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Sulfoquinovosylmonoacylglycerol inhibitory mode analysis of rat DNA polymerase β
Author(s) -
Kasai Nobuyuki,
Mizushina Yoshiyuki,
Murata Hiroshi,
Yamazaki Takayuki,
Ohkubo Tadayasu,
Sakaguchi Kengo,
Sugawara Fumio
Publication year - 2005
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2005.04848.x
Subject(s) - dna polymerase beta , dna polymerase , biochemistry , polymerase , primer (cosmetics) , dna , binding site , microbiology and biotechnology , base excision repair , chemistry , stereochemistry , biology , dna repair , organic chemistry
We have previously reported that sulfoquinovosylmonoacylglycerol (SQMG) is a potent inhibitor of mammalian DNA polymerases. DNA polymerase β (pol β) 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 β. SQMG competed with both the substrate and the template‐primer for binding to pol β. 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 β, subsequently inhibiting its catalytic activity. Fragments of SQMG such as sulfoquinovosylglycerol (SQG) and fatty acid (myristoleic acid, MA) weakly inhibited pol β 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.