Sulfoquinovosylmonoacylglycerol inhibitory mode analysis of rat DNA polymerase beta
| Autor: | Takayuki Yamazaki, Yoshiyuki Mizushina, Kengo Sakaguchi, Tadayasu Ohkubo, Hiroshi Murata, Fumio Sugawara, Nobuyuki Kasai |
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| Rok vydání: | 2005 |
| Předmět: |
Models
Molecular DNA damage DNA polymerase Electrophoretic Mobility Shift Assay DNA polymerase beta In Vitro Techniques Binding Competitive Biochemistry Fatty Acids Monounsaturated chemistry.chemical_compound Animals Electrophoretic mobility shift assay Enzyme Inhibitors Binding site Nuclear Magnetic Resonance Biomolecular Molecular Biology DNA Polymerase beta Polymerase Binding Sites biology DNA Cell Biology Base excision repair Recombinant Proteins Protein Structure Tertiary Rats Kinetics chemistry Mutagenesis Site-Directed biology.protein Glycolipids |
| Zdroj: | FEBS Journal. 272:4349-4361 |
| ISSN: | 1742-4658 1742-464X |
| Popis: | We have previously reported that sulfoquinovosylmonoacylglycerol (SQMG) is a potent inhibitor of mammalian DNA polymerases. DNA polymerase beta (pol beta) is one of the most important enzymes protecting the cell against DNA damage by base excision repair. In this study, we characterized the inhibitory action of SQMG against rat pol beta. SQMG competed with both the substrate and the template-primer for binding to pol beta. A gel mobility shift assay and a polymerase activity assay showed that SQMG competed with DNA for a binding site on the N-terminal 8-kDa domain of pol beta, subsequently inhibiting its catalytic activity. Fragments of SQMG such as sulfoquinovosylglycerol (SQG) and fatty acid (myristoleic acid, MA) weakly inhibited pol beta activity and the inhibitory effect of a mixture of SQG and MA was stronger than that of SQG or MA. To characterize this inhibition more precisely, we attempted to identify the interaction interface between SQMG and the 8-kDa domain by NMR chemical shift mapping. Firstly, we determined the binding site on a fragment of SQMG, the SQG moiety. We observed chemical shift changes primarily at two sites, the residues comprising the C-terminus of helix-1 and the N-terminus of helix-2, and residues in helix-4. Finally, based on our present results and our previously reported study of the interaction interface of fatty acids, we constructed two three-dimensional models of a complex between the 8-kDa domain and SQMG and evaluated them by the mutational analysis. The models show a SQMG interaction interface that is consistent with the data. |
| Databáze: | OpenAIRE |
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