—In reply

Work from our laboratories showed that MMR-deficient cells had defective activation of the S-phase checkpoint and that this correlated with diminished activation of Chk2 in response to ionizing radiation1. Cejka et al. now report that MMR deficiency does not result in these defects. MMR deficiency results in a mutator phenotype; hence, it is conceivable that secondary mutations, which either positively or negatively effect cellular response to genotoxic events, could be responsible for these conflicting results. Differences in cell strains, culture or treatment conditions could also contribute to these contrasting findings. In agreement with our findings, however, Franchitto et al.2 also observed defective ionizing radiation–induced phosphorylation of Chk2 in Msh2-deficient mouse embryonic fibroblasts (MEFs).

Although the engineered MMR-inducible cell line used by Cejka et al. offers a technical improvement over the MMR-deficient tumor lines complemented by stable introduction of large fragments of human chromosomes used in many studies, including ours, the isogenic 293T Lα+ and 293T Lα− lines are derivatives of the transformed human embryonic kidney line 293T, which is MMR-deficient due to epigenetic silencing of the MLH1 promoter3. Thus, it cannot be strictly ruled out that these isogenic lines, like other commonly used MMR-deficient tumor lines, contain other deleterious mutations.

Defects in cell cycle arrest and apoptosis linked to MMR deficiency are most prominently seen in response to SN1 alkylators such as N-methyl-N′-nitro-N-nitrosoguanidine and methyl-nitrosourea, the methylating compound temozolomide, the base analog 6-thioguanine and cisplatin. But it may be premature to conclude that MMR does not have any role in ionizing radiation–induced DNA damage signaling. For example, Franchitto et al.2 reported that Msh2 was required to sustain G2 arrest in response to ionizing radiation, a finding that is in agreement with studies conducted on MMR-deficient human tumor lines4,5. Furthermore, Zeng et al.6 showed that Pms2-deficient MEFs had significantly less apoptosis after irradiation than wild-type MEFs, and similar results were obtained in a study that examined Msh2-deficient mouse embryonic stem cells7.

Ionizing radiation induces a wide spectrum of damage in DNA, including base oxidation and damage to the phosphodiester backbone resulting in single- and double-strand breaks. We stated that we were unsure of the exact nature of the ionizing radiation–induced lesion(s) recognized by MMR but suggested that the presence of the oxidized purine GO could trigger assembly of an MMR complex. This possibility is supported, in part, by the observation that MSH2-MSH6 heterodimers bind to both GO:C and postreplicative GO:A base pairs in vitro8. A recent report indicates that oxidation of free nucleotide pools is an important source of GO9 but does not exclude the possibility that oxidation of guanine to GO occurs in situ in DNA. Moreover, the work of Chen and colleagues indicates that GO is detectable in genomic DNA shortly after irradiation of mitotically synchronized and asynchronous cells10. As our S-phase checkpoint assay positively indicated DNA synthesis in irradiated cells (as judged by uptake of radioactively labeled thymidine), we cannot rule out either GO incorporation into the genome as a result of postirradiation replication or oxidation in situ. A more comprehensive understanding of a potential function for MMR in response to ionizing radiation–induced DNA damage and the nature of the lesions that trigger such response will require further investigation.