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J Biol Chem. Jul 3, 2009; 284(27): 18236–18243.
Published online May 7, 2009. doi:  10.1074/jbc.M109.002584
PMCID: PMC2709338

Artemis Regulates Cell Cycle Recovery from the S Phase Checkpoint by Promoting Degradation of Cyclin E*An external file that holds a picture, illustration, etc.
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Abstract

Artemis, a member of the SNM1 gene family, is a known phosphorylation target of ATM, ATR, and DNA-PKcs. We have previously identified two serine residues in Artemis (Ser516 and Ser645) that are subject to phosphorylation by ATM and are involved in mediating recovery from the G2/M checkpoint in response to ionizing radiation. Here we show that these same sites are also phosphorylated by ATR in response to various types of replication stress including UVC, aphidicolin, and hydroxyurea. We also show that mutation of the Ser516 and Ser645 residues causes a prolonged S phase checkpoint recovery after treatment with UV or aphidicolin, and that this delayed recovery process coincides with a prolonged stabilization of cyclin E and down-regulation of Cdk2 kinase activity. Furthermore, we show that Artemis interacts with the F-box protein Fbw7, and that this interaction regulates cyclin E degradation through the SCFFbw7 E3 ubiquitin ligase complex. The interaction between Artemis and Fbw7 is regulated by phosphorylation of Ser516 and Ser645 sites that occur in response to replication stress. Thus, our findings suggest a novel pathway of recovery from the S phase checkpoint in that in response to replication stress phosphorylation of Artemis by ATR enhances its interaction with Fbw7, which in turn promotes ubiquitylation and the ultimate degradation of cyclin E.

As an arm of the DNA damage response cell cycle checkpoints maintain genomic stability by allowing time for DNA repair processing to be completed before resumption of the cell cycle (1). Much progress has been made on the elucidation of the mechanisms that lead to the detection of structural alterations in DNA and the implementation of checkpoint pathways, however, the processes by which the cell cycle reinitiates are not as well understood. Resumption of cell cycle progression after genotoxic stress is usually referred to as the recovery process. Recent findings have shown that recovery is an active process and not simply an attenuation of the initial checkpoint response (2). Perhaps the best studied recovery process is the resumption of the cell cycle from the G2 checkpoint. In this mechanism the kinase Plk1 mediates degradation of Claspin, an activator of Chk1, and Wee1, a negative regulator of Cdk1 (35). Phosphorylation of these two substrates leads to ubiquitylation by the SCFβTrcP E33 ligase, and ultimate degradation by the proteosome (6, 7). Recently it has been shown that Plk1 is activated to promote recovery by phosphorylation of its Thr210 residue by the Aurora A kinase (8), although, how completion of DNA repair results in activation of Aurora A is not clear. Other mechanisms to induce resumption of the cell cycle include dephosphorylation of p53, Chk1, and γH2AX, and the induction of Cdc25B (912).

Cdk2 and its partners cyclin E and cyclin A are crucial regulators of G1/S transition and progression through S phase. Cyclin E accumulates in late G1 as a result of E2F-mediated transcriptional regulation, which has been previously activated by cyclin D-associated kinases. During S phase cyclin E is degraded by two independent pathways. Cyclin E unbound to Cdk2 is targeted by the Cul3-based E3 ubiquitin ligase (13), whereas Cdk2-bound cyclin E is targeted by the SCFFbw7 ubiquitin ligase in a process that requires phosphorylation of cyclin E (1421). Interestingly, ectopic overexpression of cyclin E has been shown to accelerate entry into S phase, but somewhat paradoxically also slows progression through S phase (2225). One possible mechanism by which overexpression of cyclin E prolongs S phase is an interference with pre-RC assembly during G1, which ultimately leads to lower levels of replication initiation (26).

