BRCA1 facilitates microhomology-mediated end joining of DNA double strand breaks

被引:119
作者
Zhong, Q [1 ]
Chen, CF [1 ]
Chen, PL [1 ]
Lee, WH [1 ]
机构
[1] Univ Texas, Hlth Sci Ctr, Dept Mol Med, Inst Biotechnol, San Antonio, TX 78245 USA
关键词
D O I
10.1074/jbc.M200748200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
BRCA1 is critical for the maintenance of genomic stability, in part through its interaction with the Rad50.Mre11.Nbs1 complex, which occupies a central role in DNA double strand break repair mediated by nonhomologous end joining (NHEJ) and homologous recombination. BRCA1 has been shown to be required for homology-directed recombination repair. However, the role of BRCA1 in NHEJ, a critical pathway for the repair of double strand breaks and genome stability in mammalian cells, remains elusive. Here, we established a pair of mouse embryonic fibroblasts (MEFs) derived from 9.5-day-old embryos with genotypes Brca1(+/+):p53(-/-) or Brca1(-/-):p53(-/-). The Brca1(-/-):p53(-/-) MEFs appear to be extremely sensitive to ionizing radiation. T e contribution of BRCA1 in NHEJ was evaluated in these cells using three different assay systems. First, transfection of a linearized plasmid in which expression of the reporter gene required precise end joining indicated that Brca1(-/-) MEFs display a moderate deficiency when compared with Brca1(+/+) cells. Second, using a retrovirus infection assay dependent on NHEJ, a 5-10-fold reduction in retroviral integration efficiency was observed in Brca1(-/-) MEFs when compared with the Brca1(+/+) MEFs. Third, Brca1(-/-) MEFs exhibited a 50-100-fold deficiency in microhomology-mediated end-joining activity of a defined chromosomal DNA double strand break introduced by a rare cutting endonuclease I-SceI. These results provide evidence that Brca1 has an essential role in microhomology-mediated end joining and suggest a novel molecular basis for its caretaker role in the maintenance of genome integrity.
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收藏
页码:28641 / 28647
页数:7
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