| Budget Amount *help |
¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 2001: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 2000: ¥2,500,000 (Direct Cost: ¥2,500,000)
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| Research Abstract |
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.
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