Department of Medicine, University of California, San Diego, CA, USA.
Hum Mol Genet. 2010 Jul 1;19(13):2638-47. doi: 10.1093/hmg/ddq151. Epub 2010 Apr 23.
It is generally accepted that longer microsatellites mutate more frequently in defective DNA mismatch repair (MMR) than shorter microsatellites. Indeed, we have previously observed that the A10 microsatellite of transforming growth factor beta type II receptor (TGFBR2) frameshifts -1 bp at a faster rate than the A8 microsatellite of activin type II receptor (ACVR2), although both genes become frameshift-mutated in >80% of MMR-defective colorectal cancers. To experimentally determine the effect of microsatellite length upon frameshift mutation in gene-specific sequence contexts, we altered the microsatellite length within TGFBR2 exon 3 and ACVR2 exon 10, generating A7, A10 and A13 constructs. These constructs were cloned 1 bp out of frame of EGFP, allowing a -1 bp frameshift to drive EGFP expression, and stably transfected into MMR-deficient cells. Subsequent non-fluorescent cells were sorted, cultured for 7-35 days and harvested for EGFP analysis and DNA sequencing. Longer microsatellites within TGFBR2 and ACVR2 showed significantly higher mutation rates than shorter ones, with TGFBR2 A13, A10 and A7 frameshifts measured at 22.38x10(-4), 2.17x10(-4) and 0.13x10(-4), respectively. Surprisingly, shorter ACVR2 constructs showed three times higher mutation rates at A7 and A10 lengths than identical length TGFBR2 constructs but comparably lower at the A13 length, suggesting influences from both microsatellite length as well as the sequence context. Furthermore, the TGFBR2 A13 construct mutated into 33% A11 sequences (-2 bp) in addition to expected A12 (-1 bp), indicating that this construct undergoes continual subsequent frameshift mutation. These data demonstrate experimentally that both the length of a mononucleotide microsatellite and its sequence context influence mutation rate in defective DNA MMR.
一般认为，在有缺陷的 DNA 错配修复（MMR）中，较长的微卫星比较短的微卫星更频繁地发生突变。事实上，我们之前观察到转化生长因子β型 II 受体（TGFBR2）的 A10 微卫星-1bp 频率比激活素 II 受体（ACVR2）的 A8 微卫星更快地发生移框突变，尽管这两个基因在>80%的 MMR 缺陷结直肠癌中都发生了移框突变。为了在特定基因序列环境中实验确定微卫星长度对移框突变的影响，我们改变了 TGFBR2 外显子 3 和 ACVR2 外显子 10 中的微卫星长度，生成了 A7、A10 和 A13 构建体。这些构建体在 EGFP 之外的 1bp 处被克隆，使-1bp 移框驱动 EGFP 表达，并稳定转染到 MMR 缺陷细胞中。随后对非荧光细胞进行分选，培养 7-35 天，并收获用于 EGFP 分析和 DNA 测序。TGFBR2 和 ACVR2 中的较长微卫星显示出明显高于较短微卫星的突变率，TGFBR2 的 A13、A10 和 A7 移框分别测量为 22.38x10(-4)、2.17x10(-4)和 0.13x10(-4)。令人惊讶的是，较短的 ACVR2 构建体在 A7 和 A10 长度上的突变率比相同长度的 TGFBR2 构建体高 3 倍，但在 A13 长度上的突变率较低，这表明微卫星长度和序列环境都有影响。此外，TGFBR2 A13 构建体在 A11 序列（-2bp）中突变，除了预期的 A12（-1bp）之外，还表明该构建体经历了持续的后续移框突变。这些数据实验证明，单核苷酸微卫星的长度及其序列环境都影响 DNA MMR 缺陷中的突变率。
