| Project/Area Number |
08680722
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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 |
Biophysics
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| Research Institution | Osaka University |
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Principal Investigator |
YAMAGATA Yuriko Osaka University, Faculty of Pharmaceutical Sciences, Researcher, 薬学部, 助手 (40183678)
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| Project Period (FY) |
1996 – 1997
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| Project Status |
Completed (Fiscal Year 1997)
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| Budget Amount *help |
¥2,500,000 (Direct Cost: ¥2,500,000)
Fiscal Year 1997: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1996: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Keywords | natural mutations / 8-oxo-dGTPase / X-ray structure / DNA修復 |
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| Research Abstract |
Escherichia coli MutT and human MutT homologous proteins possess enzyme activity to hydrolyze 8-oxo-dGTP and 8-oxo-GTP to the corresponding nucleoside monophosphates and thus are responsible for preventing the replicational and transcriptional errors with 8-oxo-G : A mispair. In order to elucidate the molecular recognition of 8-oxo-guanine and the cleavage of the phosphodiester bond by the enzymes, we crystallized the two enzymes and carried out the X-ray diffraction study of E.coli MutT.
The MutT protein was purified and the determination of the preliminary crystallization conditions for the protein was performed using sparse matrix protocols. The crystals that were suitable for the X-ray diffraction study were obtained from the conditions with 1.6 M Na, K tartrate as precipitants, 100 mM HEPES buffer (pH7.5) and in the presence or absence of 20 mM MnCl_2. The all-Xray diffraction data were collected with Wessenberg camera on Beamlines 18B and 6B of Photon Factory in Tukuba and process
… More
ed with the programs Denzo and Scalepack. The MutT free crystals belong to the monoclinic space group P2_1 with cell dimensions a=56.5, b=73.4, c=34.5 and beta=98.7ﾟ, and contain two molecules in the asymmetric unit. The native data set up to 2.2 showed the R_<oxrgc> of 3.8%, completeness of 88.6%. Several heavy-atom compounds were tested as derivatives, but only K_2PtCl_4 was found to be useful and the X-ray date of the derivative crystal were collected. The initial SIR phases were improved by solvent flattening using the program DM in the CCP4 program suite, and the phases were gradually extended to 2.2 resolution. The free R factor was 0.30. The electron density map shows the definite boundary of molecules and some secondary structures. Now we are building the molecular model using the program TOM.The MutT crystals with Mn^<2+> have a different crystal form from MutT-free crystals. So we will determine the structure of Mn^<2+>-MutT complex by the molecular replacement method using the MutT free structure as the search model after the determination of the MutT free structure. And we will analyze the crystal of 8-oxo-dGMP-MutT complex using the soaking method.
The human MutT homologous protein was purified and crystallized. The small crystals were obtained from the conditions with PEG8000 as precipitants. We are trying to grow bigger crystals. Less
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