2006 Fiscal Year Final Research Report Summary
Induction of single DNA double strand break in animal cell genome and its monitoring methodology
| Project/Area Number |
17590279
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Pathological medical chemistry
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| Research Institution | HIROSHIMA UNIVERSITY (2006)
Kawasaki Medical School (2005) |
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Principal Investigator |
TAKATA Minoru Hiroshima University, Research Institute for Radiation Biology and Medicine, Professor, 原爆放射線医科学研究所, 教授 (30281728)
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| Co-Investigator(Kenkyū-buntansha) |
KITAO Hiroyuki Research Institute for Radiation Biology and Medicine, Postdoctoral Researcher for COE program, 原爆放射線医科学研究所, COE研究員 (30368617)
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| Project Period (FY) |
2005 – 2006
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| Keywords | DNA double strand break / DNA repair / I-SceI / homologous recombination / non-homologous end joining / estrogen receptor / tamoxifen |
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| Research Abstract |
Background :
The human genome is under constant attack by (1) exogenous DNA damage such as double stranded breaks inflicted by ionizing irradiation, and (2) endogenous DNA damage originating in stalled or collapsed replication forks. It is still unknown how DNA lesions such as DNA double strand break are actually repaired in mammalian cells. The reason for this is lack of appropriate technology to induce DSB simultaneously at the defined chromosomal site. In yeast it is feasible to create a single chromosomal DSB by using expression of HO endonuclease under the regulation of strong inducible promoter.
Methods :
In this study, we wished to develop such system using two technologies:(1) Recombinant endonuclease I-SceI tagged with membrane translocation sequence TAT, which is purifed from E.coli expression system. By adding this protein to culture medium, it is expected to translocate cell membrane and eventually be transported to the nucleus. (2) Stable expression of I-SceI fused with mutat
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ed ligand-biding domain of estrogen receptor (Mer). In normal condition, this protein is maintained in cytoplasm, but upon addition of estrogen analog tamoxifen, the protein is expected to move rapidly into the nucleus. We used chicken DT40 cell line in which recombination substrate SCneo was knocked-in to OVA locus. The SCneo has 18-bp recognition sequence for I-SceI, and I-SceI induced DSB can be repaired through homologous recombination using upstream nonfunctional neo segment as a template, conversing the cell to reo-resistant.
Results :
(1) We purified TAT-I-SceI fusion protein from E.coli, however, it precipitated and probably because of this, it was nonfunctional, In our hands, we were not able to solve this problem.
(2) We prepared three kinds of constructs (Mer-I-SceI, Mer-I-EceI-Mer, I-SceI-Mer), and expressed each of them into DT40 harboring SCneo. We could show rapid translocation of the Mer-I-EceI-Mer protein from cytoplasm to nucleus using immunocytochemistry, however, appearance of neo-resistant colony was less than expected. In all of the fusion constructs the rate of HR repair (conversion to neo resistance) was low compared to transient transfection of I-SceI. Consistently, we were able to detect DSB by ligation-mediated PCR but not by Southern blotting.
Conclusion :
We created a system that can induce chromosomal DSB by adding tamoxifen to culture media, however, its efficiency is not high enough to allow real time monitoring of DSB repair. Less
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Research Products
(15 results)
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[Journal Article] A requirement of FancL and FancD2 monoubiquitination in DNA repair.2007
Author(s)
Seki S, Ohzeki M, Uchida A, Hirano S, Matsushita N, Kitao H, Oda T, Yamashita T, Kashihara N, Tsubahara A, Takata M, Ishiai M.
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Journal Title
Genes Cells. 12(3)
Pages: 299-310
Description
「研究成果報告書概要(欧文)」より
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[Journal Article] Activation of downstream epidermal growth factor receptor (EGFR) signaling provides gefitinib-resistance in cells carrying EGFR mutation.2007
Author(s)
Uchida A, Hirano S, Kitao H, Ogino A, Rai K, Toyooka S, Takigawa N, Tabata M, Takata M, Kiura K, Tanimoto M.
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Journal Title
Cancer Sci. 98(3)
Pages: 357-63
Description
「研究成果報告書概要(欧文)」より
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[Journal Article] FAAP100 is essential for activation of the Fanconi anemia-associated DNA damage response pathway.2007
Author(s)
Ling C, Ishiai M, Ali AM, Medhurst AL, Neveling K, Kalb R, Yan Z, Xue Y, Oostra AB, Auerbach AD, Hoatlin ME, Schindler D, Joenje H, de Winter JP, Takata M, Meetei AR, Wang W.
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Journal Title
EMBO J. [Epub ahead of print]
Description
「研究成果報告書概要(欧文)」より
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[Journal Article] 53BP1 contributes to survival of cells irradiated with X-ray during G1 without Ku70 or Artemis.2006
Author(s)
Iwabuchi K, Hashimoto M, Matsui T, Kurihara T, Shimizu H, Adachi N, Ishiai M, Yamamoto K, Tauchi H, Takata M, Koyama H, Date T.
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Journal Title
Genes Cells. 11(8)
Pages: 935-48
Description
「研究成果報告書概要(欧文)」より
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[Journal Article] A FancD2-Monoubiquitin Fusion Reveals Hidden Functions of Fanconi Anemia Core Complex in DNA Repair.2005
Author(s)
Matsushita N, Kitao H, Ishiai M, Nagashima N, Hirano S, Okawa K, Ohta T, Yu DS, McHugh PJ, Hickson ID, Venkitaraman AR, Kurumizaka H, Takata M.
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Journal Title
Mol Cell. 19(6)
Pages: 841-7
Description
「研究成果報告書概要(欧文)」より
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[Journal Article] Multiple repair pathways mediate tolerance to chemotherapeutic cross-linking agents in vertebrate cells.2005
Author(s)
Nojima K, Hochegger H, Saberi A, Fukushima T, Kikuchi K, Yoshimura M, Orelli BJ, Bishop DK, Hirano S, Ohzeki M, Ishiai M, Yamamoto K, Takata M, Arakawa H, Buerstedde JM, Yamazoe M, Kawamoto T, Araki K, Takahashi JA, Hashimoto N, Takeda S, Sonoda E.
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Journal Title
Cancer Res. 65(24)
Pages: 11704-11
Description
「研究成果報告書概要(欧文)」より
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