Artemis is a member of the SNM1 gene family that is characterized by conserved metallo-β-lactamase and β-CASP domains (27). Artemis has roles in V(D)J recombination, nonhomologous end-joining mediated repair of double strand breaks, and in cell cycle regulation after DNA damage (2732). It is a known substrate both in vitro and in vivo of the phosphatidylinositol kinases ATM, ATR, and DNA-PK in response to many types of genotoxic agents (28, 30, 3235). With regard to its cell cycle functions, we have shown previously that Artemis is involved in regulating the recovery from the G2 checkpoint in response to ionizing radiation (IR) through regulation of the activation of cyclin B-Cdk1 (28, 32). Furthermore, Artemis was shown to be phosphorylated on its Ser516 and Ser645 residues by ATM, and mutation of these two sites to alanine led to a slower recovery from the G2/M checkpoint.

In prior work (32) we have also shown that Artemis is phosphorylated in vivo in response to UV irradiation, however, the functional significance of phosphorylation in response to this agent has not been elucidated. We show here that Artemis is involved in the recovery from the S phase checkpoint in response to replication fork blocking lesions, and that this function is dependent upon phosphorylation at the Ser516 and Ser645 sites by ATR. Furthermore, we show that the role of Artemis in S phase checkpoint recovery is through an involvement in the degradation of cyclin E by the SCFFbw7 E3 ligase complex. Artemis interacts with Fbw7 to promote degradation of cyclin E and to enhance recovery from S phase. Phosphorylation of Artemis on Ser516 and Ser645 promotes the interaction between Artemis and Fbw7, thus facilitating the turnover of cyclin E and S phase recovery.

EXPERIMENTAL PROCEDURES

Cells and Antibodies

HEK293 cells stably expressing Artemis alleles, tissue culture protocols, and DNA transfections were previously described (28). Mouse monoclonal antibody against cyclin E (sc-247), and rabbit polyclonal antibodies to cyclin E (sc-481), Cdk2 (sc-163), and cyclin A (sc-751) were purchased from Santa Cruz Biotechnology, Inc. Antibodies to Chk1 and phosphorylated Chk1 (pS345) were obtained from Cell Signal Technology. ATR antibodies were described previously (32). FLAG (M2)-conjugated beads were purchased from Sigma. Artemis and Artemis phospho-specific antibodies were previously described (28).

Plasmids and siRNAs

Wild-type and mutant Artemis constructs were previously described (28). FLAG-Fbw7 expression plasmids were gifts from B. Clurman (Fred Hutchinson Cancer Research Center, Seattle, WA). Artemis siRNAs were described previously (32). Cyclin E (1214563-H/904; 1214564) and ATR (1214725/747; 121426-H) siRNAs were purchased from Sigma.

Immunoblotting, Immunoprecipitation, and IP Kinase Assays

Immunoblotting, immunoprecipitation (IP), and IP kinase assays have been previously described (32). Briefly, cell lysates were prepared in RIPA buffer with protease inhibitors and 10 mm sodium fluoride, 0.2 mm sodium vanadate, 8 mm β-glycerol phosphate, and 1 mm dithiothreitol. Cell lysates were then incubated with primary antibody for 2 h and protein A/G beads for an additional 45 min at 4 °C. IP kinase assays were carried out in 10 mm MgCl2, 1 mm dithiothreitol, 70 mm NaCl, 10 μm ATP, 0.1 μg/μl histone H1 (Millipore), and 5 μCi of [γ-32P]ATP (PerkinElmer Life Sciences) at room temperature for 30 min.

In Vivo Ubiquitylation Assay

pCMV-HA-ubiquitin was transfected into HEK293 cells to facilitate detection of ubiquitin conjugates. Transfection efficiency was monitored by cotransfection of 100 ng of pEGFP-N1 (Clontech) plasmid. Twenty-four h after transfection cells were treated with MG-132 for 4 h and cell lysates were prepared for immunoblotting.

