Defective DNA replication and repair associated with decreases in deoxyribonucleotide pools in a mouse cell mutant with thermolabile ubiquitin-activating enzyme E1.

Upon shift-up in temperature, mouse tsFS20 mutant cells with thermolabile ubiquitin-activating enzyme E1 immediately stopped DNA replication and showed cell cycle arrest in S-phase. In contrast, when the cells were permeabilized with lysolecithin after culture at the nonpermissive temperature, they exhibited a normal level of replicative DNA synthesis in vitro. In agreement with this, intracellular pools of deoxyribonucleoside triphosphates were significantly reduced in the cells cultured at the nonpermissive temperature. Even under the permissive conditions, tsFS20 cells were more sensitive to hydroxyurea and alkylating agents, and induced less mutation than the wild-type cells. These results suggest that the ubiquitin system affects DNA replication and repair.

The molecular mechanisms of 5-fluoro-2'-deoxyuridine induced cell death.

The molecular mechanism of cell death induced by 5-Fluoro-2'-deoxyuridine (FUdR) was investigated. FUdR caused cell death to induce dNTP pool imbalance and following DNA double strand breaks in mouse mammary tumor FM3A cells. We isolated a new endonuclease from FUdR-treated cells, named endonuclease S, that played an important role in FUdR-induced cell death. Cells treated with FUdR showed intracellular acidification before cell death formation. We observed that the endonuclease S in acidic cells may lead the DNA fragmentation. On the other hand, we observed that protease inhibitors (such as TLCK, TPCK, PMSF, p-APMSF, Pefabloc SC and Z-Asp-CH2-DCB) blocked intracellular acidification, DNA fragmentation and FUdR-induced cell death. But the inhibitors did not affect dNTP pool imbalance in the cells. These results suggest that proteases act at the point of downstream of dNTP pool imbalance and upstream of the intracellular acidification.