title: Cell cycle-dependent induction of homologous recombination by a tightly regulated I-SceI fusion protein.
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.
authors.affiliation: Department of Medicine, Harvard Medical School and Beth Israel Deaconess Medical Center, Boston, Massachusetts, United States of America.
authors.forename: Andrea
authors.initials: A
authors.lastname: Hartlerode
authors.forename: Shobu
authors.initials: S
authors.lastname: Odate
authors.forename: Inbo
authors.initials: I
authors.lastname: Shim
authors.forename: Jenifer
authors.initials: J
authors.lastname: Brown
authors.forename: Ralph
authors.initials: R
authors.lastname: Scully
mesh_terms: D019313:BRCA1 Protein / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects / Q000235:genetics*; D045744:Cell Line, Tumor; D019457:Chromosome Breakage / Q000187:drug effects; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D011960:Receptors, Estrogen / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D011995:Recombination, Genetic / Q000187:drug effects; D016196:S Phase / Q000187:drug effects; D013629:Tamoxifen / Q000031:analogs & derivatives / Q000494:pharmacology; D013997:Time Factors
chemical_list: D019313:BRCA1 Protein; D011960:Receptors, Estrogen; D011993:Recombinant Fusion Proteins; D013629:Tamoxifen; D049452:Green Fluorescent Proteins; C016601:afimoxifene; D015252:Deoxyribonucleases, Type II Site-Specific