Identification of frame-shift intermediate mutant cells
Identification of frame-shift intermediate mutant cells
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DOI：
10.1073/pnas.0437965100
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发表时间：
2003-02-18
影响因子：
11.1
通讯作者：
Boland, CR
中科院分区：
文献类型：
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作者：
Gasche, C;Chang, CL;Boland, CR
微卫星的移码突变是聚合酶错误随时间累积的结果，随后是复制后错配修复失败。开发了一种细胞培养系统，该系统能够在最初的DNA聚合酶错误发生后，识别在单条DNA链上携带突变的中间突变细胞。构建了一种质粒，该质粒在增强型绿色荧光蛋白（EGFP）基因的翻译起始密码子之后紧接着包含13个多聚（dC - dA）·多聚（dG - dT）寡核苷酸重复序列，使EGFP基因移出其正确的阅读框。将该质粒导入人错配修复缺陷型（HCT116，hMLH1突变型）和错配修复正常型（HCT116 + chr3，hMLH1野生型）结直肠癌细胞中。在发生恢复EGFP阅读框的移码突变后，检测到表达EGFP的细胞，并识别出两个不同的荧光群体，M1（弱荧光细胞）和M2（强荧光细胞）。M1细胞数量稳定，而M2细胞随时间积累。在HCT116中，单个M2细胞产生在（CA）₁₃微卫星处有2个碱基对缺失的荧光菌落。然而，28%的单个M1细胞产生具有混合荧光模式的菌落，其同时携带（CA）₁₃和（CA）₁₂微卫星。M1细胞很可能代表携带（CA）₁₃·（GT）₁₂异源双链体的中间突变体。尽管HCT116细胞克隆中的突变率（6.2×10⁻⁴）比HCT116 + chr3（1.9×10⁻⁵）高30倍，但培养物中M1细胞的比例在HCT116（5.87×10⁻³）和HCT116 + chr3（4.13×10⁻³）之间没有显著差异，这表明中间突变体的产生不受错配修复能力的影响。
Frame-shift mutations at microsatellites occur as a time-dependent function of polymerase errors followed by failure of postreplicational mismatch repair. A cell-culture system was developed that allows identification of intermediate mutant cells that carry the mutation on a single DNA strand after the initial DNA polymerase errors. A plasmid was constructed that contained 13 repeats of a poly(dC-dA)(.)poly(dG-dT) oligonucleotide immediately after the translation initiation codon of the enhanced GFP (EGFP) gene, shifting the EGFP gene out of its proper reading frame. The plasmid was introduced into human mismatch repair-deficient (HCT116, hMLH1-mutated) and mismatch repair-proficient (HCT116+chr3, hMLH1 wild type) colorectal cancer cells. After frame-shift mutations occurred that restored the EGFP reading frame, EGFP-expressing cells were detected, and two distinct fluorescent populations, M1 (dim cells) and M2 (bright cells), were identified. M1 cell numbers were stable, whereas M2 cells accumulated over time. In HCT116, single M2 cells gave rise to fluorescent colonies that carried a 2-bp deletion at the (CA)(13) microsatellite. Twenty-eight percent of single M1 cells, however, gave rise to colonies with a mixed fluorescence pattern that carried both (CA)13 and (CA)12 microsatellites. It is likely that M1 cells represent intermediate mutants that carry (CA)(13)(.)(GT)(12) heteroduplexes. Although the mutation rate in HCT116 cell clones (6,2 x 10(-4)) was 30 times higher than in HCT116+chr3 (1.9 x 10(-5)), the proportion of M1 cells in culture did not significantly differ between HCT116 (5.87 x 10(-3))and HCT116+chr3 (4.13 X 10(-3)), indicating that the generation of intermediate mutants is not affected by mismatch-repair proficiency.