[“Metabolism Summary

Other attempts to determine the function of BRCA have centered on finding functional domains of BRCA.BRCA has been shown to interact with the basal transcriptional machinery. Cells that lack BRCA have been shown to be hypersensitive to ionizing radiation and have a decreased capacity to repair doublestranded DNA breaks. When coupled with the finding that BRCA mouse cells are deficient in TCR, all these findings suggest a role for BRCA in the cellular response to DNA damage.To determine the role of BRCA in the cellular response to DNA damage, we have developed a human genetic system.HCC is a human breast cancer cell line originally established from a family carrying a known cancercausing BRCA mutation, insC.After weeks of colony growth, the cells were stained, and colonies were counted manually.Survival of the mockexposed cells from each cell line was set equal to, and the irradiated cell survival was standardized to the survival of the mockexposed cells from each line.V cells are a cell line generated by our lab from a breast cancer patient who carries an exon BRCA mutation. The colony forming assay was performed as described previously assay was performed as described with the following changes.A, the deletion mutants generated and used to reverse the radiation sensitivity of HCC cells are depicted.BRCAD is the smallest deletion mutant able to destroy the ability of BRCA to suppress growth.BRCAD deletes a central region of BRCA but leaves the BRCT domain and the transactivation domains intact.BRCAD destroys a portion of the transactivation domain.The ability of these deletion mutants to suppress growth was measured by transfecting the mutants into HCC cells and selecting for weeks in G; BRCAD; and BRCAD. B, HCC cells were transfected with the various deletion mutants, acutely irradiated with the indicated dose of ionizing radiation hlater, and then allowed to grow for days.. HCC cells stably transfected with BRCAD are radiationresistant.To study DNA repair and the effect of BRCA on that repair, HCC cells were transfected with BRCAD. Transfected cells were selected for weeks in G, and individual colonies were propagated.Thus these antibodies are selective for only proteins expressed from the transfected BRCA mutant genes.Each experiment was performed three times with similar results each time.D, doublestand DNA repair assays were performed as described previously. Under these pulsed field gel electrophoretic conditions, damaged DNA runs as a discrete band below that of undamaged or repaired DNA.Upper panel, HCC cells show close to complete repair of doublestranded DNA breaks within hafter ionizing radiation exposure.Lower panel, HCC BRCAD cells show similar repair of doublestranded DNA breaks within hof IR exposure.Cells were then washed three times with phosphatebuffered saline, and DNA purification was performed by standard methods. Aliquots of both the bound and unbound DNA fractions were scintillation counted to determine the total amount of repaired DNA.Both bound and unbound DNA samples from each time point were then electrophoresed on a agarose gel, blotted to nylon membrane, and probed with strandspecific probes for the human DHFR gene.The functional allele in these cells contains a frameshift mutation that deletes the BRCT domain of BRCA.

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