Flow Cytometry and BrdUrd Labeling

Cell cycle analysis by flow cytometry and BrdUrd labeling were previously described (28). Briefly, cells were incubated with 10 μm BrdUrd for 15 or 30 min prior to harvesting, or released into regular medium and harvested at various time points. Cells were fixed in 70% ethanol and incubated in PBS containing 4% bovine serum albumin and 0.2% Triton X-100 for 1 h at room temperature. DNA was denatured in 2 n HCl for 20 min, washed with PBS, and resuspended in 0.1 m sodium tetraborate. Cells were exposed to monoclonal anti-BrdUrd (BD Transduction Laboratories) for 1 h, washed with PBS, and incubated with fluorescein isothiocyanate-conjugated goat antibodies to mouse IgG (Jackson Laboratories) for 30 min. After washing with PBS, cells were resuspended in PBS containing 40 μg/ml propidium iodide, and 50 μg/ml DNase-free RNase (Calbiochem). Fluorescence was measured on a FACS Calibur flow cytometer (BD Biosciences) using 488 nm laser excitation.

RESULTS

Artemis Is Phosphorylated by ATR on Ser516 and Ser645 in Response to Replication Stress

We have shown previously that in response to IR Artemis is rapidly phosphorylated on four serine residues, namely Ser516, Ser534, Ser538, and Ser645 (28). Two of these sites, Ser534 and Ser538, undergo rapid phosphorylation and dephosphorylation within 1–2 h after exposure to DNA damage, whereas the other two sites, Ser516 and Ser645, exhibit rapid and prolonged phosphorylation for up to approximately 24 h. We have also previously shown that Artemis is phosphorylated in response to UV irradiation (32), we therefore examined the kinetics of these phosphorylation events in response to UV. Phosphorylation at Ser516 and Ser645 was observable at 30 min, peaked from 2 to 12 h, and then slowly declined until 24 h (Fig. 1A). Rapid and prolonged phosphorylation of Artemis at Ser516 and Ser645 was also observed in vivo in response to aphidicolin and hydroxurea (Fig. 1B and data not shown). Thus similar to IR, Artemis also exhibits rapid and prolonged phosphorylation at Ser516 and Ser645 in response to replication stress.

FIGURE 1.
Artemis is phosphorylated by ATR on residues Ser516 and Ser645 in response to replication stress. A, immunoblot showing kinetics of Artemis phosphorylation on Ser516 and Ser645 in response to UV irradiation. HEK293 cells stably expressing GST-Artemis ...

We have also shown previously that ATM was the principal kinase for phosphorylation of Ser516 and Ser645 in response to IR (28). To determine the kinase responsible for UV-induced phosphorylation at these two sites, we examined their phosphorylation after UV in the presence of caffeine, an inhibitor of ATM and ATR. Reduced phosphorylation was observed in the cells treated with caffeine (Fig. 1C). To further identify the kinase, we used siRNA to deplete ATR, and then examined the phosphorylation at each site. siRNA-mediated knockdown of ATR significantly reduced the phosphorylation at each site (Fig. 1D). These results indicate that ATR is the major kinase responsible for Artemis phosphorylation at the Ser516 and Ser645 sites in response to UV.

Mutation of Ser516 and Ser645 Results in Prolonged S Phase Arrest

We previously reported that HEK293 cells expressing a mutant of Artemis in which the serines at Ser516 and Ser645 had been mutated to alanine residues exhibited defective recovery from the G2/M cell cycle checkpoint after IR (28, 32). Because UV induces a significant S phase arrest, we examined this Artemis mutant (termed S516A/S645A) to determine whether these two phosphorylation sites also affect the recovery from the S phase checkpoint. As shown (Fig. 2A), cells expressing the S516A/S645A mutant showed an accumulation of cells in S phase 12–18 h after UV treatment compared with cells expressing wild-type Artemis. In addition, expression of a mutant Artemis (S516D/S645D) in which the serines at 516 and 645 were mutated to aspartic acid residues to mimic phosphorylation, resulted in an essentially wild-type phenotype. To further support these results, cells were incubated with BrdUrd for 15 min, treated with UV, and returned to regular medium. Twelve h later cells expressing the S516A/S645A mutant exhibited a higher S phase content compared with cells expressing wild-type Artemis or the S516D/S645D mutant (Fig. 2B).

