Template and target-site recognition by human LINE-1 in retrotransposition.
Template and target-site recognition by human LINE-1 in retrotransposition.
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DOI：
10.1038/s41586-023-06933-5
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发表时间：
2024-02
期刊：
影响因子：
64.8
通讯作者：
Collins, Kathleen
中科院分区：
文献类型：
--
作者：
Thawani, Akanksha;Ariza, Alfredo Jose Florez;Nogales, Eva;Collins, Kathleen
长的穿插元件-1(LINE-1，以下简称L1)反转录转座子产生了近三分之一的人类基因组，是遗传多样性和人类疾病的活跃来源。L1通过一种名为靶引发逆转录的机制传播，在该机制中，编码酶(ORF2p)切割靶DNA以启动其自身或非自身RNA的逆转录。在这里，我们纯化了全长L1 ORF2p，并用模板RNA和靶点DNA生化重组了健壮的靶标启动逆转录。我们报道了完整的人L1 ORF2p与结构模板RNA结合并启动cDNA合成的冷冻电子显微镜结构。模板多聚腺苷束由五个不同的结构域以序列特异性的方式识别。其中，RNA结合结构域弯曲模板骨架，使RNA发夹茎与L1 ORF2p C-末端片段接合。此外，结构和生化重组表明了一个意想不到的靶点要求：L1ORF2p依靠上游单链DNA将相邻的双链定位在核酸内切酶活性位点上，以划破较长的DNA链，一个缺口就会产生交错的DNA断裂。我们的研究为人类基因组中正在进行的转座机制提供了洞察力，并为逆转录转座子蛋白的基因治疗工程提供了信息。人类LINE-1 ORF2p依赖于上游单链靶标DNA在内切酶活性部位定位相邻的双链，以缺口较长的DNA链，单个缺口产生交错的DNA断裂。
The long interspersed element-1 (LINE-1, hereafter L1) retrotransposon has generated nearly one-third of the human genome and serves as an active source of genetic diversity and human disease. L1 spreads through a mechanism termed target-primed reverse transcription, in which the encoded enzyme (ORF2p) nicks the target DNA to prime reverse transcription of its own or non-self RNAs. Here we purified full-length L1 ORF2p and biochemically reconstituted robust target-primed reverse transcription with template RNA and target-site DNA. We report cryo-electron microscopy structures of the complete human L1 ORF2p bound to structured template RNAs and initiating cDNA synthesis. The template polyadenosine tract is recognized in a sequence-specific manner by five distinct domains. Among them, an RNA-binding domain bends the template backbone to allow engagement of an RNA hairpin stem with the L1 ORF2p C-terminal segment. Moreover, structure and biochemical reconstitutions demonstrate an unexpected target-site requirement: L1 ORF2p relies on upstream single-stranded DNA to position the adjacent duplex in the endonuclease active site for nicking of the longer DNA strand, with a single nick generating a staggered DNA break. Our research provides insights into the mechanism of ongoing transposition in the human genome and informs the engineering of retrotransposon proteins for gene therapy. Human LINE-1 ORF2p relies on upstream single-stranded target DNA to position the adjacent duplex in the endonuclease active site for nicking of the longer DNA strand, with a single nick generating a staggered DNA break.
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影响因子：
64.8
作者：
Jumper J;Evans R;Pritzel A;Green T;Figurnov M;Ronneberger O;Tunyasuvunakool K;Bates R;Žídek A;Potapenko A;Bridgland A;Meyer C;Kohl SAA;Ballard AJ;Cowie A;Romera-Paredes B;Nikolov S;Jain R;Adler J;Back T;Petersen S;Reiman D;Clancy E;Zielinski M;Steinegger M;Pacholska M;Berghammer T;Bodenstein S;Silver D;Vinyals O;Senior AW;Kavukcuoglu K;Kohli P;Hassabis D
通讯作者：
Hassabis D
影响因子：
4.9
作者：
Hancks DC;Kazazian HH Jr
通讯作者：
Kazazian HH Jr
DOI：
10.1073/pnas.0831042100
发表时间：
2003-04-29
影响因子：
11.1
作者：
Brouha, B;Schustak, J;Kazazian, HH
通讯作者：
Kazazian, HH
影响因子：
30.8
作者：
Dewannieux, M;Esnault, C;Heidmann, T
通讯作者：
Heidmann, T
影响因子：
3.3
作者：
Adney, Emily M.;Ochmann, Matthias T.;Boeke, Jef D.
通讯作者：
Boeke, Jef D.