| Project/Area Number |
01571134
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Chemical pharmacy
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| Research Institution | Hokkaido University |
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Principal Investigator |
INOUE Hideo Hokkaido University, Fac. of Pharm. Sci., Associate Professor, 薬学部, 助教授 (80088856)
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| Project Period (FY) |
1989 – 1990
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| Project Status |
Completed (Fiscal Year 1990)
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| Budget Amount *help |
¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1990: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1989: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Keywords | E. coli RNase H / RNA-DNA hybrid / Photoaffinity labeling / Photoactive oligodeoxynucleotide / Crosslinking / Lysyl endopeptidase / Substrate recognition site / 大腸菌RNase H / RNA・DNAハイブリド / 大腸菌RNaseH |
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| Research Abstract |
Photoaffinity labeling of a substrate recognition site of E. Coli RNase H has been studied with photolabile RNA-DNA hybrid substrates. A phenyldiazirine derivative was introduced to DNA oligomer (5'-GTCATCTCC-3') at the 5'-end or the inside. In the latter case, the diazirine group was attached to C2'-modified uridine residue having an amino-linker arm which was introduced instead of T.
Irradiation of a mixture of the photoactive hybrid (the DNA was labeled with ^<32>P) and RNase H gave crosslinked product(s). The product could be separated from RNase H by cation-exchange chromatography, although it was contaminated with starting nucleic acids. The affinity-labeled product from each reaction was digested with lysyl endopeptidase. The resultant peptides were separated by reverse-phase HPLC. Peptide-mapping analysis of labeled RNase Hs showed that crosslinking reactions had occurred at peptide LEP 2, 3 and 4. It has been reported that Asp 10, Glu48 and Asp 70 were crucial for E. coli RNase H activity (these amino acid residues are located at the above peptide fragments). These data suggest that LEP 2, 3 and 4 constitute the substrate recognition site of the enzyme.
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