探討端粒結合蛋白Cdc13對於解螺旋酶Pif1活性所扮演之角色

Abstract

在真核細胞線性染色體的末端含有短片段重複訊序列的端粒DNA。在酵母菌Saccharomyces cerevisiae，端粒DNA的重複序列約為250-300個核苷酸，由TG1-3/C1-3A的重複序列所組成。端粒可以防止真核細胞染色體末端之間相互黏合，維持染色體的穩定性並且調節端粒DNA的長度。端粒酶屬於一種核醣核酸蛋白，能夠利用其中的RNA作為模板來延長G股端粒DNA。在S. cerevisiae，端粒酶的催化次單元體稱為Est2p及其模板TLC1 RNA。端粒屬於異染色質結構，由許多端粒相關蛋白組成，並且影響端粒的功能。其中，Pif1p是一種核酸解螺旋酶，能夠將ATP水解產生能量在單股DNA上由5’端往3’端的方向將端粒酶由端粒上驅趕走。先前的研究已經發現到PIF1過表現會導致端粒DNA的縮短；PIF1突變會導致端粒DNA的延長，映證了Pif1p在端粒酶的作用上扮演著負向調控的角色。Cdc13p具有單股端粒DNA結合的能力，透過與不同的端粒相關蛋白進行交互作用，Cdc13p能夠正向或負向調控端粒酶。我們建構了一套in vitro系統進行解螺旋試驗，來探討Cdc13p在Pif1p調控端粒酶功能上所扮演的角色。我們設計並合成了眾多含有TLC1序列的DNA與含有單股TG1-3的尾端雙股DNA進行黏合來模擬在生理情況下端粒酶結合至端粒DNA上的結構，來作為解螺旋酶試驗的受質。將Cdc13p與Pif1p共同作用下，我們發現Cdc13p能夠提升Pifp1解開雙股螺旋的比率，並且將受質上的間隙單股TG1-3序列置換成Cdc13p無法有效結合的序列依然可以觀察到提升的現象。除此之外，我們也比較了全片段的Cdc13p與單股結合區域Cdc13 (451-693)p以及單股結合區域突變Cdc13 (451-693)Y522Cp對於提升Pif1p解開雙股螺旋的影響。最後，我們將互補區域置換為隨機序列並且Cdc13p無法有效結合的受質中，觀察到Cdc13p仍然具有促進Pif1p功能的現象。因此，Cdc13p在不需緊密結合至DNA受質上即可促進Pif1p的活性。本篇論文的結果顯示，Cdc13p與Pif1p之間存在著交互作用，並且為Pif1p如何受調節而抑制端粒酶提供一項機制。

Telomeres are short, repetitive DNA sequence located at the terminal of eukaryotic linear chromosomes. In Saccharomyces cerevisiae, telomere DNA repeats are ~250-300 bps of TG1-3/C1-3A sequences. Telomeres are required to prevent chromosome end-to-end fusion, maintain chromosome integrity and regulate telomere elongation. Telomerase is a ribonucleoprotien that utilizes its RNA template to elongate the G-rich strand telomeric DNA. In S. cerevisiae, the telomerase catalytic subunit is Est2p and TLC1 RNA is the template. Telomeres are coated with many telomere-associated proteins to mediate telomere function. Notably, Pif1p is a single-stranded DNA (ssDNA)-dependent 5’ to 3’ helicase that utilizes ATP hydrolysis to remove telomerase from telomeres. Previously it was shown that PIF1 overexpression results in telomere shortening and mutations in PIF1 caused telomere lengthening, consistent with its negative role in regulating telomerase activity. Cdc13p is a single strand telomeric DNA binding protein. It can positively or negatively regulate telomerase through cooperating with different telomere-associated proteins. Using an in vitro reconstituted system, we aim to define the role of Cdc13p and Pif1p on regulating telomerase activity. Here we designed and synthesized different tailed-duplex DNA with the single-stranded TG1-3 annealed to oligonucleotide with the sequences similar to the template region of TLC1 RNA. The substrate mimics the structure of telomerase RNA-bound telomeres. Using purified proteins, we found Cdc13p enhanced Pif1p-induced DNA unwinding. We also compared the enhancements of Pif1p-induced DNA unwinding by Cdc13 and binding domain Cdc13 (451-693)p, binding domain mutant Cdc13 (451-693)Y522Cp. Moreover, we found Cdc13p still activated Pif1p activity by using random complementary sequence DNA substarte that Cdc13p failed to bind on. Thus, we concluded that Cdc13p stimulated Pif1p helicase activity and binding on DNA is not neccessary for its stimulating activity. Our results also implicated that a direct interaction between Cdc13p and Pif1p is involved in this stimulating activity and provided a mechanism how Pif1p is regulated to inhibit telomerase.