in vitro and in intact cells. J. Biol. Chem. 270:14192–14200.
Cerosaletti, K.M., and P. Concannon. 2003. Nibrin forkhead-associated domain and breast cancer C-terminal domain are both required for nuclear focus formation and phosphorylation. J. Biol. Chem. 278:21944–21951.
Cerosaletti, K., and P. Concannon. 2004. Independent roles for nibrin and Mre11-Rad50 in the activation and function of Atm. J. Biol. Chem. 279:38813–38819.
Cerosaletti, K., J. Wright, and P. Concannon. 2006. Active role for nibrin in the kinetics of atm activation. Mol. Cell. Biol. 26:1691–1699.
Clements, P.M., C. Breslin, E.D. Deeks, P.J. Byrd, L. Ju, P. Bieganowski, C. Brenner, M.C. Moreira, A.M. Taylor, and K.W. Caldecott. 2004. The ataxia-oculomotor apraxia 1 gene product has a role distinct from ATM and interacts with the DNA strand break repair proteins XRCC1 and XRCC4. DNA Repair (Amst.). 3:1493–1502.[CrossRef][Medline]
D'Amours, D., and S.P. Jackson. 2002. The Mre11 complex: at the crossroads of dna repair and checkpoint signalling. Nat. Rev. Mol. Cell Biol. 3:317–327.[CrossRef][Medline]
Difilippantonio, S., A. Celeste, O. Fernandez-Capetillo, H.T. Chen, B. Reina San Martin, F. Van Laethem, Y.P. Yang, G.V. Petukhova, M. Eckhaus, L. Feigenbaum, et al. 2005. Role of Nbs1 in the activation of the Atm kinase revealed in humanized mouse models. Nat. Cell Biol. 7:675–685.[CrossRef][Medline]
Difilippantonio, S., A. Celeste, M.J. Kruhlak, Y. Lee, M.J. Difilippantonio, L. Feigenbaum, S.P. Jackson, P.J. McKinnon, and A. Nussenzweig. 2007. Distinct domains in Nbs1 regulate irradiation-induced checkpoints and apoptosis. J. Exp. Med. 204:1003–1011.
Essers, J., W. Vermeulen, and A.B. Houtsmuller. 2006. DNA damage repair: anytime, anywhere? Curr. Opin. Cell Biol. 18:240–246.[CrossRef][Medline]
Falck, J., J. Coates, and S.P. Jackson. 2005. Conserved modes of recruitment of ATM, ATR and DNA-PKcs to sites of DNA damage. Nature. 434:605–611.[CrossRef][Medline]
Fernandez-Capetillo, O., A. Lee, M. Nussenzweig, and A. Nussenzweig. 2004. H2AX: the histone guardian of the genome. DNA Repair (Amst.). 3:959–967.[CrossRef][Medline]
Goldberg, M., M. Stucki, J. Falck, D. D'Amours, D. Rahman, D. Pappin, J. Bartek, and S.P. Jackson. 2003. MDC1 is required for the intra-S-phase DNA damage checkpoint. Nature. 421:952–956.[CrossRef][Medline]
Hansen, K., M. Johnell, A. Siegbahn, C. Rorsman, U. Engstrom, C. Wernstedt, C.H. Heldin, and L. Ronnstrand. 1996. Mutation of a Src phosphorylation site in the PDGF beta-receptor leads to increased PDGF-stimulated chemotaxis but decreased mitogenesis. EMBO J. 15:5299–5313.[Medline]
Horejsi, Z., J. Falck, C.J. Bakkenist, M.B. Kastan, J. Lukas, and J. Bartek. 2004. Distinct functional domains of Nbs1 modulate the timing and magnitude of ATM activation after low doses of ionizing radiation. Oncogene. 23:3122–3127.[CrossRef][Medline]
Iles, N., S. Rulten, S.F. El-Khamisy, and K.W. Caldecott. 2007. APLF (C2orf13) is a novel human protein involved in the cellular response to chromosomal DNA strand breaks. Mol. Cell. Biol. 27:3793–3803.
