Identification of breast tumor mutations in BRCA1 that abolish its function in homologous DNA recombination.
Identification of breast tumor mutations in BRCA1 that abolish its function in homologous DNA recombination.
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DOI：
10.1158/0008-5472.can-09-2850
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发表时间：
2010-02-01
期刊：
影响因子：
11.2
通讯作者：
Parvin JD
中科院分区：
文献类型：
--
作者：
Ransburgh DJ;Chiba N;Ishioka C;Toland AE;Parvin JD
很少有发表的BRCA 1基因错义突变对BRCA 1蛋白功能的影响的分析。在这项研究中，我们采用了先前描述的同源定向重组（HDR）分析BRCA 1点置换对其重组功能的影响。我们建立了HeLa衍生的细胞系，其在其基因组中整合了重组底物。在转染表达在重组底物中产生双链断裂的核酸内切酶的质粒后，HDR容易通过GFP阳性细胞的百分比评分。通过将细胞BRCA 1 mRNA特异性的RNAi与对RNAi具有抗性的BRCA 1突变体的表达相结合，我们可以用BRCA 1的选定点突变体有效地替换内源性BRCA 1蛋白，并在重组测定中测试这些突变体。我们发现BRCA 1的氨基端和羧基端约300个残基对于指导HDR是必需的。16错义突变体从BRCA 1的氨基末端进行了分析HDR的功能，我们发现，几个点突变体完全取代野生型BRCA 1和中性在这个过程中。任何单一锌配位残基的突变在该测定中是完全缺陷的。由于错义突变，包括蛋氨酸-18到苏氨酸和苏氨酸-37到精氨酸的几种蛋白质变体也被发现是重组缺陷的。因此，我们已经建立了一个强大的检测系统，用于分析BRCA 1的特定错义突变在调节同源重组过程中的影响。
There have been few published analyses of the effects of missense mutations of the BRCA1 gene on BRCA1 protein function. In this study, we adapted a previously described homology directed recombination (HDR) assay to the analysis of the effects of BRCA1 point substitutions on its function in recombination. We established a HeLa-derived cell line, which has integrated in its genome a recombination substrate. Following transfection of a plasmid that expresses the endonuclease that creates a double-stranded break in the recombination substrate, HDR is readily scored by the percentage of GFP-positive cells. By combining RNAi specific for the cellular BRCA1 mRNA with expression of BRCA1 mutants resistant to the RNAi, we could effectively replace the endogenous BRCA1 protein with selected point mutants of BRCA1 and test these in the recombination assay. We found that both, the amino- and carboxy-terminal ~300 residues of BRCA1 were essential for directing HDR. Sixteen missense mutants from the amino terminus of BRCA1 were analyzed for function in HDR, and we found that several point mutants fully replaced the wild-type BRCA1 and are neutral in this process. Mutation of any single zinc-coordinating residue was fully defective in this assay. Several protein variants due to missense mutations, including methionine-18 to threonine and threonine-37 to arginine were also found to be defective for recombination. We have thus established a robust assay system for the analysis of the effects of specific missense mutations of BRCA1 in regulating the homologous recombination process.