Umar A, Boyer J C, Thomas D C, Nguyen D C, Risinger J I, Boyd J, Ionov Y, Perucho M, Kunkel T A
Laboratory of Molecular Genetics, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709.
J Biol Chem. 1994 May 20;269(20):14367-70.
A replication error (RER+) phenotype, characterized by somatic instability in simple repeated sequences, is associated with several types of cancer. To determine if a defect in DNA replication fidelity or repair of replication errors might explain this instability, we compared both processes in cell-free extracts from RER+ endometrial and colorectal cancer cell lines to RER- cell lines. SV40 origin-dependent replication of a microsatellite sequence is highly accurate in cell extracts regardless of their RER phenotype. However, extracts from RER+ cell lines are defective in mismatch repair, while extracts of RER- cell lines are not. Lack of repair was observed when the signal (a nick) for strand-specific repair was either 3' or 5' to the mispair. One colorectal cancer cell line contained deletions in both alleles of the putative mismatch repair gene hMSH2, and one endometrial cancer cell line contained a 4-base pair duplication in one hMSH2 allele. No hMSH2 mutation was detected in the other allele or in the other five RER+ cell lines. Repair was readily detected when each of the defective extracts was mixed with a repair-proficient extract, demonstrating that no trans-acting inhibitor is present. Attempts to complement the repair deficiencies by mixing two different defective extracts identified three combinations that restored repair. The data suggest that: (i) defective repair is associated with colorectal and endometrial cancer and, by extrapolation, with other types of cancer; (ii) mutations in the hMSH2 gene, and possibly other genes, result in defective mismatch repair; (iii) the defect(s) in these lines likely involves pre-incision events or the excision step, but not the incision, polymerization, or ligation steps; and (iv) at least four functional complementation groups for mismatch repair may be involved in human cancer.
复制错误（RER+）表型的特征是简单重复序列中的体细胞不稳定性，与多种癌症类型相关。为了确定DNA复制保真度缺陷或复制错误修复是否可以解释这种不稳定性，我们将RER+子宫内膜癌细胞系和结肠直肠癌细胞系的无细胞提取物中的这两个过程与RER-细胞系进行了比较。无论其RER表型如何，微卫星序列的SV40原点依赖性复制在细胞提取物中都非常准确。然而，RER+细胞系的提取物在错配修复方面存在缺陷，而RER-细胞系的提取物则没有。当链特异性修复的信号（一个切口）在错配的3'或5'端时，观察到修复缺失。一个结肠直肠癌细胞系在假定的错配修复基因hMSH2的两个等位基因中都有缺失，一个子宫内膜癌细胞系在一个hMSH2等位基因中有一个4碱基对的重复。在另一个等位基因或其他五个RER+细胞系中未检测到hMSH2突变。当将每个有缺陷的提取物与一个修复能力强的提取物混合时，很容易检测到修复，这表明不存在反式作用抑制剂。通过混合两种不同的有缺陷提取物来补充修复缺陷的尝试确定了三种恢复修复的组合。数据表明：（i）修复缺陷与结肠直肠癌和子宫内膜癌相关，并由此推断与其他类型的癌症相关；（ii）hMSH2基因以及可能其他基因的突变导致错配修复缺陷；（iii）这些细胞系中的缺陷可能涉及切口前事件或切除步骤，但不涉及切口、聚合或连接步骤；（iv）至少四个错配修复功能互补组可能参与人类癌症。
