Xu J F, Yang Q P, Chen J Y, van Baalen M R, Hsu I C
Department of Pathology, School of Medicine, University of Maryland at Baltimore 21201, USA.
Carcinogenesis. 1996 Feb;17(2):321-6. doi: 10.1093/carcin/17.2.321.
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:C<-->T: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.
将大肠杆菌MutY基因克隆到一个经过修饰的pET-11质粒中，然后将其转染到大肠杆菌HMS174宿主中，以过量生产MutY错配修复（MR）酶。根据电泳后SDS-PAGE凝胶上的染色强度估计，在转化的HMS174细胞中，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。通过磷酸纤维素离子交换色谱法，然后是凝胶过滤色谱法，将MutY蛋白纯化至接近均一。纯化的MutY蛋白具有酶活性，可切割第一个磷酸二酯键3'端G/A错配中的A，然后切割该A的第二个磷酸二酯键的5'端。它也能切割C/A错配中的A，但程度要小得多，且该活性依赖于DNA序列。在已知或未知p53突变的人类肿瘤DNA样本中，检测了该测定法在确定DNA突变位点和性质方面的可靠性。在该测定法中，将来自正常和突变p53基因的聚合酶链反应扩增的DNA片段混合、变性并退火，以产生G/A或C/A错配，供MutY MR酶切割。测定结果揭示了已知的G:C<-->T:A突变的位点和性质。此外，该测定法还鉴定出了一个先前未知的G:C到T:A突变，该突变在一份肿瘤DNA制剂的测序分析中被误读。
