The analysis of the profile of

The analysis of the profile of protein phosphorylation in MMS-treated dabigatran etexilate confirmed that both the ATM and ATR pathways were activated in S-phase blocked cells. Interestingly, phosphorylation of Chk1 was observed 24h after MMS-treatment both in AT- and ATM-inhibited cells, whilst in the absence of ATR, no phosphorylation was detected. Chk1 phosphorylation was not noted 24h after MMS-treatment in the simultaneous absence of ATM and ATR. This result is in agreement with observations in ATM or ATR-deficient fibroblasts after HU treatment [25] but differs from those by Cuadrado et al. and Jayazeri et al. who provided evidence that both ATM and ATR are required for Chk1 phosphorylation after IR-induced DSBs [23], [51]. Therefore, the chemical structure of DSBs present after MMS-treatment may differ from those induced by IR and consequently require alternative initial processing or repair pathways. Surprisingly, only the maintenance of Chk1 phosphorylation seems to be ATR-dependent, as we did not observe decreased phosphorylation levels in the absence of both ATR and ATM 3h after DNA damage. A yet unidentified factor might therefore be responsible for the initial activation of Chk1. Phosphorylation of SMC1 24h after DNA damage in XRCC1-deficient MDA-MB-549 cells seems to require both functional ATM and ATR. In agreement with these findings, SMC1 is known to be activated by ATR or ATM depending on the type of DNA damage and in agreement with our findings, interference with its phosphorylation has been shown to abrogate the damage-induced S-phase checkpoint [27], [29]. MMS-treatment is recombinogenic and induces the formation of sister-chromatid exchanges (SCEs) and baseline SCE frequencies are elevated in cells deficient for BER components such as XRCC1 or PARP-1, implying again the generation of replication-mediated DSBs in the absence of functional BER [7], [52], [53]. Additionally, MMS-induced SCE levels in polymerase β mutant cells were higher than in wildtype cells [11]. Therefore, it seems that in BER-deficient cells, recombination is involved in the repair of MMS-induced DNA lesions. This model is supported by the observation in this study of increased levels of Rad51 foci in XRCC1-deficient MDA-MB-549 cells and in polymerase β-defective cells after MMS-treatment [10], [11]. Additionally, BRCA1 has recently been shown to co-localize to nuclear XRCC1 foci upon DNA alkylation damage [54]. Our observations can, however, only be interpreted with caution, as levels of Rad51 foci have been shown to be higher during S-phase in normal cells after IR [55]. MMS clearly leads to a variety of different lesions that activate ATM and ATR and the structure of these lesions differ from those induced by IR or UV. Given that BER machinery, replication and checkpoint proteins were found in a single complex [50], it is tempting to speculate that like in yeast and Xenopus egg extracts [56], [57], a general genome surveillance system in human cells that activates checkpoints upon the formation of N-methylated bases or SSBs does not exist, but instead, that checkpoint cascades are triggered only when the original damage encounters a replication fork resulting in its stalling or collapse and conversion into a double-strand break. In the absence of XRCC1, an accumulation of lesions triggers an ATR- and ATM-dependent S-phase arrest through the well-defined signalling cascades of these two proteins. Horton et al. [58] have recently reported that ATR signalling mediates an S-phase checkpoint after inhibition of PARP activity, another key player in the surveillance system for base damage and single-strand breaks. In a human fibroblast model expressing a kinase dead ATR, treatment with MMS and a PARP inhibitor resulted in the accumulation of cells in the S-phase together with activation of Chk1 and increased H2AX foci formation. The results presented here extend these findings and show that in addition to ATR, a functional and active ATM protein is also necessary for the cellular response to unresolved single-strand breaks.