The C-terminal Domain (CTD) of Human DNA Glycosylase NEIL1 Is Required for Forming BERosome Repair Complex with DNA Replication Proteins at the Replicating Genome DOMINANT NEGATIVE FUNCTION OF THE CTD
The C-terminal Domain (CTD) of Human DNA Glycosylase NEIL1 Is Required for Forming BERosome Repair Complex with DNA Replication Proteins at the Replicating Genome DOMINANT NEGATIVE FUNCTION OF THE CTD
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DOI：
10.1074/jbc.m115.642918
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发表时间：
2015-08-21
影响因子：
4.8
通讯作者：
Hegde, Muralidhar L.
中科院分区：
文献类型：
--
作者：
Hegde, Pavana M.;Dutta, Arijit;Hegde, Muralidhar L.
人类DNA糖基酶NEIL1最近被证明可以启动复制基因组中氧化碱基的预复制碱基切除修复（BER），从而防止诱变复制。凝胶过滤显示，细胞中很大一部分NEIL1存在于含有DNA复制和其他修复蛋白的大细胞复合体中。然而，NEIL1的相互作用如何影响其招募到复制位点进行复制前修复尚未研究。在这里，我们发现NEIL1在没有DNA的情况下通过其非保守的C端结构域（CTD）与增殖细胞核抗原夹片装载器复制因子C、DNA聚合酶delta和DNA连接酶I进行二元相互作用；复制因子C相互作用导致NEIL1活性的类似8倍的刺激。在缺乏CTD的NEIL1缺失突变体（N311）中观察到，破坏了BERosome复合体内NEIL1相互作用，不仅在体外抑制了完全的BER，而且还阻止了其染色质关联，减少了S期细胞复制焦点的招募。这表明NEIL1与复制和其他BER蛋白的相互作用是有效修复复制基因组所必需的。一致地，CTD多肽在体外修复过程中作为显性阴性抑制剂，其异位表达使人细胞对活性氧敏感。我们得出结论，BER蛋白之间的多重相互作用导致了大型复合物，这是哺乳动物细胞高效BER的关键，并且CTD相互作用可以靶向增强肿瘤细胞的药物/辐射敏感性。
The human DNA glycosylase NEIL1 was recently demonstrated to initiate prereplicative base excision repair (BER) of oxidized bases in the replicating genome, thus preventing mutagenic replication. A significant fraction of NEIL1 in cells is present in large cellular complexes containing DNA replication and other repair proteins, as shown by gel filtration. However, how the interaction of NEIL1 affects its recruitment to the replication site for prereplicative repair was not investigated. Here, we show that NEIL1 binarily interacts with the proliferating cell nuclear antigen clamp loader replication factor C, DNA polymerase delta, and DNA ligase I in the absence of DNA via its non-conserved C-terminal domain (CTD); replication factor C interaction results in similar to 8-fold stimulation of NEIL1 activity. Disruption of NEIL1 interactions within the BERosome complex, as observed for a NEIL1 deletion mutant (N311) lacking the CTD, not only inhibits complete BER in vitro but also prevents its chromatin association and reduced recruitment at replication foci in S phase cells. This suggests that the interaction of NEIL1 with replication and other BER proteins is required for efficient repair of the replicating genome. Consistently, the CTD polypeptide acts as a dominant negative inhibitor during in vitro repair, and its ectopic expression sensitizes human cells to reactive oxygen species. We conclude that multiple interactions among BER proteins lead to large complexes, which are critical for efficient BER in mammalian cells, and the CTD interaction could be targeted for enhancing drug/radiation sensitivity of tumor cells.