Jazayeri, A., J. Falck, C. Lukas, J. Bartek, G.C. Smith, J. Lukas, and S.P. Jackson. 2006. ATM- and cell cycle-dependent regulation of ATR in response to DNA double-strand breaks. Nat. Cell Biol. 8:37–45.[CrossRef][Medline]
Kanno, S., H. Kuzuoka, S. Sasao, Z. Hong, L. Lan, S. Nakajima, and A. Yasui. 2007. A novel human AP endonuclease with conserved zinc-finger-like motifs involved in DNA strand break responses. EMBO J. 26:2094–2103.[CrossRef][Medline]
Kastan, M.B., and J. Bartek. 2004. Cell-cycle checkpoints and cancer. Nature. 432:316–323.[CrossRef][Medline]
Kobayashi, J., H. Tauchi, S. Sakamoto, A. Nakamura, K. Morishima, S. Matsuura, T. Kobayashi, K. Tamai, K. Tanimoto, and K. Komatsu. 2002. NBS1 localizes to gamma-H2AX foci through interaction with the FHA/BRCT domain. Curr. Biol. 12:1846–1851.[CrossRef][Medline]
Kobayashi, J., A. Antoccia, H. Tauchi, S. Matsuura, and K. Komatsu. 2004. NBS1 and its functional role in the DNA damage response. DNA Repair (Amst.). 3:855–861.[CrossRef][Medline]
Lee, J.H., B. Xu, C.H. Lee, J.Y. Ahn, M.S. Song, H. Lee, C.E. Canman, J.S. Lee, M.B. Kastan, and D.S. Lim. 2003. Distinct functions of Nijmegen breakage syndrome in ataxia telangiectasia mutated-dependent responses to DNA damage. Mol. Cancer Res. 1:674–681.
Linding, R., L.J. Jensen, G.J. Ostheimer, M.A. van Vugt, C. Jorgensen, I.M. Miron, F. Diella, K. Colwill, L. Taylor, K. Elder, et al. 2007. Systematic discovery of in vivo phosphorylation networks. Cell. 129:1415–1426.[CrossRef][Medline]
Loizou, J.I., S.F. El-Khamisy, A. Zlatanou, D.J. Moore, D.W. Chan, J. Qin, S. Sarno, F. Meggio, L.A. Pinna, and K.W. Caldecott. 2004. The protein kinase CK2 facilitates repair of chromosomal DNA single-strand breaks. Cell. 117:17–28.[CrossRef][Medline]
Lou, Z., K. Minter-Dykhouse, S. Franco, M. Gostissa, M.A. Rivera, A. Celeste, J.P. Manis, J. van Deursen, A. Nussenzweig, T.T. Paull, et al. 2006. MDC1 maintains genomic stability by participating in the amplification of ATM-dependent DNA damage signals. Mol. Cell. 21:187–200.[CrossRef][Medline]
Lukas, C., J. Falck, J. Bartkova, J. Bartek, and J. Lukas. 2003. Distinct spatiotemporal dynamics of mammalian checkpoint regulators induced by DNA damage. Nat. Cell Biol. 5:255–260.[CrossRef][Medline]
Lukas, C., F. Melander, M. Stucki, J. Falck, S. Bekker-Jensen, M. Goldberg, Y. Lerenthal, S.P. Jackson, J. Bartek, and J. Lukas. 2004a. Mdc1 couples DNA double-strand break recognition by Nbs1 with its H2AX-dependent chromatin retention. EMBO J. 23:2674–2683.[CrossRef][Medline]
Lukas, J., C. Lukas, and J. Bartek. 2004b. Mammalian cell cycle checkpoints: signalling pathways and their organization in space and time. DNA Repair (Amst.). 3:997–1007.[CrossRef][Medline]
Lukas, C., J. Bartek, and J. Lukas. 2005. Imaging of protein movement induced by chromosomal breakage: tiny ‘local’ lesions pose great ‘global’ challenges. Chromosoma. 114:146–154.[CrossRef][Medline]
Mailand, N., and J.F. Diffley. 2005. CDKs promote DNA replication origin licensing in human cells by protecting Cdc6 from APC/C-dependent proteolysis. Cell. 122:915–926.[CrossRef][Medline]
