"探討雙股端粒結合蛋白,Rap1,之磷酸化修飾在端粒延長機制之調控"

Abstract

Rap1為一DNA結合蛋白，並在染色體末端的保護及端粒長度的調控上扮演非常重要的角色。在端粒上，Rap1除了會與Sir proteins 作用造成Telomere Position Effect外，也會與Rif proteins結合調控端粒的長度，而在本篇研究中我們發現，位在Rap1 C端的絲胺酸731之磷酸化透過影響與其他蛋白的結合，進而影響端粒的長度調控，透過yeast two-hybrid system顯示，當絲胺酸731突變為丙氨酸因而無法被加上磷酸根時，則Rap1與Rif1之間的作用增強，與Sir3之間的作用下降，因此端粒變短；而當絲胺酸731突變為天冬胺酸模擬磷酸根的負電荷時，則Rap1與Rif1之間的作用下降，與Sir3之間的作用維持不變，使得端粒變長。另一方面，透過專一性偵測絲胺酸731磷酸根的抗體發現，此磷酸修飾會在MMS treatment後與YKU80 deletion的狀況下出現，並且發現Mec1及Tel1為此磷酸修飾的kinases，暗示此磷酸修飾在細胞週期中的端粒調控機制上扮演重要角色。

Rap1 is an important DNA binding protein that protects the ends of chromosomes and maintains the homeostatic length of telomeres. Rap1 works along with its interacting proteins, Rif1 and Rif2, to execute its function in telomere maintenance. In this study, we found that phosphorylations of Rap1 at serine 731 within the C-terminal protein-interaction domain can regulate the telomere length. Mutation of serine to alanine promotes its interaction with Rif1 but reduces its interaction with Sir3 and therefore causes telomere shortening. In contrast, mutation of serine to aspartatic acid decreases the interaction with Rif1 but has no effect on the interaction with Sir3 and thus lengthens the telomere. In addition, the phosphorylation of Rap1 was found to be recognized after MMS treatment or under the condition of YKU80 deletion, and Mec1 as well as Tel1 were shown to be the kinases for the phosphorylation. All these, imply an important role of this modification during cell cycle.