Slebos R J, Taylor J A
Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA.
Biochem Biophys Res Commun. 2001 Feb 16;281(1):212-9. doi: 10.1006/bbrc.2001.4335.
Repair of DNA double-strand breaks (DSB) is essential for cell viability and genome stability. Homologous recombination repair plays an important role in DSB repair and impairment of this repair mechanism may lead to loss of genomic integrity, which is one of the hallmarks of cancer. Recent research has shown that the tumor suppressor genes p53 and BRCA1 and -2 are involved in the proper control of homologous recombination, suggesting a role of this type of repair in human cancer. We developed a novel assay based on recombination between two Green Fluorescent Protein (GFP) sequences in transiently transfected plasmid DNA. The plasmid construct contains an intact, emission-shifted, "blue" variant of GFP (BFP), with a 300 nucleotide stretch of homology to a nonfunctional copy of GFP. In the absence of homologous recombination only BFP is present, but homologous recombination can create a functional GFP. The homologous regions in the plasmid were constructed in both the direct and the inverted orientation of transcription to detect possible differences in the recombination mechanisms involved. A panel of human tumor cell lines was chosen on the basis of genetic background and chromosome integrity and tested for homologous recombination using this assay. The panel included cell lines with varying levels of karyotypic abnormalities, isogenic cell lines with normal and mutant p53, isogenic cell lines with or without DNA mismatch repair, BRCA1 and -2 mutant cell lines, and the lymphoma cell line DT40. With this assay, the observed differences between cell lines with the lowest and highest levels of recombination were about 100-fold. Increased levels of recombination were associated with mutant p53, whereas a low level of recombination was found in the BRCA1 mutant cell line. In the cell line HT1080TG, a mutagenized derivative of HT1080 with two mutant alleles of p53, high levels of recombination were found with the direct orientation but not with the inverted orientation plasmid. No difference in recombination was detected between two isogenic cell lines that only differed in DNA mismatch repair capability. We conclude that this assay can detect differences in homologous recombination capacity in cultured cell lines and that these differences follow the patterns that would be expected from the different genotypes of these cell lines. Future application in normal cells may be useful to identify genetic determinants controlling genomic integrity or to detect differences in DNA repair capacity in individuals.
DNA双链断裂（DSB）的修复对于细胞活力和基因组稳定性至关重要。同源重组修复在DSB修复中发挥重要作用，而这种修复机制的受损可能导致基因组完整性丧失，这是癌症的特征之一。最近的研究表明，肿瘤抑制基因p53以及BRCA1和BRCA2参与同源重组的适当调控，提示这种修复类型在人类癌症中发挥作用。我们基于瞬时转染质粒DNA中两个绿色荧光蛋白（GFP）序列之间的重组开发了一种新型检测方法。该质粒构建体包含一个完整的、发射光谱发生位移的“蓝色”GFP变体（BFP），其与GFP的无功能拷贝具有300个核苷酸的同源序列。在不存在同源重组的情况下，仅存在BFP，但同源重组可产生功能性GFP。质粒中的同源区域以转录的正向和反向两种方向构建，以检测所涉及的重组机制中可能存在的差异。基于遗传背景和染色体完整性选择了一组人类肿瘤细胞系，并使用该检测方法检测同源重组。该组包括具有不同核型异常水平的细胞系、具有野生型和突变型p53的同基因细胞系、具有或不具有DNA错配修复的同基因细胞系、BRCA1和BRCA2突变细胞系以及淋巴瘤细胞系DT40。通过该检测方法，观察到重组水平最低和最高的细胞系之间的差异约为100倍。重组水平的增加与突变型p53相关，而在BRCA1突变细胞系中发现重组水平较低。在HT1080TG细胞系中，它是具有两个p53突变等位基因的HT1080诱变衍生物，发现正向质粒存在高水平重组，而反向质粒则没有。在仅DNA错配修复能力不同的两个同基因细胞系之间未检测到重组差异。我们得出结论，该检测方法可检测培养细胞系中同源重组能力的差异，并且这些差异符合这些细胞系不同基因型所预期的模式。未来在正常细胞中的应用可能有助于识别控制基因组完整性的遗传决定因素或检测个体DNA修复能力的差异。
