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Construction of heteroduplex DNA and in vitro model for functional analysis of mismatch repair

机译：异源双链DNA的构建及错配修复功能分析的体外模型

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Functional deficiency of mismatch repair (MMR) system is one of the mechanisms of tumorigenesis. With the development of the investigation and the requirement from the clinical diagnosis and treatment it is necessary to build up a method to evaluate the functional status of the whole MMR system in the concerned tumors. The original ssDNA and dsDNA from wild type (wt) bacteriophage M13mp2 and its three derivates with mutation points in the lacZ alpha gene have been used to construct two kinds of hetero-duplex DNA molecules. One named del(2) has two bases deleted in the negative strand, the other has a G centre dot G mismatch base pair in the negative strand too. Introducing this heteroduplex DNA into E. coli NR9162 (mutS~- without the MMR ability on the indicator plate with x-gal and IPTG, there are three kinds of plaques, mixture plaque as the characteristic phenotype of heteroduplex DNA, blue and clear plaques. If the cell extract is mismatch repair competent the percentage of the mixture plaque will decrease after incubation with these heteroduplex DNA, the repair efficiency is expressed in percentage as 100x (1 minus the ratio of percentages of mixture plaque obtained from the extract-treated sample and untreated samples), which can imply the functional status of MMR system of certain samples. After large T-antigen-dependent SV-40 DNA replication assay cell extract from TK6, a human lymphoblastoid B-cell lymphoma cell line with MMR ability, and Lovo, a human colonic carcinoma cell line with MMR deficiency have incubated with these heteroduplex DNA. The repair efficiency of TK6 to del(2) is more than 60 percent, to G centre dot G is more than 50 percent. The Lovo efficiency to del(2) is less than 10 percent, to G centre dot G is less than 20 percent. Therefore, in this in vitro model used for functional analysis of mismatch repair of heteroduplex DNA as the repair target, TK6 can serve as the control for MMR proficiency and Lovo as the control for MMR deficiency. Using this model the tumor tissue from a case of hereditary nonpolyposis colorectal cancer (microsatellite instability high, MSI-H) was measured and lack of MMR ability was shown. And a case of sporadic rectal cancer (SRC) (microsatellite stability, MSS) maintains MMR proficiency. The results indicate that the model is sensitive and dependable. It could be used to measure the function status of MMR system in tumor cell and/or tissues. This is a reliable method to investigate the mechanic of tumorigenesis. It is meaningful in the observation of the role of MMR in the initiation and progression of concerned tumors.
机译：错配修复（MMR）系统的功能缺陷是肿瘤发生的机制之一。随着研究的发展以及临床诊断和治疗的需求，有必要建立一种评估整个MMR系统在相关肿瘤中功能状态的方法。来自野生型（wt）噬菌体M13mp2的原始ssDNA和dsDNA及其在lacZ alpha基因中具有突变点的三个衍生物已用于构建两种异源双链DNA分子。一个名为del（2）的负链删除了两个碱基，另一个在负链中删除了一个G中心点G不匹配的碱基对。将这种异源双链体DNA引入具有x-gal和IPTG的指示板上没有MMR能力的大肠杆菌NR9162（mutS〜-）中，存在三种噬菌斑，即混合噬菌斑作为异源双链DNA的特征表型，蓝色和透明噬菌斑。如果细胞提取物具有错配修复能力，则在与这些异源双链DNA孵育后，混合噬菌斑的百分比将降低，修复效率以百分数表示（100倍（1减去从提取物处理过的样品中得到的混合噬菌斑的百分比与经过大量的T抗原依赖性SV-40 DNA复制测定后，从TK6，具有MMR能力的人淋巴母细胞B细胞淋巴瘤细胞系和Lovo中提取了T抗原依赖性的SV-40 DNA复制后，可能暗示某些样品的MMR系统的功能状态。 ，具有MMR缺陷的人结肠癌细胞系已与这些异源双链DNA一起孵育，TK6对del（2）的修复效率超过60％，对G中心点G的修复效率更高。 50％。 del（2）的Lovo效率小于10％，G中心点G的Lovo效率小于20％。因此，在用于功能分析异源双链DNA错配修复的体外模型中，TK6可以作为MMR水平的对照，而Lovo可以作为MMR缺陷的对照。使用该模型，测量了遗传性非息肉性结直肠癌（微卫星不稳定性高，MSI-H）一例的肿瘤组织，显示缺乏MMR能力。一例散发性直肠癌（SRC）（微卫星稳定性，MSS）保持了MMR水平。结果表明该模型灵敏可靠。它可用于测量MMR系统在肿瘤细胞和/或组织中的功能状态。这是研究肿瘤发生机理的可靠方法。这对于观察MMR在相关肿瘤的发生和发展中的作用是有意义的。

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