dNTP Pool Imbalance Induced Endonuclease : Mechanism of All Death.

研究了细胞内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。我们之前报道过：1)5-氟脱氧尿苷（FdUrd）诱导细胞内dNTP池失衡，导致DNA双链断裂和随后的细胞死亡；2)这些DNA双链断裂和细胞死亡被环己亚胺（一种蛋白质生物合成抑制剂）抑制；3)在经过FdUrd处理的FM3A细胞的裂解液中检测到针对双链DNA的核酸内切酶活性，而未处理的细胞则没有。在本实验中，我们从fdurd处理的FM3A细胞中纯化并表征了双链内切酶。经柱层析纯化，酶纯度接近均匀。SDS-PAGE鉴定该酶分子量约为45 kDa，含有DNA。这种内切酶不需要二价金属阳离子，可以随机切割裸露的双工DNA。此外，我们还研究了FdUrd诱导的DNA断裂。fdurd诱导的DNA片段分为两类。一个是100-200 kbp的大DNA片段，另一个是核小体长度约180 bp的阶梯DNA片段。在第二种情况下，DNA断裂的类型被认为是细胞凋亡的特征。此外，我们发现核原癌基因c-fos、c-jun和c-myc的mRNA水平在fdurd处理的细胞中升高。我们得出结论，纯化的内切酶是一种新的内切酶，不同于以前表征的哺乳动物内切酶。这种酶被称为核酸内切酶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.