| Project/Area Number |
08101001
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| Research Category |
Grant-in-Aid for Specially Promoted Research
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| Allocation Type | Single-year Grants |
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| Review Section |
Chemistry
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| Research Institution | Hokkaido University |
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Principal Investigator |
OHTSUKA Eiko Graduate School of Pharmaceutical Sciences, Hokkaido University, Professor, 大学院・薬学研究科, 教授 (80028836)
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| Co-Investigator(Kenkyū-buntansha) |
SHIMADA Ichio Graduate School of Pharmaceutical Sciences, University of Tokyo, Professor, 大学院・薬学系研究科, 教授 (70196476)
SATO Yoshinori Graduate School of Pharmaceutical Sciences, University of Tokyo, Professor, 大学院・薬学系研究科, 教授 (30150014)
NIKAIDO Osamu Faculty of Pharmaceutical Sciences, Kanagawa University, Professor, 薬学部, 教授 (60019669)
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| Project Period (FY) |
1996 – 1998
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| Project Status |
Completed (Fiscal Year 1998)
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| Budget Amount *help |
¥292,000,000 (Direct Cost: ¥292,000,000)
Fiscal Year 1998: ¥58,000,000 (Direct Cost: ¥58,000,000)
Fiscal Year 1997: ¥110,000,000 (Direct Cost: ¥110,000,000)
Fiscal Year 1996: ¥124,000,000 (Direct Cost: ¥124,000,000)
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| Keywords | UV-damaged DNA / Molecular recognition / Single-chain antibody / X-ray crystallography / NMR / 光損傷DNA / 抗光損傷DNA抗体 / 合成DNA / X線結晶学 |
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| Research Abstract |
DNA damage induced by ultraviolet (UV) light, alkylating agents, radioactive decay or reactive oxygen is considered to be a principal cause of mutation, neoplastic cellular transformation and cell death. At adjacent pyridine sites in DNA, three major classes of UV-induced photoproducts, i.e. cis-syncyclobutane pyrimidine dimers (CPDs), pyrimidine (6-4) pyrimidone photoproducts [(6-4)photoproducts] and the Dewar isomers are formed. Monoclonal antibodies have been used as a means to detect and quantitative these DNA photolesions. We have been studying a series of monoclonal antibodies that specifically recognize either CPDs or (6-4) photoproducts.
In order to understand the molecular recognition of the antibodies, we have cloned and sequenced the variable region genes. Comparing these sequences revealed that all four anti-(6-4) photoproduct antibodies were highly similar to one another, although there were some differences in potential antigen-contact regions. Computer modeling have shown
… More
that most of the sequence differences occurred in potential antigen contact regions, suggesting specific positions that might account for the observed differences in binding affinities and selectivities. Oligonucleotides containing either CPDs or (6-4) photoproducts as the antigens were chemically synthesized. Single-chain Fv (scFv) derivatives of these antibodies were constructed and characterized to provide an experimental system in which structure-function relationships can be tested. These derivatives could be isolated from E.coli using metal affinity chromatographic steps and possessed the same binding specificities as the parent monoclonal antibodies. Mutagenesis studies of complementarity-determining regions (CDRs) of scFv specific for (6-4) photoproduct (64M5scFv) revealed several amino acid residues that play an important role in DNA photoproduct binding by 64M5 antibody. Three-dimensional structures of the Fab fragments for antibodies 64M2, 64M3 and 64M5 have been analyzed by use of X-ray crystallography. Among these Fabs, 64M2 Fab was obtained as a form complexed with a T(6-4)T ligand and clearly elucidated the structure of the bound ligand as well as the ligand recognition mode. Multinuclear NMR studies of interactions between UV-damaged DNA and the Fab fragments for antibodies 64M3 and 64M5 have shown the structural information on the antigen binding sites and dynamic binding mechanisms of these antibodies. Less
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