Division of labor of Y-family polymerases in translesion-DNA synthesis for distinct types of DNA damage.
Division of labor of Y-family polymerases in translesion-DNA synthesis for distinct types of DNA damage.
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DOI：
10.1371/journal.pone.0252587
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发表时间：
2021
期刊：
影响因子：
3.7
通讯作者：
Hirota K
中科院分区：
文献类型：
--
作者：
Inomata Y;Abe T;Tsuda M;Takeda S;Hirota K
生物体持续受到大量DNA损伤剂的威胁，这些DNA损伤剂影响基因组DNA。DNA复制机制在模板DNA受损时停止。停滞的复制叉通过跨损伤DNA合成聚合酶（包括Y家族聚合酶Polη、Pol I和Polκ）经由旁路复制重新启动，所述聚合酶具有掺入与受损模板相对的核苷酸的能力。为了研究这些聚合酶在体内的分工，我们从人TK 6细胞中以所有组合产生了POLη−/−、POLι−/−、POLκ−/−、双敲除（KO）和三敲除（TKO）突变体。TKO细胞表现出对紫外线（UV）、顺铂（CDDP）和甲磺酸甲酯（MMS）的超敏反应，证实了这些聚合酶在受损模板DNA的旁路复制中发挥的关键作用。POLη−/−细胞，而不是POLι−/−或POLκ−/−细胞，对UV和CDDP表现出强烈的敏感性，而TKO细胞对UV和CDDP的敏感性略高于POLη−/−细胞。另一方面，TKO细胞，但不是所有的单KO细胞，表现出显着更高的敏感性MMS比野生型细胞。DNA-纤维分析结果表明，Polη在绕过UV模拟剂4-硝基喹啉-1-氧化物和CDDP引起的损伤中起关键作用，而所有三种聚合酶在绕过MMS诱导的损伤中起互补作用。我们的研究结果表明，三个Y-家族聚合酶在旁路复制中发挥明显不同的作用，根据模板链上产生的DNA损伤的类型。
Living organisms are continuously under threat from a vast array of DNA-damaging agents, which impact genome DNA. DNA replication machinery stalls at damaged template DNA. The stalled replication fork is restarted via bypass replication by translesion DNA-synthesis polymerases, including the Y-family polymerases Polη, Polι, and Polκ, which possess the ability to incorporate nucleotides opposite the damaged template. To investigate the division of labor among these polymerases in vivo, we generated POLη−/−, POLι−/−, POLκ−/−, double knockout (KO), and triple knockout (TKO) mutants in all combinations from human TK6 cells. TKO cells exhibited a hypersensitivity to ultraviolet (UV), cisplatin (CDDP), and methyl methanesulfonate (MMS), confirming the pivotal role played by these polymerases in bypass replication of damaged template DNA. POLη−/− cells, but not POLι−/− or POLκ−/− cells, showed a strong sensitivity to UV and CDDP, while TKO cells showed a slightly higher sensitivity to UV and CDDP than did POLη−/− cells. On the other hand, TKO cells, but not all single KO cells, exhibited a significantly higher sensitivity to MMS than did wild-type cells. Consistently, DNA-fiber assay revealed that Polη plays a crucial role in bypassing lesions caused by UV-mimetic agent 4-nitroquinoline-1-oxide and CDDP, while all three polymerases play complementary roles in bypassing MMS-induced damage. Our findings indicate that the three Y-family polymerases play distinctly different roles in bypass replication, according to the type of DNA damage generated on the template strand.
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