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リボソームDNAおよびサブテロメアにおける繰り返し配列維持に係る分裂酵母Stn1の機能

山本, 唯央 京都大学 DOI:10.14989/doctor.k23589

2021.11.24

概要

Ctc1-Stn1-Ten1複合体(CST複合体)は出芽酵母、哺乳類を含む幅広い真核生物種で保存された一本鎖テロメアDNA結合蛋白質である。これまでの研究により、CST複合体は、DNA合成酵素αと結合してテロメアおよびサブテロメア領域のDNA複製に関わること、テロメア以外のゲノム領域においても機能する可能性が高いことが明らかにされていたが、具体的にどのような領域に関わるのかは不明であった。分裂酵母では、これまでにCtc1のホモログは同定されておらず、Stn1およびTen1のみが知られていた。申請者は、分裂酵母stn1遺伝子温度感受性株stn1-1を用いて、テロメア領域以外におけるStn1の機能を明らかにすることを目的に本研究を行った。

 まず、stn1-1が非制限温度下であってもデオキシリボヌクレオチド合成を阻害する複製阻害剤であるHydroxyurea(HU)に対し感受性をもつことを明らかにした。次に、これまで知られているテロメア、サブテロメア以外の領域でStn1が機能し、HU感受性の原因の一部となっているか否かを検討するために、テロメレース遺伝子trt1を欠損することでテロメアおよび多くのサブテロメア領域が欠失している環状染色体株について検討した。その結果、環状染色体をもつstn1-1にHU感受性が認められることから、申請者は、テロメア、サブテロメア以外のゲノム領域においてStn1が重要な役割を果たしていると考えた。さらに、テロメア、サブテロメアと同じように直列繰り返し配列からなるrRNA遺伝子(rDNA)領域に注目し、その長さを同一クローンについて経時的に測定すると、野生株に比べてstn1-1では長さが動的に変化し、同領域にDNA二本鎖切断マーカーであるリン酸化ヒストンH2A(γH2A)およびDNA相同組換え因子Rad52が蓄積していることを明らかにした。このことから、申請者は、stn1-1ではrDNA領域において複製フォークの停止が高頻度に起きていることを示唆した。

 分裂酵母rDNAの各リピートには複製開始点が存在し、rRNAの転写反応とそれと反対の向きに進行するDNA複製フォークの衝突が起こらないように、特定の向きをもつ複製フォークの進行を停止させるRFB(replication fork barrier)が存在し、Reb1がRFBに結合することがその停止に必要であることが分かっている。申請者はreb1欠損株では、stn1-1におけるrDNAコピー数の不安定化やγH2A蓄積が抑制されることを見出し、Stn1がRFBによるゲノム不安定化の抑制に必要であることを示した。

 分裂酵母サブテロメアも複数種類の繰り返し配列から構成されているが、申請者は、本領域においてもstn1-1ではγH2Aの蓄積やコピー数の増幅が見られ、reb1遺伝子を欠損させることでそれらの表現型が抑制されることを示した。

 以上より、申請者は、分裂酵母Stn1は、複製フォークが停止しやすい繰り返し配列を多く含むテロメア、サブテロメア、rDNA領域において、複製フォークが高頻度に停止した結果DNA二本鎖切断とDNA相同組換えによってゲノム不安定化を示すことを防ぐ役割をもつことを結論づけた。ヒトCTC1, STN1遺伝子変異は、遺伝性疾患Coats plus病の原因となることが知られている。本研究成果は本疾患の原因解明に貢献するものと期待された。

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