人間のL1レトロトランポゾンは,レトロトランポゼーションに必要な保存されたエンドヌクレアスをコードします

Q Feng¹, J V Moran, H H Kazazian

¹Department of Molecular Biology and Genetics, School of Medicine, Johns Hopkins University, Baltimore, Maryland 21205, USA.

Cell

|November 29, 1996

まとめ

研究者らは,ヒトのL1要素に重要なエンドヌクレアース (EN) ドメインを発見した. このENタンパク質は,標的部位のDNAを切断し,L1の逆転移を容易にし,逆転写をプライミングします.

科学分野:

分子生物学は分子生物学である.
遺伝学遺伝学とは
バイオケミストリーバイオケミストリー

背景:

人間のL1要素は,LTR以外のレトロトランポゾンが豊富に存在する.
L1のオープンリーディングフレーム2 (ORF2) は,逆転写酶 (RT) をコードする.
L1 ORF2 N端の機能は以前は特徴づけられていなかった.

研究の目的:

L1 ORF2.2におけるエンドヌクレアースドメインを特定し,特徴づけること.
L1エンドヌクレアゼの酵素活性と標的特異性を調査する.
L1逆転変異におけるL1内核酵素の役割を決定する.

主な方法:

L1エンドヌクレアース (L1ENp) のタンパク質浄化.
超巻きプラズミッドを用いたインビトロDNA分裂アッセイ.
L1 EN.で保存された残留物のサイト指向型変異.
L1の逆転変換効率を測定するためのアッセイ.

主要な成果:

L1 ORF2のN端で,ポリ (A) リトロトランポゾンに保存されているエンドヌクレアース (EN) ドメインが特定されました.
浄化されたL1ENpは,AP部位とは独立してDNAに5'-リン酸,3'-ヒドロキシルニークを生成する.
L1 ENpは,L1 in vivoの標的部位に似た配列を優先的に分割する.
保存されたL1 EN残基の変異により,ニッキング活動とL1の逆転移が廃止されます.

結論:

L1 ENドメインは,L1の逆転移に不可欠なエンドヌクレアースとして機能する.
L1 ENは,L1挿入のためのターゲットDNAサイトを割る可能性が高い.
L1 ENの活動は,レトロトランスポジションの際にリバーストランスクリプションをプライムするように提案されています.

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