FIGURE 2.
Mutation of Ser516 and Ser645 results in prolonged S phase arrest in response to replication stress. A, cell cycle analysis of HEK293 cells after UV treatment. Cell were treated with UV (3 J/m2), incubated for the indicated times, and analyzed by FACS. ...

To ensure that the accumulation in S phase observed after UV treatment was not the result of effects in the G1 or G2/M stages of the cell cycle, we synchronized cells at early S phase by treatment with aphidicolin, and then released them into regular medium without drug. At 12 h after release a greater accumulation was observed in the G1/S phase in cells expressing the S516A/S645A mutant compared with wild-type Artemis or the S516D/S645D mutant (Fig. 2C). In addition, we repeated this experiment, but introduced a 30-min pulse labeling of the cells with BrdUrd immediately before harvesting. Again this experiment indicated that a greater fraction of cells expressing the S516A/S645A mutant were in S phase compared with the control cell lines (Fig. 2D). Finally, we compared cells expressing the S516A/S645A mutant with those expressing the S516D/S645D mutant and found that in the absence of DNA damage the former did not exhibit a prolonged S phase (Fig. S1). Taken together, these results demonstrate that phosphorylation of Ser516 and Ser645 of Artemis facilitates recovery from the replication checkpoint in response to DNA damage.

Artemis Phosphorylation Regulates Recovery from the S Phase Checkpoint through Timely Destruction of Cyclin E

A possible explanation for the S phase accumulation in cells expressing the S516A/S645A mutant is a prolongation of the S phase checkpoint via the ATR-Chk1 pathway (36, 37). However, an examination of phosphorylation of Chk1 at Ser345, a marker of S phase checkpoint activation, after exposure of cells to aphidicolin or UV showed no significant differences among the three cell lines expressing the various forms of Artemis (Fig. S2). Thus, prolonged activation of the S phase checkpoint does not explain the accumulation of cells caused by the S516A/S645A mutant.

The target of the S phase checkpoint is the cell cycle kinase Cdk2. Using an IP kinase assay we found that the activity of this enzyme was reduced in cells expressing the S516A/S645A mutant particularly at 12 h after UV exposure when the greatest levels of S phase accumulation were observed (Fig. 3A). Thus, the Cdk2 kinase, which is principally responsible for driving cells through S phase, has reduced activity in cells expressing the S516A/S645A Artemis mutant.

FIGURE 3.
Artemis phosphorylation regulates recovery from the S phase checkpoint through timely destruction of cyclin E. A, Cdk2 activity is affected by the phosphorylation of Artemis at Ser516 and Ser645. HEK293 cells expressing wild-type (WT), S516A/S645A or ...

Cyclin E and cyclin A are the principal cyclins that regulate Cdk2 activity during the S to G2 transition (3840). Ectopic overexpression of cyclin E in specific cell lines is known to cause a delayed progression through S phase (2225), and previous studies have shown that replication stress causes an increase in cyclin E levels and a slowing of DNA replication (41). We, therefore, examined cyclin E protein levels in cells expressing wild-type Artemis, and the S516A/S645A and S516D/S645D mutants after UV irradiation or release from aphidicolin. We observed significantly higher cyclin E levels in cells expressing the S516A/S645A mutant, whereas cyclin A levels were not significantly different (Fig. 3B). Overexpression of cyclin E could slow S phase progression by competing with cyclin A for binding to Cdk2. An examination of the interaction between cyclin A and Cdk2 by co-IP analysis showed that less cyclin A was bound to Cdk2 in cells expressing the S516A/S6456A mutant (Fig. 3C). Thus a likely explanation for our findings is that the S516A/S645A mutant prevents the normal degradation of cyclin E that occurs during S phase upon recovery from the checkpoint, and this failure to degrade cyclin E interferes with the normal activation of cyclin A-Cdk2.

