酵母菌中藉由Mec1p與Tel1p磷酸化Cdc13p及非同源性末端連結機制之探討

Abstract

藉由Mec1p與Tel1p磷酸化調控端粒附著蛋白Cdc13的端粒酶吸引區域 DNA損傷反應蛋白激酶ATM與ATR在生體內(in vivo)大部份它們所磷酸化的對象都是SQ/TQ聚集在一起的序列。在出芽酵母菌中的蛋白Cdc13p具有兩個SQ/TQ聚集在一起的序列暗示著它可能可以被Mec1p與Tel1p(酵母菌中的ATM與ATR)磷酸化。在這篇研究中我們證明了Cdc13p中吸引端粒酶區域可以被Mec1p與Tel1p磷酸化。利用膠條分析方式證明Cdc13p包含了依賴Mec1p與Tel1p的轉譯後修飾。使用免疫沉澱-激酶分析我們證明了在生體外(in vitro)Mec1p可以磷酸化Cdc13p中絲胺酸225、249、255與306的位置而Tel1p則可以磷酸化絲胺酸225、249和255的位置。在生體內的表現型分析顯示出將Cdc13p中可以被Mec1p與Tel1p磷酸化的SQ序列突變會造成很多端粒及生長分面的缺陷。另外，表現Cdc13-Est1p融合蛋白在那些突變株中可以恢復正常的端粒長度及生長速度。這些結果證明Cdc13p中的端粒酶吸引區域是Mec1p及Tel1p的一個重要且新的端粒專一性的對像。 酵母菌中DNA聚合酶α、β、δ、ε和γ均參與在非同源性末端連結機制 大部分的DNA修補機制中都需要DNA聚合酶來執行核酸的修補延長。非同源性末端連結(NHEJ)參與在染色體雙股DNA斷裂修補機制中及B淋巴細胞與T淋巴細胞發育時抗原結合基多樣區域的連結。然而，在生體內(in vivo)有哪些DNA聚合酶透過NHEJ的方式來修補DNA雙股斷裂目前仍然不清楚。NHEJ修補DNA的方式包含了簡單末端連結(simple end-joining)及染色體重組(chromosomal rearrangement)。在這篇研究我們在酵母菌系統中使用生體內分析方式來探討不同的DNA聚合酶在簡單末端連結與染色體重組中所扮演的角色。本篇研究包含了酵母菌中六個主要的DNA聚合酶 (α、β、γ、δ、ε和ζ)。DNA聚合酶β被發現參與在簡單末端連結中加鹼基的步驟並且被證明它會被吸引到DNA雙股斷裂的位置。DNA聚合酶ε則被發現透過它的3’到5’外切核酸酶的活性參與在NHEJ中簡單末端連結中減鹼基的步驟。並且發現DNA聚合酶δ與γ都參與在染色體重組但不參與在簡單末端連結的機制中。總而言之，我們的研究證明了大部分的DNA聚合酶都參與在NHEJ的機制中，並且個別在細步的NHEJ機制中扮演不同的角色。

The telomerase-recruitment domain of the telomere binding protein Cdc13 is regulated by Mec1p/Tel1p-dependent phosphorylation The DNA damage-responsive protein kinases ATM and ATR phosphorylate SQ/TQ motifs that lie in clusters in most of their in vivo targets. Budding yeast Cdc13p contains two clusters of SQ/TQ motifs, suggesting that it might be a target of Mec1p/Tel1p (yeast ATR/ATM). Here we demonstrated that the telomerase-recruitment domain of Cdc13p is phosphorylated by Mec1p and Tel1p. Gel analysis showed that Cdc13p contains a Mec1/Tel1-dependent posttranslational modification. Using an immunoprecipitate (IP)-kinase assay, we showed that Mec1p phosphorylates Cdc13p on serine 225, 249, 255 and 306, and Tel1p phosphorylates Cdc13p on serine 225, 249, and 255 in vitro. Phenotypic analysis in vivo revealed that the mutations in the Cdc13p SQ motifs phosphorylated by Mec1p and Tel1p caused multiple telomere and growth defects. In addition, normal telomere length and growth could be restored by expressing a Cdc13-Est1p hybrid protein. These results demonstrate the telomerase recruitment domain of Cdc13p as an important new telomere-specific target of Mec1p/Tel1p. Involvement of DNA polymerases α,β,δ,ε and γ in non-homologous end-joining in Saccharomyces cerevisiae Most DNA repair pathways require DNA polymerase to execute the step of nucleotide extension. Non-Homologous End-Joining (NHEJ) is required to repair chromosomal double strand breaks and to assemble antigen receptor variable regions in developing B and T lymphocytes. However, the question about which DNA polymerases are involved resolving double strand breaks through NHEJ in vivo is still elusive. NHEJ pathways are observed in the both simple end joining and chromosomal rearrangement. Here we used an in vivo assay to characterize different DNA polymerases and to understand their roles in simple end joining and chromosomal rearrangement in Saccharomyces cerevisiae. All six major DNA polymerases (α,β,γ,δ,ε and ζ) in S. cerevisiae were investigated. Polβ was found to be required in adding bases in simple end joining of NHEJ and it could be physically recruited to double strand break sites. Polε involved in deleting bases through its 3’ to 5’ exonuclease activity in simple end joining of NHEJ. Moreover, both Polδ and Polγ are required in chromosomal rearrangement, but are dispensable for involved simple end joining. Altogether, our results suggest that most DNA polymerases are involved in NHEJ and each plays a distinct role in detail mechanisms of NHEJ.