Cell cycle checkpoints are biochemical signaling pathways that inhibit cell cycle progression when DNA is damaged or when antecedent events are incomplete. Checkpoint signaling networks inhibit cell cycle progression by regulating the activity or location of cyclin-dependent kinase complexes that drive cell cycle transitions (60
). For example, the ATM- and p53-dependent DNA damage G1 checkpoint inhibits progression into S phase via transactivation of the Cdk2 inhibitor p21Waf1/Cip1
. The IR-induced S checkpoint appears to inhibit replicon initiation by stimulating the ubiquitin-mediated proteolysis of Cdc25A and thereby inhibiting Cdk2. An ATR- and Chk1-dependent S checkpoint inhibits replicon initiation following treatment with low fluences of UVC. However, the downstream targets of ATR and Chk1 have not been identified, nor has the mechanism that prevents origin firing when S phase cells are damaged. The data presented in this report exclude degradation of Cdc25A and inhibition of Cdk2 as mechanisms by which UV inhibits replicon initiation. Instead, ATR- and Chk1-dependent signaling from sites of UV-induced DNA damage appears to achieve this endpoint by converging on the DDK complex.
The goal of the current study was to investigate the signaling pathway transduced by ATR kinase in human cells exposed to a low fluence of UVC. The data available to date indicate that the UVC-induced S checkpoint inhibits replicon initiation independently of either the ATM-Chk2-Cdc25A-Cdk2 or the ATM-MRN-Smc1 pathways (–). BPDE, the reactive metabolite of benzo[a]pyrene, produces the same stereotypic inhibition of replicon initiation that is observed after treatment with low-dose UVC (61
). The S checkpoint response to low-dose BPDE was also found to be dependent on ATR and Chk1 signaling (62
). Consistent with the data reported herein, Vaziri and colleagues have shown that low-dose BPDE does not induce Cdc25A degradation or the inhibition of Cdk2/cyclin E (63
). Taken together these studies suggest that low-dose UVC and BPDE inhibit replicon initiation by a mechanism distinct from that of IR.
UV-induced Cdc25A degradation has been observed in cells treated with doses of UVC ranging from 15–100 J/m2
). These high doses of UV result in saturation of nucleotide excision repair (64
), blockage of DNA chain elongation in active replicons, and inhibition of entry of cells into mitosis (65
). These stress responses induced in S phase cells by UV are reminiscent of those elicited by treatment with HU. Hence, the UV-induced Cdc25A degradation observed in normal human fibroblasts after exposure to high UVC fluences () could be associated with activation of the replication checkpoint. Cdc25A degradation was correlated also with a dose-dependent increase in UV-induced phosphorylation of Chk1 (). Work from the Harper and Draetta laboratories suggests that Cdc25A degradation, which is dependent on the F-box protein β-TrCP, requires the concerted action of both Chk1 and Chk2 (57
). DNA damage-induced Cdc25A degradation has been observed in cells treated with IR, supra-lethal doses of UVC, and HU, all of which result in the activation of both Chk1 and Chk2 (25
). This would explain the observation that a low fluence of UVC did not affect Cdc25A stability, as Chk2 phosphorylation was not observed under the same conditions ().
Cdc7/Dbf4 is a conserved kinase that has been shown to be essential for the initiation of DNA replication and a target of cell cycle checkpoints in yeast. In S. cerevisiae
Dbf4 interacts with, and is phosphorylated by Rad53 in response to treatment with HU (39
). Rad53-dependent phosphorylation results in the removal of Dbf4 from chromatin and the inhibition of Cdc7/Dbf4 kinase activity. In S. pombe,
the Dbf4 homologue Him1/Dfp1 undergoes Cds1-dependent phosphorylation after treatment with HU (37
). In Xenopus
egg extracts, Cdc7/Dbf4 complexes are targeted by ATR and Chk1 in response to DNA damage induced by either etoposide or exonuclease III treatment (41
). Upon activation, ATR, by as yet unknown mechanisms, regulates the interaction between Cdc7 and Dbf4 and thus the activity of the kinase complex. It appears that a common substrate required for activation of ATR-ATRIP includes extended regions of RPA-coated single-stranded DNA (67
). Physical blockage of the replication fork at a UV-induced photoproduct results in the uncoupling of leading- and lagging-strand synthesis and extended regions of single-stranded DNA are formed downstream from the lesion (68
). Treatment of human cells with UVC produces DNA replication intermediates that contain regions of single-stranded DNA (70
), which are similar to substrates shown to activate the ATR-Cdc7/Dbf4 checkpoint in Xenopus
egg extracts. We determined that human Chk1 could phosphorylate human Dbf4; enhancement of the in vitro phosphorylation signal was absent when Dbf4 was incubated with kinase-dead Chk1. Duncker et al. have shown that scDbf4 interacts with and is phosphorylated by Rad53 in response to treatment with HU (39
). This model of checkpoint-dependent regulation of Dbf4 appeared to be conserved as human Chk1 phosphorylated Dbf4 in vitro
and these proteins interacted in vivo
(). This interaction appeared to be specific since Dbf4 did not co-precipitate with ATR. It would seem plausible that Chk1 activated in response to blocked replication forks could interact with, and phosphorylate Dbf4 transiently, thereby regulating Cdc7/Dbf4 kinase activity. However, the evidence reported herein indicated that over-expression of Flag-Dbf4 did not abrogate the Chk1-dependent, IR-induced S checkpoint. This finding suggests that the Chk1-Dbf4 interaction is not sufficiently strong to compete with the interaction of this kinase with other substrates, such as Cdc25A.
