第三拓樸異構酶在端粒重組過程中所扮演的角色

Abstract

端粒 (telomere) 是真核生物染色體末端的特殊結構，能夠防止染色體末端被誤認為染色體損傷的訊息，抑制其不正常的融合及重組。最重要的是，核酸複製機制無法完整複製核酸末端，故每次細胞分裂時端粒的縮短可以做為緩衝，保持遺傳訊息的完整。人類腫瘤細胞可藉端粒酶 ( elomerase) 或端粒重組 (Alternative Lengthening of Telomeres) 維持端粒長度，達到無限複製分裂的永生化 (immortal) 境界。目前已研發可能的端粒酵素抑制劑，於是了解端粒重組機制以發展抑癌藥物為當前熱門議題。在缺乏端粒酵素的酵母菌中也有類似人類腫瘤的端粒重組機制，我們以酵母菌為模式生物，已發現酵母第三拓樸異構酶 (Top3p) 參與在此機制中。下一步，我們想了解第三拓樸異構酶在此機制中，究竟發揮的是其已被證實的解開 Holliday junction 的活性或是其微弱的解開染色體立體超螺旋結構的活性。 在人類骨癌細胞 Saos-2也有相同的現象：抑制人類 TOP3α即使端粒重組無法進行。有趣的是，TOP3α 被抑制的 Saos-2 細胞活化了端粒酶以繼續維持其端粒完整。我們試著探討這兩種端粒維持機制是如何互相轉換，探討轉換的過程中染色質是否重組 (chromatin remodeling)，且有無任何訊息傳導路徑 (signal transduction pathway) 參與其中。

Telomere maintenance is required for chromosome stability. Telomeres are typically replicated by telomerase. In both mammalian tumors and yeast cells that lack telomerase, telomeres are maintain by an alternative (ALT) recombination mechanism. Our previous studies in S. cerevisiae have shown that Top3p, a type IA topoisomerase, together with Sgs1p, are involved in this recombination pathway. We tested whether Top3 in Sgs1-Top3p complex functions as a Holliday junction (HJ) resolvase to remove HJ intermediates, or acts as a topoismerase to relieve the tension of supercoilings within Rap1/Rif complex-protected telomeric heterochromatin. Neither HJ resolvase nor topoismerase expressed in yeast complement the function of Sgs1-Top3p complex. As in S. cerevisiae, reduced human topoisomerase IIIα expression in Saos-2 ALT cells also showed blocked ALT pathway. More strikingly, increased telomerase expression and activity were detected. In order to find out the mechanism to turn on telomerase when ALT is blocked, we first analyzed if chromatin remodeling is involved by ChIP assay. It is possible that there is a signal transduction pathway to activate telomerase activity when ALT pathway is suppressed. We want to compare the difference of gene expression between ALT and ALT-blocked cells by the microarray approach. Howerever, we didn’t get any other ALT- blocked cell lines that also have telomerase activity by knock down topoisomerase IIIα expression.