Abstract
Double-strand break repair is executed by two major repair pathways: non-homologous end joining (NHEJ) and homologous recombination (HR). Whereas NHEJ contributes to the repair of ionizing radiation (IR)-induced double strand breaks (DSBs) throughout the cell cycle, HR acts predominantly during the S and G2 phases of the cell cycle. The rare-cutting restriction endonuclease, I-SceI, is in common use to study the repair of site-specific chromosomal DSBs in vertebrate cells. To facilitate analysis of I-SceI-induced DSB repair, we have developed a stably expressed I-SceI fusion protein that enables precise temporal control of I-SceI activation, and correspondingly tight control of the timing of onset of site-specific chromosome breakage. I-SceI-induced HR showed a strong, positive linear correlation with the percentage of cells in S phase, and was negatively correlated with the G1 fraction. Acute depletion of BRCA1, a key regulator of HR, disrupted the relationship between S phase fraction and I-SceI-induced HR, consistent with the hypothesis that BRCA1 regulates HR during S phase.
Highlights
Repair of mammalian chromosomal double-strand breaks (DSBs) entails use of one of two major repair pathways: nonhomologous end-joining (NHEJ) and homologous recombination (HR)
Expression of I-SceI induces a site-specific DSB within the reporter and stimulates HR
Recombination between the two GFP copies by either intra- or inter-chromatid HR (Figure 1A), but not single strand annealing, can generate wild type GFP, which is readily quantified by flow cytometry
Summary
Repair of mammalian chromosomal double-strand breaks (DSBs) entails use of one of two major repair pathways: nonhomologous end-joining (NHEJ) and homologous recombination (HR) (reviewed in [1,2,3,4]). These repair functions take place in relation to other chromosome processes such as transcription and DNA replication, and DNA replication alters the parameters governing DSB repair in several ways. In budding yeast, where HR is the major DSB repair pathway, repair of ionizing radiation (IR)-induced DSBs engages SCR in preference to interhomolog recombination [7]. In contrast to the constraints on HR, NHEJ can, in principle, operate at any stage of the cell cycle
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