The Polymerase Activity of Mammalian DNA Pol ζ Is Specifically Required for Cell and Embryonic Viability.
The Polymerase Activity of Mammalian DNA Pol ζ Is Specifically Required for Cell and Embryonic Viability.
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DOI：
10.1371/journal.pgen.1005759
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发表时间：
2016-01
期刊：
影响因子：
4.5
通讯作者：
Wood RD
中科院分区：
文献类型：
--
作者：
Lange SS;Tomida J;Boulware KS;Bhetawal S;Wood RD
DNA聚合酶抑制剂（polymerase，polymerase）对维持基因组稳定性非常重要。编码聚合酶催化亚基的Rev 3l基因的失活导致高频率的染色体断裂，随后在小鼠胚胎和原代细胞中致死。然而，目前尚不清楚聚合酶的DNA聚合酶活性是否是特别必要的，因为大REV 3L蛋白也作为多蛋白支架，通过多个保守的结构域进行跨病变DNA合成。我们报告说，Rev 31 cDNA拯救Rev 31空永生化小鼠成纤维细胞系的基因组不稳定性和DNA损伤敏感性。在REV 3L的聚合酶结构域中保守的催化天冬氨酸残基突变的cDNA不能拯救这些表型。为了研究REV 3L DNA聚合酶活性在体内的作用，构建了具有这种聚合酶失活改变的Rev 3l基因敲入小鼠。没有产生纯合子突变小鼠，在胚胎发生期间发生致死性。来自突变胚胎的原代成纤维细胞显示出与Rev 3l无效成纤维细胞相似的生长缺陷、升高的DNA双链断裂和顺铂敏感性。我们测试了严重的Rev 3l-/-表型是否可以通过DNA聚合酶η的缺失来挽救，正如已经用鸡DT 40细胞报道的那样。然而，Rev 31-/- Polh-/-小鼠是不存活的，并且衍生的原代成纤维细胞与Rev 31-/- Polh+/+成纤维细胞一样对DNA损伤敏感。因此，REV 3L在维持细胞活力、胚胎活力和基因组稳定性方面的功能直接依赖于其聚合酶活性，并且不能通过额外缺失pol η来改善。这些结果证实并鼓励靶向聚合酶的DNA聚合酶活性以使肿瘤对DNA损伤剂敏感的方法。翻译合成允许DNA复制在存在受损DNA的情况下发生。这个过程是由低保真DNA聚合酶（如pol nucleotide或pol η）介导的，它们可以维持基因组的稳定性。这些聚合酶的作用对限制癌症至关重要。在小鼠中，DNA聚合酶的完全缺失导致胚胎死亡，条件性缺失增强肿瘤发生。聚合酶是一种具有许多结构域的大蛋白，这些结构域与其他必需蛋白相互作用并保持聚合酶的结构完整性。目前尚不清楚聚合酶的聚合酶活性是否介导其基本活性。使用细胞培养互补系统和体内敲入小鼠，我们的工作表明，在DNA损伤的存在下，聚合酶介导的基因组稳定性的维持完全依赖于其DNA聚合酶活性。其他人已经在鸡细胞中证明了pol β和pol η的共缺失挽救了pol β依赖性表型，但是我们在小鼠和小鼠细胞培养中的工作不支持该结论。这些结果证明了聚合酶活性的生理重要性，并表明采用聚合酶反应的小分子抑制剂是使肿瘤细胞对化疗剂敏感的有效策略。
DNA polymerase ζ (pol ζ) is exceptionally important for maintaining genome stability. Inactivation of the Rev3l gene encoding the polymerase catalytic subunit causes a high frequency of chromosomal breaks, followed by lethality in mouse embryos and in primary cells. Yet it is not known whether the DNA polymerase activity of pol ζ is specifically essential, as the large REV3L protein also serves as a multiprotein scaffold for translesion DNA synthesis via multiple conserved structural domains. We report that Rev3l cDNA rescues the genomic instability and DNA damage sensitivity of Rev3l-null immortalized mouse fibroblast cell lines. A cDNA harboring mutations of conserved catalytic aspartate residues in the polymerase domain of REV3L could not rescue these phenotypes. To investigate the role of REV3L DNA polymerase activity in vivo, a Rev3l knock-in mouse was constructed with this polymerase-inactivating alteration. No homozygous mutant mice were produced, with lethality occurring during embryogenesis. Primary fibroblasts from mutant embryos showed growth defects, elevated DNA double-strand breaks and cisplatin sensitivity similar to Rev3l-null fibroblasts. We tested whether the severe Rev3l-/- phenotypes could be rescued by deletion of DNA polymerase η, as has been reported with chicken DT40 cells. However, Rev3l-/- Polh-/- mice were inviable, and derived primary fibroblasts were as sensitive to DNA damage as Rev3l-/- Polh+/+ fibroblasts. Therefore, the functions of REV3L in maintaining cell viability, embryonic viability and genomic stability are directly dependent on its polymerase activity, and cannot be ameliorated by an additional deletion of pol η. These results validate and encourage the approach of targeting the DNA polymerase activity of pol ζ to sensitize tumors to DNA damaging agents. Translesion synthesis allows DNA replication to occur in the presence of damaged DNA. This process is mediated by low-fidelity DNA polymerases (such as pol ζ or pol η) that maintain genomic stability. The action of these polymerases is crucial to limit cancer. In mice, complete deletion of DNA pol ζ leads to embryonic lethality, and conditional deletion enhances tumorigenesis. Pol ζ is a large protein with many domains that interact with other essential proteins and maintain the structural integrity of pol ζ. It is not known if the polymerase activity of pol ζ mediates its essential activities. Using a cell culture complementation system and in vivo knock-in mice, our work shows that pol ζ–mediated maintenance of genomic stability in the presence of DNA damage is absolutely dependent on its DNA polymerase activity. Others have demonstrated in chicken cells that co-deletion of pol ζ and pol η rescues the pol ζ-dependent phenotypes, but our work in mice and in mouse cell culture does not support that conclusion. These results demonstrate the physiological importance of pol ζ polymerase activity, and show that employing small-molecule inhibitors of the polymerase reaction is a valid strategy for sensitizing tumor cells to chemotherapeutic agents.