Determining the site and nature of DNA mutations with the cloned MutY mismatch repair enzyme.
Determining the site and nature of DNA mutations with the cloned MutY mismatch repair enzyme.
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DOI：
10.1093/carcin/17.2.321
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发表时间：
1996-02
期刊：
影响因子：
4.7
通讯作者：
J. Xu;Q. Yang;J. Chen;M. van Baalen;I. Hsu
中科院分区：
文献类型：
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作者：
J. Xu;Q. Yang;J. Chen;M. van Baalen;I. Hsu
将大肠杆菌 MutY 基因克隆到修饰的 pET-11 质粒中，然后将其转染到大肠杆菌 HMS174 宿主中，以过量产生 MutY 错配修复 (MR) 酶。根据电泳后 SDS-PAGE 凝胶上的染色强度估计，转化的 HMS174 细胞中大约 30-50% 的总细胞蛋白是异丙基-β-D-硫代半乳糖苷诱导的 MutY 蛋白。通过纤维素磷酸盐离子交换层析和凝胶过滤层析将 MutY 蛋白纯化至接近均质。纯化的 MutY 蛋白具有酶活性，可在第一个磷酸二酯键的 3' 端切割 G/A 错配的 A，然后在 A 的第二个磷酸二酯键的 5' 端切割。它还切割 C/A 错配的 A，但程度要小得多，并且该活性与 DNA 序列相关。在具有已知或未知 p53 突变的人类肿瘤 DNA 样本中检查了该测定法确定 DNA 突变位点和性质的可靠性。在该测定中，来自正常和突变 p53 基因的聚合酶链式反应扩增的 DNA 片段被混合、变性和退火，以产生 G/A 或 C/A 错配，以便被 MutY MR 酶切割。检测结果揭示了已知 G:CT:A 突变的位点和性质。此外，通过该测定还发现了以前未知的 G:C 到 T:A 突变，该突变在肿瘤 DNA 制剂的测序分析中被误读。
The Escherichia coli MutY gene was cloned into a modified pET-11 plasmid which was then transfected into an E.coli HMS174 host for overproduction of the MutY mismatch repair (MR) enzyme. Approximately 30-50% of the total cellular protein in the transformed HMS174 cells was isopropyl-beta-D-thiogalactoside-induced MutY protein, as estimated from the staining intensity on an SDS-PAGE gel following electrophoresis. The MutY protein was purified to near homogeneity by cellulose phosphate ion-exchange chromatography followed by gel filtration chromatography. The purified MutY protein had enzyme activities which cleaved the A of a G/A mismatch at the 3' end of the first phosphodiester bond and then the 5' end of the second phosphodiester bond of the A. It also cut the A of a C/A mismatch, but to a much lesser extent, and the activity was DNA sequence-dependent. The reliability of the assay in determining the site and nature of a DNA mutation was examined in human tumor DNA samples with known or unknown p53 mutations. In the assay, polymerase chain reaction-amplified DNA fragments from normal and mutated p53 genes were mixed, denatured and annealed to generate mismatches of G/A or C/A for cleavage by the MutY MR enzyme. The assay results revealed the site and nature of known G:CT:A mutations. In addition, a previously unknown G:C to T:A mutation, which was misread in the sequencing analysis of a tumor DNA preparation, was identified by this assay.