Jackson A L, Chen R, Loeb L A
Department of Pathology, University of Washington, Box 357705, Seattle, WA 98195, USA.
Proc Natl Acad Sci U S A. 1998 Oct 13;95(21):12468-73. doi: 10.1073/pnas.95.21.12468.
Instability of repetitive sequences, both in intronic sequences and within coding regions, has been demonstrated to be a hallmark of genomic instability in human cancer. Understanding how these mutational events arise may provide an opportunity for prevention or early intervention in cancer development. To study the source of this instability, we have identified a region of the beta-lactamase gene that is tolerant to the insertion of fragments of exogenous DNA as large as 1,614 bp with minimal loss of enzyme activity, as determined by antibiotic resistance. Fragments inserted out-of-frame render Escherichia coli sensitive to antibiotic, and compensatory frameshift mutations that restore the reading frame of beta-lactamase can be selected on the basis of antibiotic resistance. We have utilized this site to insert a synthetic microsatellite sequence within the beta-lactamase gene and selected for mutations yielding frameshifts. This assay provides for detection of one frameshift mutation in a background of 10(6) wild-type sequences. Mismatch repair deficiency increased the observed frameshift frequency approximately 300-fold. Exposure of plasmid containing microsatellite sequences to hydrogen peroxide resulted in frameshift mutations that were localized exclusively to the microsatellite sequences, whereas DNA damage by UV or N-methyl-N'-nitro-N-nitrosoguanidine did not result in enhanced mutagenesis. We postulate that in tumor cells, endogenous production of oxygen free radicals may be a major factor in promoting instability of microsatellite sequences. This beta-lactamase assay may provide a sensitive methodology for the detection and quantitation of mutations associated with the development of cancer.
内含子序列和编码区域内重复序列的不稳定性已被证明是人类癌症基因组不稳定的一个标志。了解这些突变事件是如何发生的，可能为癌症发展的预防或早期干预提供机会。为了研究这种不稳定性的来源，我们确定了β-内酰胺酶基因的一个区域，该区域能够耐受长达1614 bp的外源DNA片段的插入，且酶活性损失最小，这是通过抗生素抗性测定的。插入移码的片段会使大肠杆菌对抗生素敏感，并且可以根据抗生素抗性选择恢复β-内酰胺酶阅读框的补偿性移码突变。我们利用这个位点在β-内酰胺酶基因内插入一个合成微卫星序列，并选择产生移码的突变。该检测方法能够在10^6个野生型序列的背景中检测到一个移码突变。错配修复缺陷使观察到的移码频率增加了约300倍。含有微卫星序列的质粒暴露于过氧化氢会导致移码突变，这些突变仅局限于微卫星序列，而紫外线或N-甲基-N'-硝基-N-亚硝基胍引起的DNA损伤不会导致诱变增强。我们推测，在肿瘤细胞中，内源性氧自由基的产生可能是促进微卫星序列不稳定的一个主要因素。这种β-内酰胺酶检测方法可能为检测和定量与癌症发展相关的突变提供一种灵敏的方法。
