Hypermutable ligation of plasmid DNA ends in cells from patients with Werner syndrome.
Hypermutable ligation of plasmid DNA ends in cells from patients with Werner syndrome.
复制标题
沃纳综合征患者细胞中质粒 DNA 末端的超突变连接。
DOI：
10.1111/1523-1747.ep12371730
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发表时间：
1994
期刊：
影响因子：
--
通讯作者：
Martin,GM
中科院分区：
文献类型：
--
作者：
Rünger,TM;Bauer,C;Dekant,B;Möller,K;Sobotta,P;Czerny,C;Poot,M;Martin,GM
沃纳综合征是一种罕见的常染色体隐性遗传疾病，其特征是癌症风险增加和提示过早衰老的症状。来自这些患者的细胞表现出典型的染色体不稳定性和自发性超变性模式，具有高比率的异常大的缺失。我们研究了Werner综合征患者的三个淋巴母细胞系和三个正常供体的体内DNA连接。在我们的宿主细胞连接试验中，我们转染了线性化质粒pZ 189，并测量了由这些宿主细胞重新连接的质粒DNA末端的量，作为回收的质粒转化细菌的能力。靠近连接位点的诱变标记基因允许筛选突变。随后的突变分析提供了关于连接过程的准确性的信息。来自沃纳综合征患者的细胞在连接DNA末端方面与正常细胞一样有效。然而，突变分析显示，在DNA末端连接过程中，三种Werner综合征细胞系引入的突变是正常细胞系的2.4-4.6倍（p < 0.001）：突变率分别为69.4%、97.2%和58.7%，而正常细胞系为23.6%、21.7%和24.4%。在通过沃纳综合征细胞的过程中连接的质粒中突变频率的增加主要是由于缺失的显著增加（p <0.001）。这种易于出错的DNA连接可能是导致Werner综合征细胞自发性超变和基因组不稳定的原因，并与受影响患者的明显加速衰老和高癌症风险有关。
Werner Syndrome is a rare autosomal recessive disorder characterized by an increased cancer risk and by symptoms suggestive of premature aging. Cells from these patients demonstrate a typical pattern of chromosomal instability and a spontaneous hypermutability with a high rate of unusually large deletions. We have studied thein vivoDNA ligation in three lymphoblast cell lines from Werner syndrome patients and three from normal donors. In our host cell ligation assay we transfected linearized plasmid pZ189 and measured the amount of plasmid DNA ends rejoined by these host cells as the ability of the recovered plasmid to transform bacteria. A mutagenesis marker gene close to the ligation site allowed screening for mutations. Subsequent mutation analysis provided information about the accuracy of the ligation process. The cells from Werner syndrome patients were as effective as normal cells in ligating DNA ends. However, mutation analysis revealed that the three Werner syndrome cell lines introduced 2.4–4.6 times more mutations (p < 0.001) than the normal cell lines during ligation of the DNA ends: the mutation rates were 69.4, 97.2, and 58.7%, as compared to 23.6, 21.7, and 24.4% in the normal cell lines. These increased mutation frequencies in plasmids ligated during passage through Werner syndrome cells were mainly due to a significant (p <0.001) increase in deletions. This error-prone DNA ligation might be responsible for the spontaneous hypermutability and the genomic instability in Werner syndrome cells and related to the apparently accelerated aging and high cancer risk in affected patients.