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Structural dissection of sequence recognition and catalytic mechanism of human LINE-1 endonuclease
Nucleic Acids Research ( IF 14.9 ) Pub Date : 2021-09-09 , DOI: 10.1093/nar/gkab826
Ian Miller 1 , Max Totrov 2 , Lioubov Korotchkina 3 , Denis N Kazyulkin 3 , Andrei V Gudkov 3, 4 , Sergey Korolev 1
Affiliation  

Long interspersed nuclear element-1 (L1) is an autonomous non-LTR retrotransposon comprising ∼20% of the human genome. L1 self-propagation causes genomic instability and is strongly associated with aging, cancer and other diseases. The endonuclease domain of L1’s ORFp2 protein (L1-EN) initiates de novo L1 integration by nicking the consensus sequence 5′-TTTTT/AA-3′. In contrast, related nucleases including structurally conserved apurinic/apyrimidinic endonuclease 1 (APE1) are non-sequence specific. To investigate mechanisms underlying sequence recognition and catalysis by L1-EN, we solved crystal structures of L1-EN complexed with DNA substrates. This showed that conformational properties of the preferred sequence drive L1-EN’s sequence-specificity and catalysis. Unlike APE1, L1-EN does not bend the DNA helix, but rather causes ‘compression’ near the cleavage site. This provides multiple advantages for L1-EN’s role in retrotransposition including facilitating use of the nicked poly-T DNA strand as a primer for reverse transcription. We also observed two alternative conformations of the scissile bond phosphate, which allowed us to model distinct conformations for a nucleophilic attack and a transition state that are likely applicable to the entire family of nucleases. This work adds to our mechanistic understanding of L1-EN and related nucleases and should facilitate development of L1-EN inhibitors as potential anticancer and antiaging therapeutics.

中文翻译:

人LINE-1核酸内切酶序列识别和催化机制的结构剖析

长散布的核元素-1 (L1) 是一种自主的非 LTR 反转录转座子,占人类基因组的 20%。L1 自我繁殖会导致基因组不稳定,并且与衰老、癌症和其他疾病密切相关。L1 的 ORFp2 蛋白 (L1-EN) 的核酸内切酶结构域通过切割共有序列 5'-TTTTT/AA-3' 来启动从头 L1 整合。相反,包括结构上保守的脱嘌呤/脱嘧啶核酸内切酶 1 (APE1) 在内的相关核酸酶是非序列特异性的。为了研究 L1-EN 的序列识别和催化机制,我们解决了与 DNA 底物复合的 L1-EN 的晶体结构。这表明优选序列的构象特性驱动 L1-EN 的序列特异性和催化作用。与 APE1 不同,L1-EN 不会弯曲 DNA 螺旋,而是导致切割位点附近的“压缩”。这为 L1-EN 在逆转录转座中的作用提供了多种优势,包括促进使用带切口的 poly-T DNA 链作为逆转录的引物。我们还观察到易裂键磷酸盐的两种替代构象,这使我们能够模拟可能适用于整个核酸酶家族的亲核攻击和过渡态的不同构象。这项工作增加了我们对 L1-EN 和相关核酸酶的机制理解,并应促进 L1-EN 抑制剂作为潜在的抗癌和抗衰老治疗剂的开发。我们还观察到易裂键磷酸盐的两种替代构象,这使我们能够模拟可能适用于整个核酸酶家族的亲核攻击和过渡态的不同构象。这项工作增加了我们对 L1-EN 和相关核酸酶的机制理解,并应促进 L1-EN 抑制剂作为潜在的抗癌和抗衰老治疗剂的开发。我们还观察到易裂键磷酸盐的两种替代构象,这使我们能够模拟可能适用于整个核酸酶家族的亲核攻击和过渡态的不同构象。这项工作增加了我们对 L1-EN 和相关核酸酶的机制理解,并应促进 L1-EN 抑制剂作为潜在的抗癌和抗衰老治疗剂的开发。
更新日期:2021-09-09
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