Endonuclease EEPD1 Is a Gatekeeper for Repair of Stressed Replication Forks.
Endonuclease EEPD1 Is a Gatekeeper for Repair of Stressed Replication Forks.
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DOI：
10.1074/jbc.m116.758235
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发表时间：
2017-02-17
期刊：
影响因子：
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通讯作者：
Hromas RA
中科院分区：
文献类型：
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作者：
Kim HS;Nickoloff JA;Wu Y;Williamson EA;Sidhu GS;Reinert BL;Jaiswal AS;Srinivasan G;Patel B;Kong K;Burma S;Lee SH;Hromas RA
复制并不像人们曾经想象的那样连续，DNA 损伤经常会阻碍复制叉。应激复制叉的异常修复可能导致细胞死亡或基因组不稳定，并最终转化为恶性肿瘤。应激复制叉最常通过同源重组 (HR) 进行修复，该重组从 5' 末端切除开始，由核酸外切酶复合物介导，其中之一包含 Exo1。然而，Exo1 需要游离的 5'-DNA 末端来发挥作用，而这些末端通常不存在于非反向停滞复制叉中。为了生成自由的 5' 末端，必须切割停滞的复制叉。尽管几种候选核酸内切酶参与了停滞复制叉的切割以允许末端切除，但这种核酸内切酶的身份仍然难以捉摸。在这里，我们展示了 5'-核酸内切酶 EEPD1 在滞后亲本链和未复制的 DNA 亲本双链之间的连接处切割复制叉。这种切割创建了 Exo1 进行 5' 末端切除和 HR 起始所需的结构。我们观察到 EEPD1 和 Exo1 组成型相互作用，并且 Exo1 在没有 EEPD1 的情况下很难修复停滞的复制叉。因此，EEPD1 通过介导复制叉分裂来执行复制叉修复的看门人功能，从而允许启动 HR 介导的修复并重新启动受压的复制叉。
Replication is not as continuous as once thought, with DNA damage frequently stalling replication forks. Aberrant repair of stressed replication forks can result in cell death or genome instability and resulting transformation to malignancy. Stressed replication forks are most commonly repaired via homologous recombination (HR), which begins with 5′ end resection, mediated by exonuclease complexes, one of which contains Exo1. However, Exo1 requires free 5′-DNA ends upon which to act, and these are not commonly present in non-reversed stalled replication forks. To generate a free 5′ end, stalled replication forks must therefore be cleaved. Although several candidate endonucleases have been implicated in cleavage of stalled replication forks to permit end resection, the identity of such an endonuclease remains elusive. Here we show that the 5′-endonuclease EEPD1 cleaves replication forks at the junction between the lagging parental strand and the unreplicated DNA parental double strands. This cleavage creates the structure that Exo1 requires for 5′ end resection and HR initiation. We observed that EEPD1 and Exo1 interact constitutively, and Exo1 repairs stalled replication forks poorly without EEPD1. Thus, EEPD1 performs a gatekeeper function for replication fork repair by mediating the fork cleavage that permits initiation of HR-mediated repair and restart of stressed forks.