轉譯後修飾在酵母菌DNA解旋酶Sgs1功能上扮演的角色探討

Abstract

當RecQ DNA 解旋酶─BLM及WRN發生突變時，會導致基因體的不穩定、提早老化並有易罹患癌症的徵兆。酵母菌中的RecQ 解旋酶 Sgs1 以調控基因重組的方式來維持基因體的完整性。其他物種中的同源蛋白─BLM及WRN，已被發現會受到多種轉譯後修飾，如類泛素化(sumoylation)、磷酸化(phosphorylation)及乙醯化(acetylation)。本研究確定了Sgs1發生類泛素化的時機，並釐清這樣的修飾在DNA重組修復上所扮演的角色。我發現 Sgs1 類泛素化發生在Lysine621 的位置，且特別會在DNA雙股斷裂發生時進行這樣的修飾。Sgs1的類泛素化會促進端粒重組，但不影響Sgs1的其他功能，如DNA受損的修復、重組、調控top3基因突變造成的生長遲緩及rDNA的重組。我們的研究證實了Sgs1的類泛素化調控了端粒重組的結果。 另外，我們也發現Sgs1會被磷酸化，且受到細胞週期素依賴性蛋白激酶(Cdk1)及細胞週期的調控。我們發現Sgs1磷酸化的程度在S 期最多，而在G1時期則最少，在Cdk1受損時這樣的磷酸化變化則會消失。利用激酶活性偵測實驗，我們證實了Sgs1會直接地被Cdk1磷酸化。這份研究中我證明了Sgs1會受到類泛素化及磷酸化的修飾，並會將我的結果衍生以期能夠更了解這些修飾是怎樣影響Sgs1的功能。

Mutations in genes encoding WRN and BLM RecQ DNA helicases lead to genome instability, premature aging and cancer predisposition syndromes. The Saccharomyces cerevisiae Sgs1 RecQ helicase safeguards genome integrity through its function in DNA recombination. RecQ homologues, WRN and BLM were all previously shown to be undergoing several post-translational modifications (PTMs), such as sumoylation, phosphorylation and acetylation. In this report, we identify the conditions under which Sgs1 sumoylation is induced, and clarify the role of this modification in different types of recombinational repair. We found that the Lysine 621 residue is critical for sumoylation in vivo and in vitro. We demonstrate that Sgs1 is specifically sumoylated under the stress of double strand breaks (DSBs). Sumoylation of Sgs1 promotes telomere-telomere recombination. In contrast, sumoylation of Sgs1 is dispensable for other functions of Sgs1, including damage recovery, homologous recombination, regulation of top3 slow growth and rDNA recombination. Our observations indicate that sumoylation on Sgs1 modulates the outcome in telomere recombination. Moreover, we also observed that Sgs1 is phosphorylated in a Cdk1- and cell cycle-dependent manner. We showed that Sgs1 is hyperphosphorylated during S phase and is usually under-phosphorylated in G1 stage. The Cdk1-crippling backgrounds led to diminishment of Sgs1 hyperphosphorylation during S phase. By in vitro kinase assay, I also demonstrated that Sgs1 is directly phosphorylated by Cdk1 in vitro. Here I provided solid evidence that Sgs1 is sumoylated and phosphorylated, and will apply my data to further understood the detail mechanism how these modifications regulate Sgs1 functions.