dNTP Imbalance and DNA Double Strand Breaks in Mouse FM3A Cells and the Mechanism of Cell Death

研究了三磷酸脱氧核糖核苷（dNTP）池失衡诱导小鼠FM3A细胞死亡的机制。我们以前报道过5-氟脱氧尿苷（FdUrd）可以诱导细胞内dNTP池失衡，随后导致DNA双链断裂和随后的细胞死亡。我们现在已经发现fdurd诱导的DNA片段可以分为两类。一种是大的DNA片段，大小为100-200 kbp，与哺乳动物基因组中的复制单位相对应；另一种是较短的DNA片段，核小体长度的间距，是细胞凋亡的特征。此外，我们还从fdurd处理的FM3A细胞裂解液中纯化了引起双链断裂的内切酶。在fdurd处理的FM3A细胞的裂解物中检测到这种内切酶活性，但在未处理的细胞中检测不到。利用含有dna的SDS-PAGE对该内切酶进行纯化，其分子量估计约为40 kDa。该酶的最适pH值为6.0 ~ 6.5，不需要二价金属阳离子即可发挥活性。核酸内切酶对DNA的切割产生5'-磷酸化末端。具有这些特性的哺乳动物细胞的内切酶尚未在文献中描述。我们怀疑这种内切酶（我们称之为内切酶S）在dNTP失衡死亡中起着重要作用，这一过程在许多方面类似于细胞凋亡，即细胞自杀反应。

The mechanism of deoxyribonucleoside triphosphate (dNTP) pool imbalanceinduced cell death in cultured mouse FM3A cells was studied. We reported previously that 5-fluorodeoxyuridine (FdUrd) can induce intracellular dNTP pool imbalance followed by DNA double-strand breaks and subsequent cell death. We have now found that the FdUrd-induced DNA fragments can be separated into two classes. One was large DNA fragments with sizes of 100-200 kbp, which corresponded to the replication units in mammalian genomes, and the other was shorter DNA fragments with a pitch of nucleosome length, characteristic for apoptosis. In addition, we have purified the double-strand break causing endonuclease from the lysate of FdUrd-treated FM3A cells. This endonuclease activity was detectable in the lysate of FdUrd-treated FM3A cells but not in untreated cells. The endonuclease was purified to near homogeneity and its molecular mass was estimated to be approximately 40 kDa by use of a DNA-containing SDS-PAGE.The endonuclease exhibited an optimal pH of 6.0-6.5 and did not require divalent metal cations for its activity. The cleavage of DNA by the endonuclease produced 5'-phosphoryl termini. Endonuclease of mammalian cells having these properties have not been described in the literature. We suspect that this endonuclease (which we termed Endonuclease S) plays an important role in the dNTP imbalance death, a process in many ways similar to 'apoptosis', the cellular suicide response.