Boyer J C, Farber R A
Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, 27599, USA.
Cancer Res. 1998 Sep 1;58(17):3946-9.
Dinucleotide repeats, because of their repetitive nature, are prone to frameshift mutations, most likely via a DNA-polymerase slippage mechanism. Mutation rates in microsatellite DNA sequences are high in mismatch repair-defective cells. In normal cells, only estimates of maximal rates of mutation in microsatellites have been possible previously, because of the low sensitivity of screening assays for mutations in endogenous sequences. We have measured the spontaneous mutation rate of a dinucleotide repeat in diploid human foreskin fibroblasts. In our system, the mutation target is a (CA)17 repeat contained within a stably integrated plasmid. The repeat disrupts the reading frame of a neomycin (neo) resistance gene within the plasmid. Cells containing frameshift mutations in the CA repeat that correct the reading frame of the neo gene are selected using the neo analogue G418. This system of measuring microsatellite mutation rates is highly sensitive, because there is a specific target within which mutations can be selected. Fluctuation analysis of cells containing the target DNA yielded mutation rates of <3.1 x 10(-8) to 44.8 x 10(-8) mutations/cell/generation. This is the first report of a direct measurement of a spontaneous mutation rate of a microsatellite sequence in normal human cells.
由于其重复性质，二核苷酸重复序列容易发生移码突变，最有可能是通过DNA聚合酶滑动机制。在错配修复缺陷的细胞中，微卫星DNA序列的突变率很高。在正常细胞中，由于对内源序列突变的筛选检测灵敏度较低，以前只能估计微卫星的最大突变率。我们已经测量了二倍体人包皮成纤维细胞中二核苷酸重复序列的自发突变率。在我们的系统中，突变靶点是一个稳定整合质粒中包含的(CA)17重复序列。该重复序列破坏了质粒中新霉素(neo)抗性基因的阅读框。使用neo类似物G418筛选出在CA重复序列中发生移码突变从而校正neo基因阅读框的细胞。这种测量微卫星突变率的系统非常灵敏，因为存在一个可以选择突变的特定靶点。对含有靶DNA的细胞进行波动分析，得出的突变率为<3.1×10(-8)至44.8×10(-8)个突变/细胞/代。这是首次直接测量正常人细胞中微卫星序列自发突变率的报告。
