Analysis of the maintenance and repair mechanism of the mitochondrial DNA using fission yeast.

线粒体的维护和修复机制！对线粒体DNA进行了研究，主要以裂殖酵母(Schizosaccharmyces Pombe)为模式生物。一种可能的机制是线粒体的碱基切除修复(v-ZR)系统。另一种可能是线粒体！内切酶如内切酶G(Endo G)降解并消除受损的线粒体DNA，以维持正常的线粒体DNA。我们分析了与核BER有关的DNA糖基酶(NTH1)的亚细胞定位，发现该蛋白的N末端序列具有对线粒体和核的分选信号。接下来，我们发现Endo G的活性是由线粒体调节的！磷脂、多胺和单链DNA结合蛋白等因素。为了研究线粒体核酸内切酶的生理功能，我们分离了裂解酵母线粒体内切酶SpNUCI的CDNA*，它由322个氨基酸组成，与芽殖酵母NUC1和哺乳动物内切酶G有显著的同源性。通过在酵母中表达SpNUCI-绿色荧光蛋白融合蛋白，证实了酶在线粒体中的定位。核酸内切酶是通过截断蛋白质的氨基末端区域而被激活的，这表明该酶被编码为一个非活性前体。该酶降解单链DNA和RNA，其活性依赖于Mg2(Mn2)。在野生型细胞、Sm/d缺失突变体和SpNUCI过表达细胞中未观察到表型变化。然而，在生长的死亡阶段，Spnuci的数量增加。此外，DNA损伤剂处理可诱导SpNUCI的表达。这些结果表明，SpNUCI参与了细胞的程序性死亡。

Maintenance and repair mechanism of the mitochondria! DNA (mtDNA) was studied, mainly using fission yeast (Schizosaccharomyces pombe) as a model organism. One possible mechanism is base excision repair v-ZR) system in mitochondria. Another possibility, a mitochondria! endonuciease such as endonuclease G (endo G) degrades and eliminates the damaged mtDNA for maintenance of normal mtDNA. We analysed subcellular localization of a DNA glycosylase (NTH1) involved in nuclear BER : and found that the N-terminal sequence of the protein has sorting signals to both mitochondria and nuclei. Next, we found that the activity of endo G is modulated by mitochondria! factors such as phospholipids, polyamines and single-strandedk DNA-binding protein. To study the physiological roles of mitochondrial endonuclease, we isolated the CDNA* of the fission yeast mitochondrial endonuciease SpNUCI, which consists of 322 amino acids and has a significant homology with the budding yeast NUC1 and mammalian endonuclease G. Comparison of the CDNA sequence with the genomic sequence showed that the gene consists of three exons and two introns and spans 1.31 kb. The enzyme localization in mitochondria was demonstrated by expressing the SpNUCI- green fluorescent protein fusion in the yeast. The endonuclease was activated by truncation of the amino terminal region of the protein, indicating the enzyme is encoded as an inactive precursor. The active enzyme degraded single-stranded DNA and RNA, the activity being dependent on Mg2+ (Mn2+). Phenotypic change was not observed among wild type cells, Sm/d-lacking mutant, and SpNUCI-overexpressing cells. However, he amount of SpNUCI increased at death phase in the growth. Moreover, the treatment of the DNA damage agent induced expression of SpNUCI. These result suggested that the SpNUCI is involved in programmed cell death.