title: Mutations in the FHA-domain of ectopically expressed NBS1 lead to radiosensitization and to no increase in somatic mutation rates via a partial suppression of homologous recombination.
abstract: Ionizing radiation induces DNA double-strand breaks (DSBs). Mammalian cells repair DSBs through multiple pathways, and the repair pathway that is utilized may affect cellular radiation sensitivity. In this study, we examined effects on cellular radiosensitivity resulting from functional alterations in homologous recombination (HR). HR was inhibited by overexpression of the forkhead-associated (FHA) domain-mutated NBS1 (G27D/R28D: FHA-2D) protein in HeLa cells or in hamster cells carrying a human X-chromosome. Cells expressing FHA-2D presented partially (but significantly) HR-deficient phenotypes, which were assayed by the reduction of gene conversion frequencies measured with a reporter assay, a decrease in radiation-induced Mre11 foci formation, and hypersensitivity to camptothecin treatments. Interestingly, ectopic expression of FHA-2D did not increase the frequency of radiation-induced somatic mutations at the HPRT locus, suggesting that a partial reduction of HR efficiency has only a slight effect on genomic stability. The expression of FHA-2D rendered the exponentially growing cell population slightly (but significantly) more sensitive to ionizing radiation. This radiosensitization effect due to the expression of FHA-2D was enhanced when the cells were irradiated with split doses delivered at 24-h intervals. Furthermore, enhancement of radiation sensitivity by split dose irradiation was not seen in contact-inhibited G0/G1 populations, even though the cells expressed FHA-2D. These results suggest that the FHA domain of NBS1 might be an effective molecular target that can be used to induce radiosensitization using low molecular weight chemicals, and that partial inhibition of HR might improve the effectiveness of cancer radiotherapy.
authors.affiliation: Department of Biological Sciences, Faculty of Science, Ibaraki University, Bunkyo 2-1-1, Mito, Ibaraki 310-8512, Japan.
authors.forename: Maki
authors.initials: M
authors.lastname: Ohara
authors.affiliation: Department of Biological Sciences, Faculty of Science, Ibaraki University, Bunkyo 2-1-1, Mito, Ibaraki 310-8512, Japan.
authors.forename: Yumi
authors.initials: Y
authors.lastname: Funyu
authors.affiliation: Department of Biological Sciences, Faculty of Science, Ibaraki University, Bunkyo 2-1-1, Mito, Ibaraki 310-8512, Japan.
authors.forename: Shunsuke
authors.initials: S
authors.lastname: Ebara
authors.affiliation: Department of Biological Sciences, Faculty of Science, Ibaraki University, Bunkyo 2-1-1, Mito, Ibaraki 310-8512, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Sakamoto
authors.affiliation: Department of Biological Sciences, Faculty of Science, Ibaraki University, Bunkyo 2-1-1, Mito, Ibaraki 310-8512, Japan.
authors.forename: Ryota
authors.initials: R
authors.lastname: Seki
authors.affiliation: Department of Biological Sciences, Faculty of Science, Ibaraki University, Bunkyo 2-1-1, Mito, Ibaraki 310-8512, Japan.
authors.forename: Kenta
authors.initials: K
authors.lastname: Iijima
authors.affiliation: Department of Biological Sciences, Faculty of Science, Ibaraki University, Bunkyo 2-1-1, Mito, Ibaraki 310-8512, Japan.
authors.forename: Akiko
authors.initials: A
authors.lastname: Ohishi
authors.affiliation: Department of Genome Repair Dynamics, Radiation Biology Center, Kyoto University, Yoshida-Konoe Cho, Sakyo-ku, Kyoto 606-8501 Japan.
authors.forename: Junya
authors.initials: J
authors.lastname: Kobayashi
authors.affiliation: Department of Genome Repair Dynamics, Radiation Biology Center, Kyoto University, Yoshida-Konoe Cho, Sakyo-ku, Kyoto 606-8501 Japan.
authors.forename: Kenshi
authors.initials: K
authors.lastname: Komatsu
authors.affiliation: Department of Biological Sciences, Faculty of Science, Ibaraki University, Bunkyo 2-1-1, Mito, Ibaraki 310-8512, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Tachibana
authors.affiliation: Department of Biological Sciences, Faculty of Science, Ibaraki University, Bunkyo 2-1-1, Mito, Ibaraki 310-8512, Japan htauchi@mx.ibaraki.ac.jp.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Tauchi
mesh_terms: D000818:Animals; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics*; D002460:Cell Line; D006224:Cricetinae; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair / Q000235:genetics / Q000528:radiation effects; D006367:HeLa Cells; D059765:Homologous Recombination; D006801:Humans; D009154:Mutation; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics*; D017434:Protein Structure, Tertiary; D011836:Radiation Tolerance / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
chemical_list: D018797:Cell Cycle Proteins; C112156:NBN protein, human; D009687:Nuclear Proteins; D011994:Recombinant Proteins
keywords: NBS1; homologous recombination; radiosensitization; split dose irradiation