Site-directed mutational analysis of structural interactions of low molecule compounds binding to the N-terminal 8 kDa domain of DNA polymerase beta
| Autor: | Kengo Sakaguchi, Shinji Kamisuki, Keisuke Ohta, Shizuka Murakami, Yoshiyuki Mizushina, Kei Ichi Takata, Nobuyuki Kasai, Fumio Sugawara, Seisuke Kimura |
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| Rok vydání: | 2006 |
| Předmět: |
Models
Molecular Lithocholic acid DNA polymerase Biophysics Biochemistry Fatty Acids Monounsaturated chemistry.chemical_compound Animals Electrophoretic mobility shift assay Amino Acids Enzyme Inhibitors Molecular Biology Nuclear Magnetic Resonance Biomolecular DNA Polymerase beta chemistry.chemical_classification biology Fatty acid Cell Biology DNA-binding domain Peptide Fragments Amino acid Protein Structure Tertiary Rats Molecular Weight chemistry biology.protein Mutagenesis Site-Directed Lithocholic Acid DNA Nervonic acid Protein Binding |
| Zdroj: | Biochemical and biophysical research communications. 350(1) |
| ISSN: | 0006-291X |
| Popis: | We previously reported the mode of inhibition of DNA polymerase {beta} (pol. {beta}) by long chain fatty acids and a bile acid, involving binding analyses to the N-terminal 8-kDa DNA binding domain. Here we describe a site-directed mutational analysis in which the key amino acids (L11, K35, H51, K60, L77, and T79), which are direct interaction sites in the domain, were substituted with K, A, A, A, K, and A, respectively. And their pol. {beta} interactions with a C24-long chain fatty acid, nervonic acid (NA), and a bile acid, lithocholic acid (LCA), were investigated by gel mobility shift assay and NMR spectroscopy. In the case of K35A, there was complete loss of DNA binding activity while K60A hardly has any activity. In contrast the other mutations had no appreciable effects. Thus, K35 and K60 are key amino acid sites for binding to template DNA. The DNA binding activities of L11K, H51A, and T79A as well as the wild type were inhibited by NA to the same extent. T79A demonstrated a disturbed interaction with LCA. {sup 1}H-{sup 15}N HSQC NMR analysis indicated that despite their many similarities, the wild-type and the mutant proteins displayed some significant chemical shift differences. Not onlymore » were the substituted amino acid residues three-dimensionally shifted, but some amino acids which are positioned far distant from the key amino acids showed a shift. These results suggest that the interaction surface was significantly distorted with the result that LCA could not bind to the domain. These findings confirm our previous biochemical and 3D structural proposals concerning inhibition by NA and LCA.« less |
| Databáze: | OpenAIRE |
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