Artemis Depletion Stabilizes Cyclin E and Delays Recovery from the S Phase Checkpoint

As shown above, HEK293 cells that overexpress the S516A/S645A Artemis mutant exhibit a delayed progression through S phase after replication stress. To directly demonstrate that Artemis is required for normal recovery from the S phase checkpoint, we depleted endogenous Artemis by siRNA, exposed the cells to aphidicolin for 14 h, and then released them into regular medium. As shown (Fig. 4, A and B), depletion of Artemis resulted in a slower recovery from the aphidicolin treatment similar to that observed with overexpression of the S516A/S645A mutant. We next determined whether depletion of Artemis also resulted in stabilization of cyclin E as observed above with overexpression of the S516A/S645A mutant. Upon release from aphidicolin cyclin E is strongly stabilized in cells depleted of Artemis compared with cells treated with a control siRNA (Fig. 4C). These findings suggest that Artemis is required for degradation of cyclin E during recovery from the replication stress checkpoint, and that the S516A/S645A mutant acts as a dominant negative in this pathway.

FIGURE 4.
Depletion of Artemis stabilizes cyclin E and delays recovery from the S phase checkpoint. A, depletion of Artemis from HEK293 cells prolongs S phase arrest in response to replication stress. HEK293 cells were transfected with the indicated siRNAs. Forty ...

Artemis Regulates Cyclin E Abundance through SCFFbw7-mediated Ubiquitylation

To elucidate the mechanism by which Artemis affects cyclin E abundance, we first examined the effect of the S516A/S645A mutation on cyclin E protein stability. Cycloheximide was added to HEK293 cells stably expressing wild-type Artemis or the S516A/S645A mutant 30 min before treatment with UV. Samples were collected at the indicated times and cyclin E levels were determined by immunoblotting (Fig. 5A). The Artemis S516A/S645A mutant exhibited a reduced cyclin E turnover compared with wild-type Artemis. Because cyclin E is degraded through ubiquitin-mediated proteolysis, we next assessed the ubiquitylation of cyclin E in vivo. In the presence of the proteosome inhibitor MG-132, levels of ubiquitinated cyclin E were far lower in cells expressing the S516A/S645A mutant compared with cells expressing wild-type Artemis (Fig. 5B). This result demonstrates that ubiquitylation of cyclin E is impaired by the expression of the S516A/S645A Artemis mutant.

FIGURE 5.
Artemis regulates cyclin E stability via SCFFbw7-mediated ubiquitylation. A, Artemis phosphorylation affects cyclin E stabilization at the post-translational level. HEK293 cells expressing wild-type or the S516A/S645A mutant were irradiated with UV (3 ...

Cyclin E is a substrate of the Skp1-Cul1-F-box (SCF) E3 ubiquitin ligase complex, and the F-box protein or substrate receptor for cyclin E is Fbw7/Cdc4 (16). Because Artemis affects cyclin E ubiquitylation in vivo we examined whether Artemis interacts with Fbw7. Fbw7 has three isoforms α, β, and γ, two of which, α and γ, have been implicated in the ubiquitylation of cyclin E, although the functions of these isoforms are distinctly different (21). To assay for an interaction between Artemis and Fbw7, we cotransfected HEK293 cells with GST-Artemis and FLAG-Fbw7 α, β, or γ isoforms, and performed reciprocal co-IPs. Interestingly, Artemis co-IPed with the Fbw7 α and γ isoforms, but not with the β isoform (Fig. 5C), which indicates that the interaction is specific for the former two isoforms. Because the phosphorylation state of Artemis affects the stabilization of cyclin E, we examined the interaction between Artemis and the Fbw7 α and γ isoforms after UV irradiation. As shown (Fig. 5D), UV irradiation increased the co-IP between these proteins. Furthermore, prior treatment of the cells with wortmannin decreased the interaction suggesting that phosphorylation of Artemis helped to promote the interaction with Fbw7 α and γ. To confirm this possibility, the co-IP wild-type and S516A/S645A or S516D/S645D mutants of Artemis with Fbw7 α and γ were compared. As shown (Fig. 5E), after UV irradiation the wild-type protein interacted more efficiently than did the S516A/S645A mutant protein (upper panel). Interestingly, the S516D/S645D mutant appeared to exhibit an enhanced interaction particularly in the untreated samples (lower panel). Note, however, that phosphorylation of Artemis is not necessary for the interaction between Fbw7 isoforms and cyclin E (Fig. S3), suggesting that Artemis is required subsequent to cyclin E recruitment.

