Members of the PIKK family of kinases, including ATM, ATR, and DNA-PKcs, are activated in response to various types of DNA damage. It is generally accepted that ATM and ATR are required for signal transduction upon DSBs and replication stress, respectively, whereas DNA-PKcs primarily participates in DSB repair. The simplicity of this model has been challenged by a growing body of evidence suggesting that these PIKK kinases have overlapping roles in the resolution of different types of DNA damage 8; 11; 12
. In the current study, we provide further evidence for the involvement of ATM and DNA-PKcs kinases in response to UV-induced replication stress. We particularly focused on KAP-1 Ser824 and DNA-PKcs Ser2056 phosphorylation as these phosphorylation events are absolutely dependent on ATM and DNA-PKcs, respectively 17; 21; 22
. ATM and DNA-PKcs dependent phosphorylation events were induced by UV with delayed kinetics. ATM-mediated Chk2 and KAP-1 phosphorylations as well as DNA-PKcs Ser2056 autophosphorylation increased slightly at 1 hour and reached peaks at 4 to 8 hours after UV irradiation. These delayed kinetics were in contrast to the rapid increases in levels of ATR-dependent phosphorylations. Clearly, there are distinct phases of signaling events upon UV-induced replication stress: ATR is activated immediately, whereas ATM and DNA-PKcs kinases are activated at later time points. The delayed kinetics or late activation of ATM and DNA-PKcs kinases also coincided with a dramatic increase of UV-induced γH2AX (), which occurred predominantly in response to UV-induced replication stress 16; 23
. Furthermore, UV-induced γH2AX at 8 hours overlapped with positive TUNEL staining (). These evidence are consistent with the previous study that unrepaired UV photolesions are converted into DNA breaks during DNA replication 2
. Taken together, these results imply that the late-phase activation of ATM and DNA-PKcs kinases is due to DSB formation at sites of collapsed replication forks.
ATM is known to play an important role in S-phase checkpoint upon IR treatment or DSB induction 7; 20
; however, it was not clear whether ATM is involved in S-phase checkpoint upon replication stresses. The late activation of ATM-Chk2 signaling pathway upon UV irradiation suggests that ATM compensates for the decreased activity of the ATR pathway at late time points in order to maintain vigilant cell-cycle checkpoint regulation in the presence of stalled replication forks. This notion is supported by our finding that S-phase progression in ATM-defective cells is faster than that in ATM-proficient cells following UV irradiation (), indicating a defective S-phase checkpoint or radioresistant DNA synthesis (RDS) phenotype in ATM-deficient cells. By titration of UV doses, we found that 0.5-1 J/m2
of UV irradiation is sufficient to induce ATM-mediated Chk2 phosphorylation in HeLa cells (Supplemental Fig. 5
). Even after this minimal UV dose, we observed a rapid degradation of Cdc25A in S-phase-synchronized HeLa cells (data not shown). Cdc25A degradation after IR treatment is one of the key events in ATM-mediated S-phase checkpoint 20; 24
and ATM-Chk2 pathway activation after UV treatment is likely required for UV-induced Cdc25A degradation. In addition to its role in S-phase checkpoint response, ATM has also been implicated in regulation of G2-M checkpoint after IR 25
. The increase of BrdU-positive G1 population suggests that UV-irradiated AT5 cells are able to continue cell cycle beyond G2-M phases (), thus confirming that ATM activity is also required for G2-M checkpoint upon replication stresses.
In addition to cell cycle checkpoint regulation, the increase of ATM activity at late time points after UV irradiation may contribute to and sustain some of the S/TQ phosphorylations initiated by ATR. It is well established that ATM and ATR share many common downstream targets 7
. While ATR is essential for the initial increase of SMC1 and H2AX phosphorylations after UV treatment, our results show that there are no significant differences in phosphorylation levels between wild-type and ATR-deficient cells at 8 hours after UV irradiation ( and ). It is likely that overlapping activities of ATR and ATM kinases are required for proper maintenance of these phosphorylation events in response to UV irradiation. Thus, defects in kinases or lack of S/TQ phosphorylations will lead to an increase of cellular sensitivity toward UV irradiation 10; 26
The late activation of DNA-PKcs may contribute to DSB repair activity through the nonhomologous DNA end joining (NHEJ) pathway. Cells lacking functional DNA-PK components are known to have elevated sensitivity toward UV irradiation 9
and we reported previously that DNA-PKcs phosphorylations at the Thr2609 cluster are required for cellular resistance against UV irradiation 11
. Furthermore, a recent study indicated that the DNA polymerase inhibitor aphidicolin induces a surge of DSBs in replicating cells and that DNA-PKcs is required for the repair of these DSBs 27
. This data suggests that DNA-PKcs kinase is important for the repair of replication-associated DSBs. Additionally, DNA-PKcs activity may be involved in cell cycle checkpoint regulation after UV irradiation as it was reported that DNA-PKcs-deficient cells exhibit a radioresistant DNA synthesis (RDS) phenotype 28
, an indication that these cells are defective in intra-S checkpoint. However, the precise role of DNA-PKcs in replication stress-induced checkpoint regulation remains to be clarified.
Although our results clearly demonstrate that UV-induced ATM and DNA-PKcs phosphorylations occur predominantly in replicating cells with stalled replication forks, we do not rule out the possibility that ATM and DNA-PKcs phosphorylations may be involved in the nucleotide excision repair (NER) mechanism as we did observe a slight increase in ATM Ser1981 phosphorylation in G1-phase synchronized cell population after UV irradiation (). The induction of ATM Ser1981 phosphorylation in G1-phase may participate in the NER mechanism as ATM deficient cells are sensitive to UV irradiation and are defective in repair of UV photolesions 10
. Similarly, it was reported that the NER pathway can initiate checkpoint activation in non-proliferative cells 29
and is required for UV-induced γH2AX in G1-phase, although maximum induction of γH2AX occurs during S-phase 23
. This is consistent with our results that UV irradiation leads to a significant induction of KAP-1 and Chk2 phosphorylations in S-phase synchronized cells ( and Supplemental Fig. 2
). In XPC-proficient and XPC-deficient cells there was no clear difference in levels of UV-induced ATM and KAP-1 phosphorylations and that KAP-1 phosphorylation occurred in BrdU-positive replicating cells in the presence and absence of XPC (). Taken together, these data suggest that replication stress is the main cause of activation of the ATM signaling pathway in response to UV irradiation.
In summary, our current study revealed the involvement of ATM and DNA-PKcs in the replication stress response. ATM and DNA-PKcs were both activated by UV irradiation at late time points when DSB formation occurs. In conjunction with early onset of ATR activity, increase of ATM and DNA-PKcs activities likely contribute to cell cycle checkpoint regulation and DSB repair for the maintenance of genomic stability.