R-loops promote trinucleotide repeat deletion through DNA base excision repair enzymatic activities
R-loops promote trinucleotide repeat deletion through DNA base excision repair enzymatic activities
复制标题
R 环通过 DNA 碱基切除修复酶活性促进三核苷酸重复删除
DOI：
10.1074/jbc.ra120.014161
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发表时间：
2020-10-02
影响因子：
4.8
通讯作者：
Liu, Yuan
中科院分区：
文献类型：
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作者：
Laverde, Eduardo E.;Lai, Yanhao;Liu, Yuan
三核苷酸重复序列（TNR）的扩增和缺失导致40多种神经退行性疾病，并与癌症相关。TNR可以经历由DNA损伤和修复以及基因转录介导的体细胞不稳定性。最近的研究已经指出了R环在引起TNR扩增和缺失中的作用，并且已经表明碱基切除修复（BER）可以导致酵母中R环的CAG重复缺失。然而，R环中的BER如何介导TNR不稳定性仍然是未知的。在这项研究中，使用生物化学的方法，我们研究BER酶活性和它们对TNR环的影响。我们发现，AP核酸内切酶1切割TNR环的非模板链上的脱碱基位点，产生含有RNA：DNA杂合体的双瓣中间体，其随后抑制TNR的聚合酶β（pol β）合成。这刺激了参与R环的TNR的瓣状核酸内切酶1（FEN 1）切割。此外，我们发现FEN 1也有效地切割RNA链，促进pol β环/发夹旁路合成和通过BER解析TNR环。因此，这导致由pol β合成的TNR比由FEN 1去除的TNR少，从而导致重复缺失。我们的研究结果表明，TNR的R-环优先导致重复删除BER期间，通过破坏添加和删除TNR之间的平衡。我们的发现为治疗和预防重复扩张性疾病和癌症开辟了新的途径。
Trinucleotide repeat (TNR) expansion and deletion are responsible for over 40 neurodegenerative diseases and associated with cancer. TNRs can undergo somatic instability that is mediated by DNA damage and repair and gene transcription. Recent studies have pointed toward a role for R-loops in causing TNR expansion and deletion, and it has been shown that base excision repair (BER) can result in CAG repeat deletion from R-loops in yeast. However, it remains unknown how BER in R-loops can mediate TNR instability. In this study, using biochemical approaches, we examined BER enzymatic activities and their influence on TNR R-loops. We found that AP endonuclease 1 incised an abasic site on the nontemplate strand of a TNR R-loop, creating a double-flap intermediate containing an RNA:DNA hybrid that subsequently inhibited polymerase beta (pol beta) synthesis of TNRs. This stimulated flap endonuclease 1 (FEN1) cleavage of TNRs engaged in an R-loop. Moreover, we showed that FEN1 also efficiently cleaved the RNA strand, facilitating pol beta loop/hairpin bypass synthesis and the resolution of TNR R-loops through BER. Consequently, this resulted in fewer TNRs synthesized by pol beta than those removed by FEN1, thereby leading to repeat deletion. Our results indicate that TNR R-loops preferentially lead to repeat deletion during BER by disrupting the balance between the addition and removal of TNRs. Our discoveries open a new avenue for the treatment and prevention of repeat expansion diseases and cancer.