Induction of microsatellite instability by oxidative DNA damage
Induction of microsatellite instability by oxidative DNA damage
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DOI：
10.1073/pnas.95.21.12468
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发表时间：
1998-10-13
影响因子：
11.1
通讯作者：
Loeb, LA
中科院分区：
文献类型：
--
作者：
Jackson, AL;Chen, R;Loeb, LA
重复序列的不稳定性，无论是在内含子序列还是在编码区，都已被证明是人类癌症基因组不稳定的标志。了解这些突变事件是如何发生的，可能会为预防或早期干预癌症的发展提供机会。为了研究这种不稳定性的来源，我们已经确定了β-内酰胺酶基因的一个区域，该区域可以耐受长达1,614个核苷酸的外源DNA片段的插入，而酶活性的损失最小，这是由抗生素耐药性决定的。插入框外的片段使大肠杆菌对抗生素敏感，可以根据抗生素耐药性选择恢复β-内酰胺酶阅读框的代偿性移码突变。我们利用这个位点在β-内酰胺酶基因中插入了一个合成的微卫星序列，并对产生移码的突变进行了选择。这种检测方法可以在10(6)个野生型序列的背景中检测一个移码突变。错配修复缺陷使观察到的移码频率增加了近300倍。含有微卫星序列的质粒暴露在过氧化氢中会导致移码突变，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-亚硝基胍对DNA的损伤不会导致更强的突变。我们推测，在肿瘤细胞中，氧自由基的内源性产生可能是促进微卫星序列不稳定的主要因素。这种β-内酰胺酶检测可能为检测和量化与癌症发展相关的突变提供了一种灵敏的方法。
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 p-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 P-lactamase can be selected on the basis of antibiotic resistance. We have utilized this site to insert a synthetic microsatellite sequence within the p-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 approximate to 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 p-lactamase assay may provide a sensitive methodology for the detection and quantitation of mutations associated with the development of cancer.