Functional assay for BRCA1: mutagenesis of the COOH-terminal region reveals critical residues for transcription activation.
Functional assay for BRCA1: mutagenesis of the COOH-terminal region reveals critical residues for transcription activation.
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发表时间：
2000-05
期刊：
影响因子：
11.2
通讯作者：
F. Hayes;C. Cayanan;D. Barillà;A. Monteiro
中科院分区：
文献类型：
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作者：
F. Hayes;C. Cayanan;D. Barillà;A. Monteiro
乳腺癌和卵巢癌易感基因产物BRCA 1是一种肿瘤抑制因子，但其精确的生化功能仍不清楚。BRCA 1 COOH末端作为一个转录激活结构域，该区域的生殖系癌症易感性突变会消除转录激活，而良性多态性则不会。这些结果提高了BRCA 1转录激活的丧失对肿瘤发生至关重要的可能性。因此，鉴定BRCA 1参与转录激活的残基将有助于理解为什么特定的种系错义突变是有害的，并可能提供更可靠的症状前风险评估。BRCA 1 COOH末端（氨基酸1560-1863）由两个BRCT组成，前面是一个可能是非小叶的区域。我们结合了定点诱变和随机诱变，然后在酵母中进行了功能性转录测定：（a）易错PCR诱导的随机诱变产生了8个导致功能丧失的独特错义突变，其中6个针对犬、小鼠、大鼠和人BRCA 1中保守的疏水残基;（B）随机插入可变五肽盒产生了21个插入突变体。所有的五肽插入NH 2-末端的BRCT保留野生型活性，而在BRCT中的插入，除了少数例外，是有害的;和（c）定点诱变用于表征五个已知的种系突变，并进行删除分析的COOH末端。缺失分析显示，最COOH-末端疏水簇（I1855，L1854和Y1853）的完整性是活性所必需的。我们的结论是BRCT结构域的完整性是至关重要的转录激活和疏水残基可能是重要的BRCT功能。因此，基于酵母的转录激活测定可以成功地用于为BRCA 1的结构-功能分析提供工具，并可能构成BRCA 1功能测定的基础。
The breast and ovarian cancer susceptibility gene product BRCA1 is a tumor suppressor, but its precise biochemical function remains unknown. The BRCA1 COOH terminus acts as a transcription activation domain, and germ-line cancer- predisposing mutations in this region abolish transcription activation, whereas benign polymorphisms do not. These results raise the possibility that loss of transcription activation by BRCA1 is crucial for oncogenesis. Therefore, identification of residues involved in transcription activation by BRCA1 will help understand why particular germ-line missense mutations are deleterious and may provide more reliable presymptomatic risk assessment. The BRCA1 COOH terminus (amino acids 1560-1863) consists of two BRCTs preceded by a region likely to be nonglobular. We combined site-directed and random mutagenesis, followed by a functional transcription assay in yeast: (a) error-prone PCR-induced random mutagenesis generated eight unique missense mutations causing loss of function, six of which targeted hydrophobic residues conserved in canine, mouse, rat, and human BRCA1; (b) random insertion of a variable pentapeptide cassette generated 21 insertion mutants. All pentapeptide insertions NH2-terminal to the BRCTs retained wild-type activity, whereas insertions in the BRCTs were, with few exceptions, deleterious; and (c) site-directed mutagenesis was used to characterize five known germ-line mutations and to perform deletion analysis of the COOH terminus. Deletion analysis revealed that the integrity of the most COOH-terminal hydrophobic cluster (I1855, L1854, and Y1853) is necessary for activity. We conclude that the integrity of the BRCT domains is crucial for transcription activation and that hydrophobic residues may be important for BRCT function. Therefore, the yeast-based assay for transcription activation can be used successfully to provide tools for structure-function analysis of BRCA1 and may form the basis of a BRCA1 functional assay.