| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2005: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 2004: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Research Abstract |
The telomere is an essential nucleoprotein structure that protects the ends of eukaryotic linear chromosomes. The telomere DNA consists of a unique repeat that is synthesized by telomerase, which is a specialized reverse transcriptase. Due to problems with end-replication, cells that lack telomerase activity are unable to maintain the original telomere length after replication. Therefore, successive rounds of replication in telomerase-deficient cells generate unstable telomere-less chromosomes, which subsequently cause impairment of cell proliferation.
The maintenance of telomeric DNA requires additional factors, at least in yeasts, including a wide range of proteins that are involved in DNA metabolism. Using the chromain immunoprecipitation assay, we showed that Tel1p and Mec1p, PIKK family proteins in Saccharomyces cerevisiae, were recruited to the telomeres at specific times in the cell cycle, in a mutually exclusive manner. Mec1p interacted with the telomeres during late S phase, and this interaction required its own kinase activity, which peaked during the S phase. Moreover, we showed that the MRX (Mre11-Rad50-Xrs2) complex, which functions in DNA double-strand break repair, was recruited to the telomeres in the late S phase, and that its loading was related to the extension of single-stranded telomere tails during this period. MRX was required for the late S phase-specific recruitment of Mec1 (ATR) to the telomeres. Mec1, in turn, contributed to the assembly of the telomerase regulators Cdc13 and Est1 at the telomere ends. Based on these results, we proposed a model for the hierarchical assembly of telomere replication proteins in the late S phase, which involves triggering by the loading of MRX onto the chromosome termini. The recruitment of DNA repair-related proteins to the telomeres at particular times in the cell cycle suggests that the normal terminus of a chromosome is recognized as a DNA double-strand break once per cell cycle.
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