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大学・研究所にある論文を検索できる 「The MRE11 nuclease promotes homologous recombination not only in DNA double-strand break resection but also in post-resection in human TK6 cells」の論文概要。リケラボ論文検索は、全国の大学リポジトリにある学位論文・教授論文を一括検索できる論文検索サービスです。
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The MRE11 nuclease promotes homologous recombination not only in DNA double-strand break resection but also in post-resection in human TK6 cells

Shimizu, Naoto 京都大学 DOI:10.14989/doctor.k23091

2021.03.23

概要

DNA 二重鎖切断 (DSB) は、１つでも発生後一定時間再結合されないまま残ると細胞自殺を起こす。DSB の発生が、放射線治療の作用機序である。 DSB は、非相同末端結合経路と相同組換え経路によって修復（再結合）される。非相同末端経路は、細胞周期を通じて DSB 修復を行うのに対し、相同組換え経路は S/G2 期で活性化する。相同組換え欠損細胞では、放射線に超感受性を示すことから、DSB 修復おける相同組換えの役割は極めて大きい。相同組換えの修復効率は、放射線の治療効果を決定する。

相同組換えは、DSB 切断端の5’末端からヌクレアーゼによる削り込みを受ける(ステップ１)ことで開始される。削り込みよってできる3’末端が突出した一本鎖DNA は、RAD51蛋白質依存的に姉妹染色分体の相同領域に侵入する(ステップ２)。侵入した一本鎖 DNAの 3’末端から DNA 合成が起こる。DNA 合成後、 DNA 四本鎖からなる中間体、Joint molecule 構造が生じる。このJoint molecule 構造は、構造特異的エンドクレアーゼ GEN1などによって切断され(ステップ３)、相同組換えが完結する。

MRE11 ヌクレアーゼは、相同組換え修復の初期反応である DSB 切断端の5’末端の削り込み反応に関与すると言われている。マウスのMRE11 ヌクレアーゼ活性欠損MEF 細胞は、MRE11 正常細胞に比べ、削り込み反応が50%程度減弱する。以前の報告で、50%程度の削り込み反応低下では、相同組換え頻度に影響を与えないことが示されている。酵母及び哺乳類細胞におけるMRE11 ヌクレアーゼ活性欠損及び遺伝子欠損の表現型は、極めて重篤な相同組換え欠損を示す。したがって、削り込み反応におけるMRE11 の機能だけで、MRE11 変異による重篤な組換え欠損が説明できるかは不明である。本研究の目的は、 相同組換えにおける MRE11 の DSB 切断端の 5’末端の削り込み反応以外の機能を解明し、 MRE11 変異による重篤な組換え欠損を示す原因を明らかにすることである。

本研究では、MRE11 を条件的に欠損(MRE11-/-)あるいはヌクレアーゼ活性欠損 (MRE11-/H129N)することが可能なヒト TK6 細胞を使った。相同組換え進行を調べるために、DSB 発生に応答して形成されるRAD51 foci の経時変化を調べた。RAD51 は、姉妹染色分体の相同領域に侵入する反応(ステップ２)に必須であり、相同組換え進行を解析する分子マーカーである。MRE11-/-及びMRE11-/H129N 細胞は、放射線照射後、RAD51 foci形成が野生型細胞と同程度であった。一方で、これらの細胞では、RAD51 foci 消失が遅れた。これらのことは、5’末端の削り込み反応以降の相同組換え過程にMRE11 が重要な機能を担っていることを意味していた。また MRE11-/-及びMRE11-/H129N 細胞では、野生型に比べ、相同組換え中間体(Joint molecule 構造)であるIso-chromatid break が顕著に増加した。GEN1 ヌクレアーゼの過剰発現によって、放射線照射によって増加した Iso-chromatid break 形成が抑制された。このことから、TK6 細胞において、MRE11 ヌクレアーゼは、 5’末端の削り込み反応以降に形成される組換え中間体の解消(ステップ３)に重要な役割を果たしていることがわかった。将来、相同組換えに複数の機能を担う MRE11 を阻害することで、放射線治療の効果を高めることが期待される。

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