Finally, as shown above (Fig. 1), ATR is required for the phosphorylation of Artemis at Ser516 and Ser645 in response to replication stress. We, therefore, examined whether ATR affected the interaction between Artemis and Fbw7 isoforms, and whether it affected the ubiquitylation of cyclin E. As shown (Fig. 6, A and B), in both cases depletion of ATR by siRNA reduced Artemis-Fbw7 interactions and reduced cyclin E ubiquitylation to levels observed with the expression of the S516A/S645A mutant after exposure to UV irradiation. Taken together, these findings indicate that Artemis is involved in the ubiquitylation of cyclin E by the SCFFbw7 E3 ligase through an interaction with the α and γ isoforms of Fbw7, and that phosphorylation of Artemis on residues Ser516 and Ser645 by ATR positively regulates this association.

FIGURE 6.
ATR is required for the Artemis-mediated degradation of cyclin E. A, depletion of ATR by siRNA reduces the interaction between Artemis and Fbw7 isoforms α and γ. HEK293 cells were transfected with GST-Artemis and with either control or ...

DISCUSSION

As we and others have shown previously Artemis is a substrate of both ATM and ATR, and is phosphorylated by at least one of these kinases in response to various types of genotoxic stress (28, 30, 3235, 4245). In this report we demonstrate that Artemis is phosphorylated at residues Ser516 and Ser645 in response to UV irradiation or aphidocolin exposure by the ATR kinase. Cells that express a mutant of Artemis with these residues converted to alanine exhibit a prolonged S phase and a failure to recover normally from the S phase checkpoint. There are a number of possible mechanisms that could lead to this phenotype. One is that a failure to repair UV-induced lesions would lead to a prolonged checkpoint response. However, whereas Artemis has been reported to have a role in the repair of double strand breaks, no such function has been ascribed for Artemis in the repair of photolesions. Furthermore, Artemis-deficient cells are not sensitive to fork blocking adducts such as those induced by mitomycin C or etoposide (31, 35). In addition, our results indicate that there is not a prolonged activation of Chk1 in cells expressing the S516A/S645A mutant. Rather, our results indicate that the abnormal recovery from the S phase checkpoint is due to stabilization of cyclin E that preferentially occurred in cells expressing the mutant form of Artemis in comparison to the wild-type protein. Previous studies have shown that ectopic overexpression of cyclin E results in a more rapid transition from the G1 to S phase, but a slower progression through S phase (2225). Thus, the abnormal stabilization of cyclin E observed due to expression of the S516A/S645A mutant likely accounts for the failure of cells to properly recover from the S phase checkpoint, and indicates that phosphorylation of Artemis at Ser516 and Ser645 is necessary for degradation of cyclin E after genotoxic stress. Depletion of Artemis by siRNA also resulted in the same phenotypic consequences, thus adding support for this model. Our results further show that the reduced degradation of cyclin E during S phase checkpoint recovery in cells expressing the S516A/S645A mutant results in impaired Cdk2 activity concomitant with a reduced interaction between Cdk2 and cyclin A. Thus, high levels of cyclin E appear to interfere with the association between Cdk2 and cyclin A, which is required for reinitiation of DNA replication during checkpoint recovery.

