単一分子DNAポリメラーゼI (Klenow断片) ナノ回路によるデオキシヌクレオシドトリホスファートアナログの漸進的組み込み
Kaitlin M Pugliese1, O Tolga Gul1, Yongki Choi1
1Departments of †Chemistry, §Physics and Astronomy, and ⊥Molecular Biology and Biochemistry, University of California, Irvine, California 92697, United States.
Journal of the American Chemical Society
|July 7, 2015
まとめ
DNAポリメラーゼは,改変されたデオキシリボヌクレオシドトリフォスファート (dNTPs) を組み込むことができるが,単一分子研究により,塩基組み込み中の酵素動態が明らかになる. これは,新生塩基対のダイナミックな安定性チェックメカニズムを示唆している.
科学分野:
- バイオケミストリー バイオケミストリー
- 分子生物学は分子生物学である.
- ナノテクノロジー ナノテクノロジー
背景:
- DNAポリメラーゼは,デオキシリボヌクレオシドトリホスファート (dNTP) の正確な認識のための洗練されたメカニズムを持っています.
- それにもかかわらず,彼らは様々なdNTPアナログに対して顕著な耐性を示しています.
- 分子レベルでこの耐性を理解することは,DNA複製と修復の研究に不可欠です.
研究 の 目的:
- dNTPアナログと相互作用するDNAポリメラーゼIのKlenow断片 (KF) の単分子ダイナミクスを調査する.
- 単一壁の炭素ナノチューブフィールド効果トランジスタ (SWCNT-FETs) を使用して,これらのアナログの酵素の収納を解決します.
- 改変されたdNTPに対するDNAポリメラーゼ耐性の基礎となるメカニズムを解明する.
主な方法:
- 個々のKF分子とSWCNT-FETを結び付け,リアルタイムで単一分子電流測定を行う.
- ネイティブおよびアナログのdNTPのベース組み込み中の現在の変化 (τclosedとtopen) を分析する.
- ナノ回路を用いて,ポリメリゼーション中の代替酵素構成を検出する.
主要な成果:
- 閉じた状態 (τclosed) の持続時間は,dNTPのアナログ組み込みの影響を受けませんでした.
- dNTP アナログの組み込み率は,主に分子認識を反映したオープン状態の持続時間 (τopen) によって影響を受けました.
- 特定のアナログ (α-thio-dNTPs,6-Cl-2APTP,2-thio-dTTP,2-thio-dCTP) は,よりゆっくりと組み込まれ,代替酵素構成が誘発されました.
結論:
- DNAポリメラーゼKFは,dNTPアナログに対する耐性を示すが,認識 (τopen) は速度制限のステップである.
- 酵素は,新生塩基対の安定性を評価するために,O-ヘリックス回転を含むダイナミックな安定性チェックメカニズムを使用します.
- このメカニズムは,アロステリック効果と組み合わせて,非ネイティブの塩基対を収容することを可能にし,すべてのイベントを酵素閉塞に起因する以前のモデルとは異なる.
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