dNTP Pool Imbalance Induced Endonuclease : Mechanism of All Death.

研究了细胞内脱氧核苷三磷酸（dNTP）库失衡诱导小鼠FM 3A细胞死亡的机制。5-氟脱氧尿苷（FdUrd）可诱导细胞内dNTP池失衡，导致DNA双链断裂和细胞死亡;放线菌酮（cycloheximide）可抑制FdUrd诱导的DNA双链断裂和细胞死亡;在这个实验中，我们已经从FdUrd处理的FM 3A细胞中纯化并表征了双链核酸内切酶。用柱层析法将酶纯化至接近均一。通过含有DNA的SDS-PAGE，核酸内切酶的分子量约为45 kDa。核酸内切酶不需要二价金属阳离子，并且随机切割裸双链体DNA。此外，我们还研究了FdUrd诱导的DNA片段化。FdUrd诱导的DNA片段分为两类。一个是100-200 kbp的大片段，另一个是180 bp左右的梯状DNA片段，其节距与核小体长度相当。在第二种情况下，DNA片段化被认为是细胞凋亡的特征类型。此外，我们发现，核原癌基因，c-fos，c-jun和c-myc的mRNA水平增加，在FdUrd处理的细胞。我们得出结论，纯化的核酸内切酶是一种新的核酸内切酶，不同于以前的特点哺乳动物核酸内切酶。这种酶被称为内切核酸酶K。

The mechanism of intracellular deoxyribonucleoside triphosphates (dNTP) pool imbalance-induced cell death in mouse FM3A cells was studied. We previously reported that 1) 5-fluoro-deoxyuridine (FdUrd) induced intracellular dNTP pool imbalance followed by DNA double strand breaks and subsequent cell death, 2) these DNA double strand breaks and cell death were inhibited by cycloheximide, an inhibitor of protein biosynthesis, 3) an endonuclease activity toward double stranded DNA was detected in the lysate ofFdUrd-treated FM3A cells but not untreated cells. In this experiment, we have purified and characterized the double-stranded endonuclease from FdUrd-treated FM3A cells. The enzyme which was purified to near homogeneity by column chromatographys. The molecular mass of the endonuclease was approximately 45 kDa by SDS-PAGE that contained DNA. The endonuclease required no divalent metal cations and cleaved naked duplex DNA at random. In addition we have investigated the DNA fragmentation induced by FdUrd. FdUrd-induced DNA fragments were separated into two classes. One was large DNA fragment with size of 100-200 kbp, and other was ladder DNA fragment with pitch of nucleosome length, approximately 180 bp. In second case the DNA fragmentation was of the type considered characteristic of apoptosis. Furthermore, we found that mRNA levels of nuclear proto-oncogenes, c-fos, c-jun and c-myc, increased in FdUrd-treated cells. We conclude that the purified endonuclease is a novel endonuclease, distinct from previously characterized mammalian endonucleases. The enzyme, which was called endonuclease K.