Three Human Pol ι Variants with Impaired Polymerase Activity Fail to Rescue H2O2 Sensitivity in POLI-Deficient Cells.,Chemical Research in Toxicology

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Three Human Pol ι Variants with Impaired Polymerase Activity Fail to Rescue H2O2 Sensitivity in POLI-Deficient Cells.
Chemical Research in Toxicology ( IF 3.7 ) Pub Date : 2020-07-08 , DOI: 10.1021/acs.chemrestox.0c00127
Mina Yeom ₁ , Jin-Kyung Hong ₁ , Jae-Kwon Kim ₁ , F Peter Guengerich ₂ , Jeong-Yun Choi ₁

Affiliation

Human Y-family DNA polymerase (pol) ι is involved in translesion DNA synthesis (TLS) and base excision repair (BER) of oxidative DNA damage. Genetic variations may alter the function of pol ι and affect cellular susceptibility to oxidative genotoxic agents, but their effects remain unclear. We investigated the impacts of 10 human missense germline variations on pol ι function by biochemical and cell-based assays. Both polymerase and deoxyribose phosphate (dRP) lyase activities were determined utilizing recombinant pol ι (residues 1–445) proteins. The K209Q, K228I, and Q386R variants showed 4- to 53-fold decreases in specificity constants (k_cat/K_m) for dCTP insertion opposite G and 8-oxo-7,8-dihydroguanine compared to the wild-type. The R126C and K345E variants showed wild-type-like polymerase activity, although these two variants (as well as the R209Q, K228I, and Q386R variants) showed greater than 6-fold decreases in dRP lyase activity compared to the wild-type. A CRISPR/Cas9-mediated POLI knockout conferred higher sensitivity to H₂O₂ in human embryonic kidney (HEK293) cells. Exogenous expression of the full-length wild-type, R126C, and K345E variants fully rescued the H₂O₂ sensitivity in POLI-deficient cells, while full-length R209Q, K228I, and Q386R variants did not rescue the sensitivity. Our results indicate that the R126C and K345E variants (having wild-type-like polymerase activity, albeit impaired in dRP lyase activity) could fully rescue the H₂O₂ sensitivity in POLI-deficient cells, while the R209Q, K228I, and Q386R variants, all impaired in polymerase and dRP lyase activity, failed to rescue the sensitivity, indicating the relative importance of TLS-related polymerase function of pol ι rather than its BER-related dRP lyase function in protection from oxidative stress. The possibility exists that the hypoactive pol ι variants increase the individual susceptibility to oxidative genotoxic agents.

中文翻译：

聚合酶活性受损的三种人类 Polι 变体未能挽救 POLI 缺陷细胞对 H2O2 的敏感性。

人类 Y 家族 DNA 聚合酶 (pol) ι 参与跨损伤 DNA 合成 (TLS) 和氧化性 DNA 损伤的碱基切除修复 (BER)。遗传变异可能会改变 pol ι 的功能并影响细胞对氧化性基因毒剂的敏感性，但它们的影响仍不清楚。我们通过生化和基于细胞的分析研究了 10 种人类错义种系变异对 pol ι 功能的影响。聚合酶和磷酸脱氧核糖 (dRP) 裂解酶活性均使用重组 pol ι（残基 1-445）蛋白测定。K209Q、K228I 和 Q386R 变体的特异性常数降低了 4 到 53 倍 ( k _cat / K _m) 与野生型相比，用于与 G 和 8-oxo-7,8-二氢鸟嘌呤相对的 dCTP 插入。R126C 和 K345E 变体显示出类似野生型的聚合酶活性，尽管与野生型相比，这两种变体（以及 R209Q、K228I 和 Q386R 变体）的 dRP 裂解酶活性降低了 6 倍以上。CRISPR/Cas9 介导的 POLI 敲除使人胚胎肾 (HEK293) 细胞对 H ₂ O _{2具有更高的敏感性。}全长野生型、R126C 和 K345E 变体的外源表达完全挽救了POLI中的 H ₂ O ₂敏感性-缺陷细胞，而全长 R209Q、K228I 和 Q386R 变体没有挽救敏感性。我们的结果表明，R126C 和 K345E 变体（具有野生型样聚合酶活性，尽管 dRP 裂解酶活性受损）可以完全挽救 POLI 缺陷细胞中的 H 2 _O 2_敏感性，而 R209Q、K228I 和 Q386R 变体，聚合酶和 dRP 裂解酶活性均受损，未能挽救敏感性，表明 polι 的 TLS 相关聚合酶功能而不是其 BER 相关 dRP 裂解酶功能在保护免受氧化应激方面的相对重要性。存在低活性的 pol ι 变体增加个体对氧化性基因毒物的敏感性的可能性。

更新日期：2020-08-17

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