PMC:138691 / 3111-7457 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/138691","sourcedb":"PMC","sourceid":"138691","source_url":"http://www.ncbi.nlm.nih.gov/pmc/138691","text":"Measuring DSB-induced recombination frequency in BRCA2-defective cells\nPierce et al have designed a set of recombination substrates for measuring the level of homologous recombination in vivo (Fig. 1) [25]. The DNA substrate contains a pair of mutated GFP genes (GFP encodes the easily detected green fluorescent protein), one of which contains a restriction site for I-SceI, a yeast intron encoded endonuclease with an 18 base pair recognition site. Transient transfection of an I-SceI expression vector results in the production of a DSB in the first mutated copy of GFP. One or both DNA ends formed by the break invade(s) the homologous sequence in the second mutant GFP copy, resulting in repair of the DSB via a homology-mediated gene conversion event. The configuration of the GFP construct is such that homology-mediated repair often results in the formation of a functional copy of GFP. Such events can be detected by fluorescence-activated cell sorting analysis by virtue of their expression of GFP.\nFigure 1 Recombination substrates used for assaying homology-directed repair. Cutting at the I-SceI site within the mutant GFP (SceGFP) results in a double-strand break that can be repaired through homologous gene conversion using a 3'-truncated copy of GFP as sequence donor. The mechanism results in the formation of a functional copy of the GFP gene. The model shown assumes gene conversion occurs via the synthesis-dependent annealing mechanism. Moynahan et al [24] have used such a GFP recombination substrate to demonstrate that cells with defective BRCA2 protein are deficient in their ability to repair the I-SceI-induced DSB through homologous recombination. Expression of I-SceI resulted in 1 out of 1400 cells producing GFP via homologous recombination in the human pancreatic tumor cell line CAPAN-1. CAPAN-1 cells carry a deletion of BRCA2 on one homolog and codes for a protein truncated at amino acid 1981 on the other homolog. The authors indicate that the level of I-SceI-induced recombination in CAPAN-1 is over 100-fold less than that seen using other (BRCA2+) human tumor cell lines. The different lines examined, however, are very likely to differ genetically from CAPAN-1 cells not only at the BRCA2 locus, but also at a very large number of additional loci. This raises the possibility that some or even all of the recombinational repair defect seen in CAPAN-1 could be due to mutations at non-BRCA2 loci.\nWhile Moynahan et al were careful to point out this problem, two sets of results argue against the possibility that the recombinational repair deficiency of CAPAN-1 cells is completely independent of its defect in BRCA2. First, in the same study [24], recombination in a mouse ES cell line that expresses only truncated BRCA2 protein was measured. This line was found to have lower recombination efficiency than isogenic cells expressing full-length BRCA2 (the defect observed was about fivefold to sixfold). Second, in an independent study, Powell was able to compare derivatives of CAPAN-1 that differed only in their ability to express full-length BRCA2 protein (S. Powell, personal communication, 2001). In these experiments, the derivative expressing full-length BRCA2 yielded 10-fold more recombinants than controls expressing only truncated BRCA2 (S. Powell, personal communication, 2001). While the experimental design of these experiments was somewhat different from that of Moyna-han et al [24], the results raise the possibility that CAPAN-1 cells have more than one mutation that lowers the efficiency of recombinational repair relative to that observed in other human cell lines. Conversely, the BRCA2 defect in CAPAN-1 could be fully responsible for the 100-fold defect in recombination if the level of BRCA2 complementation observed by Powell was incomplete. Furthermore, the discrepancy between the 100-fold difference between CAPAN-1 and the other human lines as compared with the fivefold to sixfold difference between the Brca2lex1/lex2cells and Brca2+/+ ES cells might simply be accounted for by the fact that Brca2lex1/lex2 is not a null allele. Additional studies are likely to shed light on the efficiency of BRCA2-independent recombination pathways. Taken together, the results indicate that BRCA2 is indeed required for high levels of recombinational repair in both human and 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