Mutation rates of TGFBR2 and ACVR2 coding microsatellites in human cells with defective DNA mismatch repair.
Mutation rates of TGFBR2 and ACVR2 coding microsatellites in human cells with defective DNA mismatch repair.
复制标题
DOI：
10.1371/journal.pone.0003463
复制
发表时间：
2008
期刊：
影响因子：
3.7
通讯作者：
Carethers JM
中科院分区：
文献类型：
--
作者：
Chung H;Young DJ;Lopez CG;Le TA;Lee JK;Ream-Robinson D;Huang SC;Carethers JM
微卫星不稳定性通过在肿瘤抑制基因（如TGFBR2和ACVR2）的编码微卫星处产生移码突变来促进结肠肿瘤发生。因此，在DNA错配修复（MMR）缺陷型肿瘤中，通过这些转化生长因子β（TGFβ）家族受体的信号传导被阻断。这些突变是如何实时发生的以及这些人类编码序列的突变率此前尚未得到研究。我们利用具有不同MMR缺陷（hMLH1 -/ -、hMSH6 -/ -、hMSH3 -/ -以及MMR正常）的细胞系来测定突变率。构建了一些质粒，其中TGFBR2的第3外显子和ACVR2的第10外显子在增强型绿色荧光蛋白（EGFP）基因的翻译起始密码子之后立即被克隆为移码+1bp，使得 -1bp的移码突变能够驱动EGFP表达。通过中断编码微卫星序列构建了抗突变质粒，以防止移码突变。建立了包含部分TGFBR2和ACVR2的稳定细胞系，对非荧光细胞进行分选，培养7 - 35天，并在特定时间点收获用于流式细胞术突变检测和DNA测序。DNA测序显示荧光细胞中存在 -1bp的移码突变（TGFBR2中为A9，ACVR2中为A7）。鉴定出两个不同的荧光群体，M1（暗淡，代表异源双链体）和M2（明亮，代表完全突变体），其中M2部分随时间积累。与hMSH6缺陷相比，hMLH1缺陷时TGFBR2和ACVR2微卫星的突变率分别高11倍（5.91×10⁻⁴）和15倍（2.18×10⁻⁴）。在hMLH1和hMSH6缺陷中，TGFBR2微卫星的突变率比ACVR2微卫星高约3倍。TGFBR2和ACVR2微卫星序列的 -1bp移码突变率取决于人类MMR背景。
Microsatellite instability promotes colonic tumorigenesis through generating frameshift mutations at coding microsatellites of tumor suppressor genes, such as TGFBR2 and ACVR2. As a consequence, signaling through these TGFβ family receptors is abrogated in DNA Mismatch repair (MMR)-deficient tumors. How these mutations occur in real time and mutational rates of these human coding sequences have not previously been studied. We utilized cell lines with different MMR deficiencies (hMLH1−/−, hMSH6−/−, hMSH3−/−, and MMR-proficient) to determine mutation rates. Plasmids were constructed in which exon 3 of TGFBR2 and exon 10 of ACVR2 were cloned +1 bp out of frame, immediately after the translation initiation codon of an enhanced GFP (EGFP) gene, allowing a −1 bp frameshift mutation to drive EGFP expression. Mutation-resistant plasmids were constructed by interrupting the coding microsatellite sequences, preventing frameshift mutation. Stable cell lines were established containing portions of TGFBR2 and ACVR2, and nonfluorescent cells were sorted, cultured for 7–35 days, and harvested for flow cytometric mutation detection and DNA sequencing at specific time points. DNA sequencing revealed a −1 bp frameshift mutation (A9 in TGFBR2 and A7 in ACVR2) in the fluorescent cells. Two distinct fluorescent populations, M1 (dim, representing heteroduplexes) and M2 (bright, representing full mutants) were identified, with the M2 fraction accumulating over time. hMLH1 deficiency revealed 11 (5.91×10−4) and 15 (2.18×10−4) times higher mutation rates for the TGFBR2 and ACVR2 microsatellites compared to hMSH6 deficiency, respectively. The mutation rate of the TGFBR2 microsatellite was ∼3 times higher in both hMLH1 and hMSH6 deficiencies than the ACVR2 microsatellite. The −1 bp frameshift mutation rates of TGFBR2 and ACVR2 microsatellite sequences are dependent upon the human MMR background.
登录
查看更多内容
影响因子：
3.7
作者：
Marra, G;Boland, CR
通讯作者：
Boland, CR
影响因子：
29.4
作者：
Jung, Barbara H.;Beck, Stayce E.;Carethers, John M.
通讯作者：
Carethers, John M.
影响因子：
64.5
作者：
FISHEL, R;LESCOE, MK;KOLODNER, R
通讯作者：
KOLODNER, R
影响因子：
158.5
作者：
Hampel, H;Frankel, WL;Papadopoulos, N
通讯作者：
Papadopoulos, N
DOI：
10.1073/pnas.0437965100
发表时间：
2003-02-18
影响因子：
11.1
作者：
Gasche, C;Chang, CL;Boland, CR
通讯作者：
Boland, CR