Mailand, N., S. Bekker-Jensen, J. Bartek, and J. Lukas. 2006. Destruction of Claspin by SCFbetaTrCP restrains Chk1 activation and facilitates recovery from genotoxic stress. Mol. Cell. 23:307–318.[CrossRef][Medline]
Mailand, N., S. Bekker-Jensen, H. Faustrup, F. Melander, J. Bartek, C. Lukas, and J. Lukas. 2007. RNF8 ubiquitylates histones at DNA double-strand breaks and promotes assembly of repair proteins. Cell. 131:887–900.[CrossRef][Medline]
Nyberg, K.A., R.J. Michelson, C.W. Putnam, and T.A. Weinert. 2002. Toward maintaining the genome: DNA damage and replication checkpoints. Annu. Rev. Genet. 36:617–656.[CrossRef][Medline]
Olsen, J.V., B. Blagoev, F. Gnad, B. Macek, C. Kumar, P. Mortensen, and M. Mann. 2006. Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Cell. 127:635–648.[CrossRef][Medline]
Petrini, J.H., and T.H. Stracker. 2003. The cellular response to DNA double-strand breaks: defining the sensors and mediators. Trends Cell Biol. 13:458–462.[CrossRef][Medline]
Shiloh, Y. 2003. ATM and related protein kinases: safeguarding genome integrity. Nat. Rev. Cancer. 3:155–168.[CrossRef][Medline]
Stewart, G.S., B. Wang, C.R. Bignell, A.M. Taylor, and S.J. Elledge. 2003. MDC1 is a mediator of the mammalian DNA damage checkpoint. Nature. 421:961–966.[CrossRef][Medline]
Stracker, T.H., J.W. Theunissen, M. Morales, and J.H. Petrini. 2004. The Mre11 complex and the metabolism of chromosome breaks: the importance of communicating and holding things together. DNA Repair (Amst.). 3:845–854.[CrossRef][Medline]
Stracker, T.H., M. Morales, S.S. Couto, H. Hussein, and J.H. Petrini. 2007. The carboxy terminus of NBS1 is required for induction of apoptosis by the MRE11 complex. Nature. 447:218–221.[CrossRef][Medline]
Stucki, M., and S.P. Jackson. 2006. gammaH2AX and MDC1: anchoring the DNA-damage-response machinery to broken chromosomes. DNA Repair (Amst.). 5:534–543.[CrossRef][Medline]
Stucki, M., J.A. Clapperton, D. Mohammad, M.B. Yaffe, S.J. Smerdon, and S.P. Jackson. 2005. MDC1 directly binds phosphorylated histone H2AX to regulate cellular responses to DNA double-strand breaks. Cell. 123:1213–1226.[CrossRef][Medline]
Spycher, C., E.S. Miller, K. Townsend, L. Pavic, N.A. Morrice, P. Janscak, G.S. Stewart, and M. Stucki. 2008. Constitutive phosphorylation of MDC1 physically links the MRE11–RAD50–NBS1 complex to damaged chromatin. J. Cell Biol. 181:227–240.
Tauchi, H., J. Kobayashi, K. Morishima, S. Matsuura, A. Nakamura, T. Shiraishi, E. Ito, D. Masnada, D. Delia, and K. Komatsu. 2001. The forkhead-associated domain of NBS1 is essential for nuclear foci formation after irradiation but not essential for hRAD50[middle dot]hMRE11[middle dot]NBS1 complex DNA repair activity. J. Biol. Chem. 276:12–15.
Zhao, S., W. Renthal, and E.Y. Lee. 2002. Functional analysis of FHA and BRCT domains of NBS1 in chromatin association and DNA damage responses. Nucleic Acids Res. 30:4815–4822.
Zhou, B.B., and S.J. Elledge. 2000. The DNA damage response: putting checkpoints in perspective. Nature. 408:433–439.[CrossRef][Medline]
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Facebook 
Reddit 
Technorati 
Twitter What's this?
Related Article
Related In this Issue article
This article has been cited by other articles:
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
|
