【作者】 张登宏；

【导师】 周金秋；

【作者基本信息】 中国科学院研究生院（上海生命科学研究院） ， 生物化学与分子生物学， 2006， 博士

【摘要】 端粒是真核生物线状染色体的末端结构,由端粒DNA和其结合蛋白组成。它在维持基因组的稳定、防止染色体DNA末端融合和降解中起着重要的作用,并与癌症、衰老密切相关。端粒DNA重复序列的延伸是由特化的反转录酶即端粒酶完成的。端粒酶的核心组分是由具有催化活性的蛋白质亚基(hTERT)和RNA亚基(hTR)组成,hTR为合成端粒DNA重复序列提供模板。以前的研究发现芽殖酵母Pif1解旋酶通过抑制端粒酶途径来抑制端粒DNA的加长。端粒DNA复制的基本机制从酵母到人都高度保守,而类Pif1基因在多种生物中都广泛存在。因而,本研究的主要目的是鉴定人Pif1蛋白,是否也参与了端粒DNA的复制和维持。本工作首先克隆了人Pif1的cDNA。一级结构及生化分析表明,人Pif1蛋白是大肠杆菌RecD潜在的同源蛋白;具有依赖于ATP的5’至3’的DNA解旋酶活性。在端粒酶阳性的HT1080细胞中过表达野生型hPif1蛋白可导致细胞端粒DNA缩短,抑制hPif1过表达可使端粒DNA长度恢复。人Pif1对端粒DNA长度的影响需要其ATPase/解旋酶活性,因为丧失ATPase/解旋酶活性的突变体过表达不影响端粒DNA的长度。在端粒酶阴性的GM487细胞中过表达hPif1不影响端粒变化,提示hPif1蛋白对端粒DNA的效应是通过端粒酶通路介导的。体外生化实验还显示,重组Pif1蛋白可通过降低端粒酶的进行性从而直接抑制端粒酶的活性,而且,hPif1解旋酶能够解开DNA/RNA杂合双螺旋。染色质免疫沉淀还证实hPif1结合在端粒DNA上。基于这些实验结果,我们推测hPif1通过把端粒酶RNA从端粒DNA上解离下来而抑制端粒酶介导端粒DNA的加长。类RecD解旋酶在生物进化过程中已获得新功能。更多还原

【Abstract】 Telomeres are nucleoprotein structures that are positioned at eukaryotic linearchromosomal ends. The primary role of telomeres is to insulate the chromosomal endsfrom both fusion with other ends and from nucleolytic digestion. The stablemaintenance of telomeres contributes to genome stability, and correlates with cancerand aging. The telomerase is a specialized reverse transcriptase that extends the 3’ endof the G-rich strand of telomeres.The Saccharomyces Pif1 helicase inhibits telomere lengthening through thetelomerase pathway. Pif1-like genes are found in diverse organisms. Because thefundamental mechanism of telomere replication is highly conserved from yeast tomammals, we want to know whether hPif1 exists, and what the role of hPif1 plays inhuman. Here, we identified a human helicase, hPif1, that inhibits telomerase activity.The primary sequence and biochemical analysis suggest that hPif1 is a potentialhomologue of Escherichia coli RecD, an ATP-dependent 5’ to 3’ DNA helicase.Ectopic expression of wild-type, but not the ATPase/helicase deficient hPif1, causestelomere shortening in HT1080 cells. hPif1 inhibits human telomerase activityprobably due to reducing the repeat addition processivity of telomerase in vitro. hPif1could unwind DNA/RNA duplex and preferentially binds telomeric DNA in vitro andin vivo. We propose that the inhibitory mechanism of hPif1 on telomerase involvesunwinding of the DNA/RNA duplex formed by telomerase RNA and telomeric DNA.RecD homologues in eukaryotes may have evolved gaining additional functions.更多还原