Cyclin E is ubiqutinated and ultimately degraded during S phase by the SCFFbw7 E3 ligase complex and the 26 S proteosome (16). Of the three alternative splice isoforms of Fbw7 only two, α and γ, participate in the ubiquitylation of cyclin E, although with distinct functions (21). Fbw7α is required for the recruitment of Pin1 and cyclin E to the SCFFbw7γ complex to promote prolyl cis/trans isomerization of cyclin E. The isomerized cyclin E is then competent for multiubiquitylation by the SCFFbw7γ complex. Our results also show that expression of wild-type Artemis, but not the S516A/S645A mutant, induces strong ubiquitylation of cyclin E in vivo. Furthermore, Artemis interacts with the Fbw7α and Fbw7γ isoforms, but not with the Fbw7β isoform, in vivo, and this interaction is enhanced by UV irradiation. In addition, consistent with the reduced degradation of cyclin E observed in cells expressing the S516A/S645A mutant, the interaction between the Fbw7α and Fbw7γ isoforms with this mutant protein were reduced, whereas the interactions appeared to be enhanced in cells expressing the S516D/S645D mutant. Nevertheless, the degradation of cyclin E was not enhanced by the S516D/S645D mutant, thus suggesting that Artemis is necessary, but not sufficient, for triggering checkpoint recovery. This is a reasonable scenario because phosphorylation of Artemis occurs rapidly after UV irradiation, whereas the degradation of cyclin E during checkpoint recovery occurs hours later. Thus, our findings indicate that in addition to its well recognized role in the initiation of the replication checkpoint, ATR, via Artemis, also regulates recovery from the checkpoint. This mechanism is highly similar to our published findings showing that ATM regulates recovery from the G2/M checkpoint after IR via phosphorylation of Artemis (28, 32).

A trivial explanation for these findings, namely that Artemis is a substrate of the SCFFbw7 complex and that its overexpression competes with cyclin E is not supported by our results. Overexpression of wild-type Artemis causes increased ubiquitylation of cyclin E and increased interaction with Fbw7α and Fbw7γ, results, which are contrary to the predictions of this model. Rather, Artemis appears to interact with the SCFFbw7 complex to promote ubiquitylation of cyclin E, perhaps acting as a substrate specificity factor to enhance rapid degradation of cyclin E during checkpoint recovery. This model is further supported by our finding that DNA damage-induced phosphorylation of Artemis by ATR enhances its interaction with Fbw7 and the multiubiquitylation of cyclin E. Interestingly, it has recently been shown that cyclin E is a direct phosphorylation substrate of ATR in response to DNA damage indicating that ATR may regulate cyclin E through multiple pathways (46).

Artemis has, in cooperation with DNA-PKCs, a defined role in the cleavage of DNA hairpins at coding joints during V(D)J recombination (29, 44, 47). We have previously shown that Artemis also functions in the recovery from the G2/M checkpoint after IR treatment via regulation of the activation of cyclin B-Cdk1 (28), which showed initially that Artemis has a role in cell cycle regulation. Here we have demonstrated an additional function for Artemis in the recovery from the S phase checkpoint via regulation of cyclin E. Furthermore, we show that the mechanism of this regulation of cyclin E by Artemis is as an interacting partner of the SCFFbw7 E3 ligase complex. Taken together, these findings define a dramatically novel role for Artemis as a component of the ubiquitin-proteosome system.

Supplementary Material

Supplemental Data:

*This work was supported, in whole or in part, by National Institutes of Health NCI Grants CA096574 and CA097175 (to R. J. L.). This work was also supported by DNA sequencing resources from Cancer Center Support Grant CA16672.

An external file that holds a picture, illustration, etc.
Object name is sbox.jpg The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S3.

3The abbreviations used are:

E3
ubiquitin-protein isopeptide ligase
IR
ionizing radiation
siRNA
small interfering RNA
IP
immunoprecipitation
HA
hemagglutinin
PBS
phosphate-buffered saline
GST
glutathione S-transferase
FACS
fluorescence-activated cell sorter.

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