Role of Brca1 in Nonhomologous DNA End Joining
Final rept. 11 Aug 2003-10 Aug 2004
VIRGINIA COMMONWEALTH UNIV RICHMOND
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To examine the possible role of the breast cancer susceptibility factor BRCA1 in the nonhomologous end joining pathway of DNA double-strand break repair, an assay to detect both accurate and inaccurate end joining events in intact cells was devised. A fusion gene of green fluorescent protein EGFP and neomycin resistance nec was constructed with a linker containing cleavage sites for the enzymes BcgI and 1-Scel. EcgI induces two simultaneous breaks, excising a 34-bp segment of DNA. Accurate repair of such a break would be detected as a 34-bp deletion that would put neo back in-frame and confer G4l8 resistance. Inaccurate repair events of both BcgI- and 1-Scel-induced breaks would similarly be detected. A lentivirus vector was constructed to introduce the fusion gene into human cells, and transductants of MCF-7 breast tumor cells were isolated. Although development of the assay system is not yet complete, proof-of-principle experiments showed that deletions could indeed be generated by 1-Scel-induced cleavage and detected as reactivation of the neo gene. Intracellular DNA cleavage by BcgI, introduced into cells by electroporation was also demonstrated, although it remains to be determined whether such cleavage will be sufficient to detect repair and assess repair accuracy.
- Medicine and Medical Research