title: Monoacetylcurcumin: a new inhibitor of eukaryotic DNA polymerase lambda and a new ligand for inhibitor-affinity chromatography.
abstract: We previously reported that a phenolic compound, curcumin (diferuloylmethane), was a selective inhibitor of DNA polymerase lambda (pol lambda) in vitro [Y. Mizushina, M. Hirota, C. Murakami, T. Ishidoh, S. Kamisuki, N. Shimazaki, M. Takemura, M. Perpelescu, M. Suzuki, H. Yoshida, F. Sugawara, O. Koiwai, K. Sakaguchi, Some anti-chronic inflammatory compounds are DNA polymerase lambda-specific inhibitors, Biochem. Pharmacol. 66 (2003) 1935-1944.]. We also found that monoacetylcurcumin ([1E,4Z,6E]-7-(4''-acetoxy-3''-methoxyphenyl)-5-hydroxy-1-(4'-hydroxy-3'-methoxyphenyl)hepta-1,4,6-trien-3-on), a chemically synthesized derivative of curcumin, was a stronger pol lambda inhibitor than curcumin, achieving 50% inhibition at a concentration of 3.9microM. Monoacetylcurcumin did not influence the activities of replicative pols such as alpha, delta, and epsilon, and showed no effect even on the activity of pol beta, the three-dimensional structure of which is thought to be highly similar to that of pol lambda. The compound-induced inhibition of pol lambda activity was non-competitive with respect to both the DNA template-primer and the dNTP substrate. Monoacetylcurcumin did not inhibit the activity of the C-terminal catalytic domain of pol lambda including the pol beta-like core, in which the BRCT motif was deleted. The compound did not influence the activities of prokaryotic pols or other mammalian DNA metabolizing enzymes such as calf primase of pol alpha, calf terminal deoxynucleotidyl transferase, human telomerase, human immunodeficiency virus type-1 reverse transcriptase, T7 RNA polymerase, T4 polynucleotide kinase, and bovine deoxyribonuclease I. Therefore, we concluded that monoacetylcurcumin is a selective inhibitor of pol lambda and could be used as a chromatographic ligand to purify pol lambda. We then made a monoacetylcurcumin-conjugated column with epoxy-activated Sepharose 6B. In the column, pol lambda of full length was selectively adsorbed and eluted.
authors.affiliation: Laboratory of Food and Nutritional Sciences, Department of Nutritional Science, Kobe-Gakuin University, Nishi-ku, Kobe, Hyogo 651-2180, Japan. mizushin@nutr.kobegakuin.ac.jp
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Mizushina
authors.forename: Tomomi
authors.initials: T
authors.lastname: Ishidoh
authors.forename: Toshifumi
authors.initials: T
authors.lastname: Takeuchi
authors.forename: Noriko
authors.initials: N
authors.lastname: Shimazaki
authors.forename: Osamu
authors.initials: O
authors.lastname: Koiwai
authors.forename: Kouji
authors.initials: K
authors.lastname: Kuramochi
authors.forename: Susumu
authors.initials: S
authors.lastname: Kobayashi
authors.forename: Fumio
authors.initials: F
authors.lastname: Sugawara
authors.forename: Kengo
authors.initials: K
authors.lastname: Sakaguchi
authors.forename: Hiromi
authors.initials: H
authors.lastname: Yoshida
mesh_terms: D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods*; D003474:Curcumin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D019951:DNA Polymerase beta / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D004331:Drosophila melanogaster; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D020128:Inhibitory Concentration 50; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D015394:Molecular Structure; D051381:Rats
chemical_list: D004791:Enzyme Inhibitors; D007527:Isoenzymes; D008024:Ligands; C506547:monoacetylcurcumin; C415367:DNA polymerase beta2; D019951:DNA Polymerase beta; D003474:Curcumin