Defective repair capacity of variant proteins of the DNA glycosylase NTHL1 for 5-hydroxyuracil, an oxidation product of cytosine
Defective repair capacity of variant proteins of the DNA glycosylase NTHL1 for 5-hydroxyuracil, an oxidation product of cytosine
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DOI：
10.1016/j.freeradbiomed.2018.12.010
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发表时间：
2019-02-01
影响因子：
7.4
通讯作者：
Sugimura, Haruhiko
中科院分区：
文献类型：
--
作者：
Shinmura, Kazuya;Kato, Hisami;Sugimura, Haruhiko
NTHL1基因编码DNA糖基化酶，参与碱基切除修复，该基因的双等位基因突变导致NTHL1相关息肉病（NAP），这是一种以结直肠息肉病和多种类型癌症为特征的遗传性疾病。然而，到目前为止，还没有对变异NTHL1蛋白进行适当的功能表征。在此，我们报告了变异NTHL1蛋白的功能评估，以帮助准确诊断NAP。首先，我们研究了用胞嘧啶氧化产物5-羟基尿嘧啶（5OHU）进行评价是否合适。在supF正向突变实验中，5OHU引起了人类细胞中突变频率的增加，并且在5OHU诱导的突变中，C -> T突变占主导地位。此外，在DNA切割活性测定中，5OHU被NTHL1和其他四种DNA糖基酶（SMUG1, NEIL1， TDG和UNG2）切除。当建立过表达这五种DNA糖基酶的人细胞时，发现包括NTHL1在内的五种DNA糖基酶均具有抑制5ohu诱导突变的能力。基于上述结果，我们使用含5ohu的DNA底物或穿梭质粒对8种NTHL1变体进行了功能评估。DNA裂解活性分析表明，NTHL1、Q90X、Y130X、R153X和Q287X的突变体，而R19Q、V179I、V217F和G286S的突变体对5OHU和其他两个氧化损伤碱基的修复活性不佳。此外，supF正向突变试验显示，四种截断型NTHL1变体在人类细胞中抑制5ohu诱导突变的能力降低。这些结果表明，NTHL1突变体Q90X、Y130X、R153X和Q287X在5OHU修复中存在缺陷，而R19Q、V179I、V217F和G286S不存在缺陷，编码这些突变体的等位基因被认为是NAP的致病基因。
The NTHL1 gene encodes DNA glycosylase, which is involved in base excision repair, and biallelic mutations of this gene result in NTHL1-associated polyposis (NAP), a hereditary disease characterized by colorectal polyposis and multiple types of carcinomas. However, no proper functional characterization of variant NTHL1 proteins has been done so far. Herein, we report functional evaluation of variant NTHL1 proteins to aid in the accurate diagnosis of NAP. First, we investigated whether it would be appropriate to use 5-hydroxyuracil (5OHU), an oxidation product of cytosine, for the evaluation. In the supF forward mutation assay, 5OHU caused an increase of the mutation frequency in human cells, and the C -> T mutation was predominant among the 5OHU-induced mutations. In addition, in DNA cleavage activity assay, 5OHU was excised by NTHL1 as well as four other DNA glycosylases (SMUG1, NEIL1, TDG, and UNG2). When human cells overexpressing the five DNA glycosylases were established, it was found that each of the five DNA glycosylases, including NTHL1, had the ability to suppress 5OHU-induced mutations. Based on the above results, we performed functional evaluation of eight NTHL1 variants using 5OHU-containing DNA substrate or shuttle plasmid. The DNA cleavage activity assay showed that the variants of NTHL1, Q90X, Y130X, R153X, and Q287X, but not R19Q, V179I, V217F, or G286S, showed defective repair activity for 5OHU and two other oxidatively damaged bases. Moreover, the supF forward mutation assay showed that the four truncated-type NTHL1 variants showed a reduced ability to suppress 5OHU-induced mutations in human cells. These results suggest that the NTHL1 variants Q90X, Y130X, R153X, and Q287X, but not R19Q, V179I, V217F, or G286S, were defective in 5OHU repair and the alleles encoding them were considered to be pathogenic for NAP.