Over-expression of epitope-tagged Dbf4 attenuated the UVC-induced S checkpoint (). These data are in agreement with observations made in yeast and Xenopus
systems. In yeast, over-expression of a 248-aminoacid fragment of scDbf4 was shown to override Rad53-dependent HU-induced inhibition of activation of late origins (39
). Further, the ATR-dependent S checkpoint in Xenopus
egg extracts was reversed by increasing the concentration of recombinant Cdc7/Dbf4 (41
). In the same system, the addition of recombinant Cdc25A to egg extracts reversed the ATM-dependent S checkpoint response induced by linear DNA molecules (resembling a DNA DSB) (42
). Transient over-expression of Cdc25A in HeLa cells also was shown to reverse the IR-induced S checkpoint (25
). In the present study, pretreatment of diploid human fibroblasts with LLnL increased the basal level of Cdc25A, which reversed the IR-induced S checkpoint, but not the response to UVC (). By analogy to the evidence that Cdc25A over-expression attenuated the S checkpoint response to IR, the attenuation of the S checkpoint response to UVC by over-expression of Dbf4 strongly implicates DDK as an effector in the ATR/ATRIP/Claspin/Timeless/Chk1 signaling pathway.
Considering the ability of exogenous Dbf4 to override the S checkpoint, it was expected that endogenous Cdc7 would form active complexes with Flag-Dbf4. Cdc7 was present in Flag-Dbf4 immuno-precipitates, irrespective of the type of induced DNA damage (). These data imply that the ability of Flag-Dbf4 to reverse the S checkpoint was associated with a stable interaction between Cdc7 and Dbf4 in the presence of induced checkpoint signaling. Over-expression of Dbf4 may render ineffective the ATR/Chk1-induced checkpoint signaling that inhibits DDK complexes. An excess of active DDK complexes is then available to activate the Pre-RCs. The observation that an excess of Dbf4 did not attenuate the S checkpoint response to IR suggests that at least one intermediate signaling step might exist between Chk1 and DDK. If the balance between Chk1 and Dbf4 abundance is tilted in the opposite direction by over-expressing Chk1, DNA synthetic activity is almost completely inhibited (27
). These observations are consistent with a model in which ATM and ATR actively monitor DNA synthesis and regulate replicon initiation by constitutively regulating the activity of CDK (ATM) and DDK (ATR) (71
Schematic representation of S checkpoint signaling in human cells.
The data presented here support a model that low fluences of UVC activate an ATR-dependent S checkpoint that inhibits replicon initiation by regulating DDK. It is worth considering, however, that ATR and Chk1 might inhibit replicon initiation by regulating the activity of the pre-replication complex itself. Cortez et al. have shown that the MCM complex is a target of ATM and ATR-dependent phosphorylation in response to IR or UV-induced DNA damage (72
). MCM2 is phosphorylated by ATR on serine 108 in response to UV irradiation. However, the biological significance of this phosphorylation has not yet been demonstrated. Ishimi et al. have shown that MCM4 is phosphorylated in an ATR- and Chk1-dependent manner following treatment of cells with UV and HU, and this phosphorylation may inhibit DNA replication by inactivating the MCM helicase (73
). Recently, another connection between checkpoint signaling and the pre-replication complex was observed. Tsao et al. have shown that UV-induced activation of ATR and Chk1 requires the interaction between Rad17 and Mcm7 (74
). Depletion of either Rad17 or Mcm7 by RNAi-mediated knockdown inhibits ATR-dependent phosphorylation of Chk1 and attenuates the UV-induced S checkpoint. Other potential targets of the S checkpoint include TopBP1 and Mcm10, proteins that are required for the binding of Cdc45 to origins of DNA replication (16
). However, under conditions in which replicon initiation is inhibited by ~50% there was no significant decrease in either TopBP1 or Mcm10 chromatin binding (data not shown). Additionally, Dunphy and colleagues have reported on a Dbf4-related factor (Drf1) (75
). In Xenopus
egg extracts, Drf1 binds DNA following DNA damage and replication stress in an ATR-dependent manner. Drf1 binding to DNA is thought to inhibit replicon initiation by preventing the association of Cdc45 with origins of DNA replication. However, little is known about the role of Drf1 in human cells. Taken together, it appears that ATR and Chk1 can regulate proteins involved in replicon initiation, but whether these responses are essential components of the DNA damage S checkpoint remains to be determined.
In summary, the S checkpoint that inhibits replicon initiation in response to sub-lethal irradiation with UVC is biochemically distinct from that induced by IR. The ATR- and Chk1-dependent S checkpoint response to UVC inhibits replicon initiation without degradation of Cdc25A or inhibition of Cdk2/cyclin E. Chk1 phosphorylated Dbf4 in vitro and over-expression of Dbf4 reversed the UVC-induced S checkpoint, suggesting that Chk1 may regulate DDK to control the rates of replicon initiation in human cells. This regulation, however, may include an intermediate between Chk1 kinase and DDK. Taken together, these data suggest that human cells have evolved multiple mechanisms to inhibit replicon initiation in response to DNA damage ().