端粒重組需要拓樸異構酶的參與

Abstract

端粒 (Telomere) 是動態DNA蛋白質複合體，能夠保護線狀染色體，避免整個基因組不穩定以及染色體末端之間互相融合。大部分端粒DNA都是由端粒酶 (Telomerase) 合成。癌細胞可以藉由端粒酶或是替代重組機制來延長端粒 (Alternative Lengthening of Telomere)。要去了解ALT分子機制並非單是為了研究端粒問題，ALT是目前以端粒酶為目標療法失敗或是癌細胞得到抗藥性主因。本篇研究分別在酵母菌以及人類細胞中去探討端粒複製重組機制，我們已經知道在酵母菌裡拓樸異構酶2和3(Topoisomerase II and III) 都是ALT所必須。之前研究指出Rif1p，Rif2p以及Rap1p會黏附在端粒區域並使端粒成為緊密異染色質結構。在酵母菌裡端粒進行重組延長時，在重組叉前進時兩端都會產生張力。拓樸異構酶可以解開在重組叉之前高度正螺旋結構，拓樸異構酶3可以解開重組叉之後高度負螺旋結構。將RIF1基因剔除會使端粒變鬆散，不會產生張力時重組就不需要拓樸異構酶2和3了。在培養基加入拓樸異構酶毒物，etoposide，能夠導致ALT細胞端粒縮短。目前尚無藥物能夠抑制ALT，而我們也將尋找能夠抑制酵母菌Type II重組機制以及ALT機制的拓樸異構酶毒物/抑制物。

Telomeres are dynamic DNA-protein complexes that protect the ends of linear chromosomes from genome instability and end fusion. Most telomeric DNA is synthesized by the enzyme telomerase. Cancer cells can elongate telomere either through telomerase reactivation or through an alternative recombination pathway for telomere lengthening (ALT). The importance of understanding the molecular mechanism of ALT goes far beyond solving an interesting problem in telomere biology, as alternative ALT is one of the main reasons for current therapeutic failures and/or it acquired resistances in the telomerase-inhibition-based anticancer therapy. In this study, we investigate telomere recombination in two model systems, budding yeast and mammalian. We have shown that topoisomerase 2 and 3 are all required for the ALT in yeast cells. Previous reports have shown that RIF1, RIF2 and RAP1 bind telomere regions and make telomere regions compact (heterochromatin). During telomere recombination in yeast cells, tensions form before and after recombination forks. Topoisomerase 2 resolves hyperpositively supercoiled DNA before recombination forks and topoisomerase 3 resolves hypernegatively supercoiled DNA after recombination forks. Knock out RIF1 conferred telomere regions loose. Topoisomerase 2 and 3 are not required for telomere recombination when supercoiled DNA is not formed. Adding topoisomerase 2 poison, etoposide, in medium could also conferred telomere shortening in human ALT cells. So far there is no drug developed to inhibit ALT，so we are looking forward to find Top2p poisons or inhibitors can inhibit both Type II recombination and ALT pathway.